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Sample records for capsid protein vp1

  1. The Pseudorabies Virus VP1/2 Tegument Protein Is Required for Intracellular Capsid Transport†

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    Luxton, G.W. Gant; Lee, Joy I-Hsuan; Haverlock-Moyns, Sarah; Schober, Joseph Martin; Smith, Gregory Allan

    2006-01-01

    Transport of capsids in cells is critical to alphaherpesvirus infection and pathogenesis; however, viral factors required for transport have yet to be identified. Here we provide a detailed examination of capsid dynamics during the egress phase of infection in Vero cells infected with pseudorabies virus. We demonstrate that the VP1/2 tegument protein is required for processive microtubule-based transport of capsids in the cytoplasm. A second tegument protein that binds to VP1/2, UL37, was nec...

  2. Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

    Science.gov (United States)

    Rodgers, R. E.; Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

  3. Expression of enterovirus 71 capsid protein VP1 in Escherichia coli and its clinical application

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    Mei Shi

    2013-12-01

    Full Text Available The VPl gene of enterovirus 71 (EV71 was synthesized, construct a recombinant plasmid pET15b/VP1 and expressed in E. coli BL21. The recombinant VP1 protein could specifically react with EV71-infected patient sera without the cross-reaction with serum antibodies of coxsackievirus A16 (CA16, A4, A5, B3 and B5 as well as echovirus 6. In acute and convalescent phases, IgM and IgG antibodies of 182 serum samples were detected by ELISA with recombinant VP1 protein as a coated antigen. The results showed that the sensitivity, specificity, positive predictive value (PPV and negative predictive value (NPV of IgM antibodies in serum samples for the diagnosis of EV71 infection were 90.1, 98.4, 98.8 and 88.7%, respectively; similarly, those of IgG antibodies in serum samples were 82.4, 89.1, 91.5 and 78.1%, respectively. Five of 80 samples (6.25% from CA16infected patients were detected positive by ELISA with recombinant VP1 protein in which indicated the cross reactions and 0 of 5 samples from patients infected with other enteroviruses including CA4, CA5, CB3, CB5 and echovirus 6. Therefore, the recombinant VP1 protein of EV7l may provide a theoretical reference for establishing an effective antibody screening of IgM for EV71-infected patients with clinically suspected hand, foot, and mouth disease (HFMD.

  4. A novel finding for enterovirus virulence from the capsid protein VP1 of EV71 circulating in mainland China.

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    Liu, Yongjuan; Fu, Chong; Wu, Suying; Chen, Xiong; Shi, Yingying; Zhou, Bingfei; Zhang, Lianglu; Zhang, Fengfeng; Wang, Zhihao; Zhang, Yingying; Fan, Chengpeng; Han, Song; Yin, Jun; Peng, Biwen; Liu, Wanhong; He, Xiaohua

    2014-04-01

    Enterovirus 71 (EV71) is a neurotropic virus that causes various clinical manifestations in young children, ranging from asymptomatic to fatal. Different pathotypes of EV71 notably differ in virulence. Several virulence determinants of EV71 have been predicted. However, these reported virulence determinants could not be used to identify the EV71 strains of subgenotype C4, which mainly circulate in China. In this study, VP1 sequences of 37 EV71 strains from severe cases (SC-EV71) and 192 EV71 strains from mild cases (MC-EV71) in mainland China were analyzed to determine the potential virulence determinants in the capsid protein VP1 of EV71. Although most SC-EV71 strains belonged to subgenotype C4a, no specific genetic lineages in C4a were correlated with EV71 virulence. Interestingly, amino acid substitutions at nine positions (H22Q, P27S, N31S/D, E98K, E145G/Q, D164E, T240A/S, V249I, and A289T) were detected by aligning the VP1 sequences of the SC-EV71 and MC-EV71 strains. Moreover, both the constituent ratios of the conservative or mutated residues in the MC-EV71 and SC-EV71 strains and the changes in the VP1 3D structure resulting from these mutations confirmed that the conservative residues (22H, 249V, and 289A) and the mutated residues (27S, 31S/D, 98K, 145G/Q, 164E, and 240A/S) might be potential virulence determinants in VP1 of EV71. Furthermore, these results led to the hypothesis that VP1 acts as a sandwich switch for viral particle stabilization and cellular receptors attachment, and specific mutations in this protein can convert mild cases into severe cases. These findings highlight new opportunities for diagnostic and therapeutic interventions.

  5. Antigenic heterogeneity of capsid protein VP1 in foot-and-mouth disease virus (FMDV serotype Asia1

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    Alam SM

    2013-08-01

    Full Text Available SM Sabbir Alam,1 Ruhul Amin,1 Mohammed Ziaur Rahman,2 M Anwar Hossain,1 Munawar Sultana11Department of Microbiology, University of Dhaka, Dhaka, Bangladesh; 2International Centre for Diarrhoeal Disease Research, Dhaka, BangladeshAbstract: Foot and mouth disease virus (FMDV, with its seven serotypes, is a highly contagious virus infecting mainly cloven-hoofed animals. The serotype Asia1 occurs mainly in Asian regions. An in-silico approach was taken to reveal the antigenic heterogeneities within the capsid protein VP1 of Asia1. A total of 47 VP1 sequences of Asia1 isolates from different countries of South Asian regions were selected, retrieved from database, and were aligned. The structure of VP1 protein was modeled using a homology modeling approach. Several antigenic sites were identified and mapped onto the three-dimensional protein structure. Variations at these antigenic sites were analyzed by calculating the protein variability index and finding mutation combinations. The data suggested that vaccine escape mutants have derived from only few mutations at several antigenic sites. Five antigenic peptides have been identified as the least variable epitopes, with just fewer amino acid substitutions. Only a limited number of serotype Asia1 antigenic variants were found to be circulated within the South Asian region. This emphasizes a possibility of formulating synthetic vaccines for controlling foot-and-mouth disease by Asia1 serotypes.Keywords: protein modeling, antigenic sites, sequence variation

  6. Alignment of capsid protein VP1 sequences of all human rhinovirus prototype strains: conserved motifs and functional domains.

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    Laine, Pia; Blomqvist, Soile; Savolainen, Carita; Andries, Koen; Hovi, Tapani

    2006-01-01

    An alignment was made of the deduced amino acid sequences of the entire capsid protein VP1 of all human rhinovirus (HRV) prototype strains to examine conserved motifs in the primary structure. A set of previously proposed crucially important amino acids in the footprints of the two known receptor molecules was not conserved in a receptor group-specific way. In contrast, VP1 and VP3 amino acids in the minor receptor-group strains corresponding to most of the predicted ICAM-1 footprint definitely differed from those of the ICAM-1-using major receptor-group strains. Previous antiviral-sensitivity classification showed an almost-complete agreement with the species classification and a fair correlation with amino acids aligning in the antiviral pocket. It was concluded that systematic alignment of sequences of related virus strains can be used to test hypotheses derived from molecular studies of individual model viruses and to generate ideas for future studies on virus structure and replication.

  7. Inhibition of enterovirus 71 (EV-71 infections by a novel antiviral peptide derived from EV-71 capsid protein VP1.

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    Chee Wah Tan

    Full Text Available Enterovirus 71 (EV-71 is the main causative agent of hand, foot and mouth disease (HFMD. In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC(50 values ranging from 6-9.3 µM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71.

  8. High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

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    You Bang-Jau

    2011-07-01

    Full Text Available Abstract Background Chicken anemia virus (CAV, the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. Results Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay. Conclusions Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.

  9. Recombinant viral capsid protein VP1 suppresses migration and invasion of human cervical cancer by modulating phosphorylated prohibitin in lipid rafts.

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    Chiu, Ching-Feng; Peng, Jei-Ming; Hung, Shao-Wen; Liang, Chi-Ming; Liang, Shu-Mei

    2012-07-28

    Recombinant capsid protein VP1 (rVP1) of foot-and-mouth disease virus inhibits invasion/metastasis of cancer cells. Here we studied its mechanism of action on human cervical cancer cells. The inhibition of cell invasion by rVP1 was accompanied with reduction in phosphatidylinositol (3,4,5)-triphosphate (PIP3), phospho-Akt S473, phosphorylated prohibitin (phospho-PHB) T258 in lipid rafts, dissociation of phospho-PHB T258 with Raf-1 and the inactivation of Raf-1/ERK. Addition of PIP3 or overexpression of constitutively active Akt and raft-anchored PHB T258 but not PHB T258I mutant protein reversed the inhibitory effects of rVP1. rVP1 inhibited cervical tumor growth and metastasis, and prolonged survival in xenograft mouse models. These results suggest that rVP1 inhibits cancer metastasis via de-phosphorylation of Akt and PHB T258 in lipid rafts to downregulate Raf/ERK signaling.

  10. Progressive Multifocal Leukoencephalopathy (PML) Development Is Associated With Mutations in JC Virus Capsid Protein VP1 That Change Its Receptor Specificity

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    Reid, Carl; Testa, Manuela; Brickelmaier, Margot; Bossolasco, Simona; Pazzi, Annamaria; Bestetti, Arabella; Carmillo, Paul; Wilson, Ewa; McAuliffe, Michele; Tonkin, Christopher; Carulli, John P.; Lugovskoy, Alexey; Lazzarin, Adriano; Sunyaev, Shamil; Simon, Kenneth; Cinque, Paola

    2011-01-01

    Progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease caused by JC virus (JCV) infection of oligodendrocytes, may develop in patients with immune disorders following reactivation of chronic benign infection. Mutations of JCV capsid viral protein 1 (VP1), the capsid protein involved in binding to sialic acid cell receptors, might favor PML onset. Cerebrospinal fluid sequences from 37/40 PML patients contained one of several JCV VP1 amino acid mutations, which were also present in paired plasma but not urine sequences despite the same viral genetic background. VP1-derived virus-like particles (VLPs) carrying these mutations lost hemagglutination ability, showed different ganglioside specificity, and abolished binding to different peripheral cell types compared with wild-type VLPs. However, mutants still bound brain-derived cells, and binding was not affected by sialic acid removal by neuraminidase. JCV VP1 substitutions are acquired intrapatient and might favor JCV brain invasion through abrogation of sialic acid binding with peripheral cells, while maintaining sialic acid–independent binding with brain cells. PMID:21628664

  11. Molecular Evolution and Genetic Analysis of the Major Capsid Protein VP1 of Duck Hepatitis A Viruses: Implications for Antigenic Stability.

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    Ma, Xiuli; Sheng, Zizhang; Huang, Bing; Qi, Lihong; Li, Yufeng; Yu, Kexiang; Liu, Cunxia; Qin, Zhuoming; Wang, Dan; Song, Minxun; Li, Feng

    2015-01-01

    The duck hepatitis A virus (DHAV), a member of the family Picornaviridae, is the major cause of outbreaks with high mortality rates in young ducklings. It has three distinctive serotypes and among them, serotypes 1 (DHAV-1) and 3 (DHAV-3) were recognized in China. To investigate evolutionary and antigenic properties of the major capsid protein VP1 of these two serotypes, a primary target of neutralizing antibodies, we determined the VP1 coding sequences of 19 DHAV-1 (spanning 2000-2012) and 11 DHAV-3 isolates (spanning 2008-2014) associated with disease outbreaks. By bioinformatics analysis of VP1 sequences of these isolates and other DHAV strains reported previously, we demonstrated that DHAV-1 viruses evolved into two genetic lineages, while DHAV-3 viruses exhibited three distinct lineages. The rate of nucleotide substitution for DHAV-1 VP1 genes was estimated to be 5.57 x 10(-4) per site per year, which was about one-third times slower than that for DHAV-3 VP1 genes. The population dynamics analysis showed an upward trend for infection of DHAV-1 viruses over time with little change observed for DHAV-3 viruses. Antigenic study of representative DHAV-1 and DHAV-3 strains covering all observed major lineages revealed no detectable changes in viral neutralization properties within the serotype, despite the lack of cross-neutralization between serotypes 1 and 3 strains. Structural analysis identified VP1 mutations in DHAV-1 and DHAV-3 viruses that underpin the observed antigenic phenotypes. Results of our experiments described here shall give novel insights into evolution and antigenicity of duck picornaviruses.

  12. Expression, purification and characterization of the capsid protein VP1 of enterovirus 71 in E.coli%肠道病毒71型外壳蛋白VP1的可溶性表达、纯化及活性鉴定

    Institute of Scientific and Technical Information of China (English)

    周亚萍; 史伟峰; 朱荫昌; 曹利民; 朱思梅; 史梅; 李坤; 姜庆波

    2011-01-01

    目的 克隆、表达和鉴定肠道病毒71型(EV71)VP1基因,得到可溶性的蛋白VP1,为制备EV71的抗体和诊断试剂的开发打下基础.方法 优化EV71 VP1蛋白基因,克隆并构建重组表达质粒pET15b/VP1,转化大肠杆菌BL21.使用NP亲和层析柱对重组蛋白进行纯化,并用Western blotting检测目的 蛋白.以重组蛋白VP1为抗原,ELISA检测抗原活性.结果 重组蛋白在大肠杆菌中可以高效表达,SDS-PAGE显示其相对分子质量为36 000,与预计大小一致.ELISA实验证实,重组蛋白具有良好的抗原性.结论 本研究成功克隆和表达了EV71 VP1蛋白,并得到可溶性的蛋白,对肠道病毒71型诊断试剂的开发有进一步潜在的应用价值.%Objective To clone, express and characterize the recombinant protein of the capsicl protein VP1 of enterovirus 71 (EV71) , and lay the foundation for preparation of antibodies and diagnose reagents of EV71. Methods The VP1 gene was cloned into pET15b vector to construct the recombinant plasmid pET15b/VPl. Recombinant VP1 protein was expressed in E.coli BL21 and purified by metal (Ni2+) chelating affinity chromatography.The recombinant protein was determined by Western blot.ELJSA was used to determine the antigenicity of the recombinant protein. Results The recombinant VP1 protein can be over expressed in E.coli. The molecular mass was estimated as 36 kDa by SDS-PAGE, and was consistent with the expected size. The antigenicity of the recombinant protein was demonstrated by ELISA. Conclusion The capsid protein VP1 of EV71 was successful cloned and expressed, which could be useful for developing diagnose reagents of EV71.

  13. Species-specific and cross-reactive IgG1 antibody binding to viral capsid protein 1 (VP1 antigens of human rhinovirus species A, B and C.

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    Jua Iwasaki

    Full Text Available BACKGROUND: Human rhinoviruses (HRV are associated with upper and lower respiratory illnesses, including severe infections causing hospitalization in both children and adults. Although the clinical significance of HRV infections is now well established, no detailed investigation of the immune response against HRV has been performed. The purpose of this study was to assess the IgG1 antibody response to the three known HRV species, HRV-A, -B and -C in healthy subjects. METHODS: Recombinant polypeptides of viral capsid protein 1 (VP1 from two genotypes of HRV-A, -B and -C were expressed as glutathione S-transferase (GST fusion proteins and purified by affinity and then size exclusion chromatography. The presence of secondary structures similar to the natural antigens was verified by circular dichroism analysis. Total and species-specific IgG1 measurements were quantitated by immunoassays and immunoabsorption using sera from 63 healthy adults. RESULTS: Most adult sera reacted with the HRV VP1 antigens, at high titres. As expected, strong cross-reactivity between HRV genotypes of the same species was found. A high degree of cross-reactivity between different HRV species was also evident, particularly between HRV-A and HRV-C. Immunoabsorption studies revealed HRV-C specific titres were markedly and significantly lower than the HRV-A and HRV-B specific titres (P<0.0001. A truncated construct of HRV-C VP1 showed greater specificity in detecting anti-HRV-C antibodies. CONCLUSIONS: High titres of IgG1 antibody were bound by the VP1 capsid proteins of HRV-A, -B and -C, but for the majority of people, a large proportion of the antibody to HRV-C was cross-reactive, especially to HRV-A. The improved specificity found for the truncated HRV-C VP1 indicates species-specific and cross-reactive regions could be defined.

  14. Production of a recombinant capsid protein VP1 from a newly described polyomavirus (RacPyV for downstream use in virus characterization

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    Molly E. Church

    2016-06-01

    Full Text Available Here we describe the methods for production of a recombinant viral capsid protein and subsequent use in an indirect enzyme linked immunosorbent assay (ELISA, and for use in production of a rabbit polyclonal antibody. These reagents were utilized in development and optimization of an ELISA, which established the extent of exposure of free ranging raccoons to a newly described polyomavirus (RacPyV [1]. Production of a polyclonal antibody has allowed for further characterization of RacPyV, including immunohistochemistry and immunocytochemistry techniques, in order to answer questions about pathogenesis of this virus.

  15. The cryo-electron microscopy structure of feline calicivirus bound to junctional adhesion molecule A at 9-angstrom resolution reveals receptor-induced flexibility and two distinct conformational changes in the capsid protein VP1.

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    Bhella, David; Goodfellow, Ian G

    2011-11-01

    Caliciviridae are small icosahedral positive-sense RNA-containing viruses and include the human noroviruses, a leading cause of infectious acute gastroenteritis and feline calicivirus (FCV), which causes respiratory illness and stomatitis in cats. FCV attachment and entry is mediated by feline junctional adhesion molecule A (fJAM-A), which binds to the outer face of the capsomere, inducing a conformational change in the capsid that may be important for viral uncoating. Here we present the results of our structural investigation of the virus-receptor interaction and ensuing conformational changes. Cryo-electron microscopy and three-dimensional image reconstruction were used to solve the structure of the virus decorated with a soluble fragment of the receptor at subnanometer resolution. In initial reconstructions, the P domains of the capsid protein VP1 and fJAM-A were poorly resolved. Sorting experiments led to improved reconstructions of the FCV-fJAM-A complex both before and after the induced conformational change, as well as in three transition states. These data showed that the P domain becomes flexible following fJAM-A binding, leading to a loss of icosahedral symmetry. Furthermore, two distinct conformational changes were seen; an anticlockwise rotation of up to 15° of the P domain was observed in the AB dimers, while tilting of the P domain away from the icosahedral 2-fold axis was seen in the CC dimers. A list of putative contact residues was calculated by fitting high-resolution coordinates for fJAM-A and VP1 to the reconstructed density maps, highlighting regions in both virus and receptor important for virus attachment and entry.

  16. Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice.

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    Wang, Man; Jiang, Shuai; Wang, Yefu

    2013-01-01

    Human enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is also associated with serious neurological diseases in children. Currently, there are no effective antiviral drugs or vaccines against EV71 infection. VP1, one of the major immunogenic capsid proteins of EV71, is widely considered to be the candidate antigen for an EV71 vaccine. In this study, VP1 of EV71 was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris, and purified by Ni-NTA affinity chromatography. Immunogenicity and vaccine efficacy of the recombinant VP1 were assessed in mouse models. The results showed that the recombinant VP1 could efficiently induce anti-VP1 antibodies in BALB/c mice, which were able to neutralize EV71 viruses in an in vitro neutralization assay. Passive protection of neonatal mice further confirmed the prophylactic efficacy of the antisera from VP1 vaccinated mice. Furthermore, VP1 vaccination induced strong lymphoproliferative and Th1 cytokine responses. Taken together, our study demonstrated that the yeast-expressed VP1 protein retained good immunogenicity and was a potent EV71 vaccine candidate.

  17. Intein-mediated backbone cyclization of VP1 protein enhanced protection of CVB3-induced viral myocarditis

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    Qi, Xingmei; Xiong, Sidong

    2017-01-01

    CVB3 is a common human pathogen to be highly lethal to newborns and causes viral myocarditis and pancreatitis in adults. However, there is no vaccine available for clinical use. CVB3 capsid protein VP1 is an immunodominant structural protein, containing several B- and T-cell epitopes. However, immunization of mice with VP1 protein is ineffective. Cyclization of peptide is commonly used to improve their in vivo stability and biological activity. Here, we designed and synthesizd cyclic VP1 protein by using engineered split Rma DnaB intein and the cyclization efficiency was 100% in E. coli. As a result, the cyclic VP1 was significantly more stable against irreversible aggregation upon heating and against carboxypeptidase in vitro and the degradation rate was more slowly in vivo. Compared with linear VP1, immunization mice with circular VP1 significantly increased CVB3-specific serum IgG level and augmented CVB3-specific cellular immune responses, consequently afforded better protection against CVB3-induced viral myocarditis. The cyclic VP1 may be a novel candidate protein vaccine for preventing CVB3 infection and similar approaches could be employed to a variety of protein vaccines to enhance their protection effect. PMID:28148910

  18. Detection of polyomavirus major capsid antigen (VP-1 in human pilomatricomas Detección del antígeno mayor de la cápside de poliomavirus (VP-1 en pilomatricomas humanos

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    Norberto A. Sanjuán

    2010-04-01

    Full Text Available The family Polyomaviridae is composed of small, non-enveloped, double-stranded DNA viruses widely used to study cell transformation in vitro and tumor induction in vivo. The development of pilomatricomas in mice experimentally infected with polyomavirus led us to detect the viral major capsid protein VP-1 in human pilomatricomas. This tumor, even uncommon, is one of the most frequent benign hair follicle tumors in humans and is composed of proliferating matrix cells that undergo keratinization, and form cystic neoplasms. The detection of VP-1 was performed using the peroxidase-antiperoxidase technique in paraffin-embedded slides with a specific primary serum. Adjacent slides treated with normal rabbit serum as a primary were employed as internal control. Positive and negative controls were also employed as well as slides of lesions caused by human papillomavirus to rule out any unspecific cross-reactivity. In 4 out of 10 cases polyomavirus VP-1 was clearly detected in nuclei of human pilomatricomas proliferating cells, in a patchy pattern of distribution. The controls confirmed the specificity of the immunocytochemical procedure. These results could indicate either an eventual infection of the virus in already developed tumors or alternatively, a direct involvement of polyomavirus in the pathogenesis of some pilomatricomas. The recent discovery of a new human polyomavirus associated with Merkel cell carcinomas has been a strong contribution to better understand the pathogenesis of some human uncommon skin cancers. Hopefully the results reported in this work will encourage further research on the role of polyomavirus in other human skin neoplasms.La familia Poliomaviridae está compuesta por virus oncogénicos pequeños, no envueltos, con ADN de doble cadena. En un modelo experimental murino pudimos desarrollar pilomatricomas inducidos por la inoculación de virus polioma. Eso nos llevó a estudiar la posibilidad de que otro virus polioma

  19. Characterization of the DNA binding properties of polyomavirus capsid protein

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    Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

  20. Processing of the VP1/2A Junction Is Not Necessary for Production of Foot-and-Mouth Disease Virus Empty Capsids and Infectious Viruses: Characterization of “Self-Tagged” Particles

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    Gullberg, Maria; Polacek, Charlotta; Bøtner, Anette;

    2013-01-01

    the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction...... is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines....

  1. Natural type 3/type 2 intertypic vaccine-related poliovirus recombinants with the first crossover sites within the VP1 capsid coding region.

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    Yong Zhang

    Full Text Available BACKGROUND: Ten uncommon natural type 3/type 2 intertypic poliovirus recombinants were isolated from stool specimens from nine acute flaccid paralysis case patients and one healthy vaccinee in China from 2001 to 2008. PRINCIPAL FINDINGS: Complete genomic sequences revealed their vaccine-related genomic features and showed that their first crossover sites were randomly distributed in the 3' end of the VP1 coding region. The length of donor Sabin 2 sequences ranged from 55 to 136 nucleotides, which is the longest donor sequence reported in the literature for this type of poliovirus recombination. The recombination resulted in the introduction of Sabin 2 neutralizing antigenic site 3a (NAg3a into a Sabin 3 genomic background in the VP1 coding region, which may have been altered by some of the type 3-specific antigenic properties, but had not acquired any type 2-specific characterizations. NAg3a of the Sabin 3 strain seems atypical; other wild-type poliovirus isolates that have circulated in recent years have sequences of NAg3a more like the Sabin 2 strain. CONCLUSIONS: 10 natural type 3/type 2 intertypic VP1 capsid-recombinant polioviruses, in which the first crossover sites were found to be in the VP1 coding region, were isolated and characterized. In spite of the complete replacement of NAg3a by type 2-specific amino acids, the serotypes of the recombinants were not altered, and they were totally neutralized by polyclonal type 3 antisera but not at all by type 2 antisera. It is possible that recent type 3 wild poliovirus isolates may be a recombinant having NAg3a sequences derived from another strain during between 1967 and 1980, and the type 3/type 2 recombination events in the 3' end of the VP1 coding region may result in a higher fitness.

  2. Expression and subcellular targeting of canine parvovirus capsid proteins in baculovirus-transduced NLFK cells.

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    Gilbert, Leona; Välilehto, Outi; Kirjavainen, Sanna; Tikka, Päivi J; Mellett, Mark; Käpylä, Pirjo; Oker-Blom, Christian; Vuento, Matti

    2005-01-17

    A mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protein is a prerequisite for efficient targeting of VP2 to the nucleus. The baculovirus vectors were functional and the genes of interest efficiently introduced to this CPV susceptible mammalian cell line. Thus, we show evidence that the system could be utilized to study targeting of the CPV capsid proteins.

  3. Amino acid sequences mediating vascular cell adhesion molecule 1 binding to integrin alpha 4: homologous DSP sequence found for JC polyoma VP1 coat protein

    Directory of Open Access Journals (Sweden)

    Michael Andrew Meyer

    2013-07-01

    Full Text Available The JC polyoma viral coat protein VP1 was analyzed for amino acid sequences homologies to the IDSP sequence which mediates binding of VLA-4 (integrin alpha 4 to vascular cell adhesion molecule 1. Although the full sequence was not found, a DSP sequence was located near the critical arginine residue linked to infectivity of the virus and binding to sialic acid containing molecules such as integrins (3. For the JC polyoma virus, a DSP sequence was found at residues 70, 71 and 72 with homology also noted for the mouse polyoma virus and SV40 virus. Three dimensional modeling of the VP1 molecule suggests that the DSP loop has an accessible site for interaction from the external side of the assembled viral capsid pentamer.

  4. Amino Acid Sequences Mediating Vascular Cell Adhesion Molecule 1 Binding to Integrin Alpha 4: Homologous DSP Sequence Found for JC Polyoma VP1 Coat Protein.

    Science.gov (United States)

    Meyer, Michael Andrew

    2013-01-01

    The JC polyoma viral coat protein VP1 was analyzed for amino acid sequences homologies to the IDSP sequence which mediates binding of VLA-4 (integrin alpha 4) to vascular cell adhesion molecule 1. Although the full sequence was not found, a DSP sequence was located near the critical arginine residue linked to infectivity of the virus and binding to sialic acid containing molecules such as integrins (3). For the JC polyoma virus, a DSP sequence was found at residues 70, 71 and 72 with homology also noted for the mouse polyoma virus and SV40 virus. Three dimensional modeling of the VP1 molecule suggests that the DSP loop has an accessible site for interaction from the external side of the assembled viral capsid pentamer.

  5. EV71结构蛋白VP1的原核表达及VP1单克隆抗体的制备%Expression of structural protein VP1 of enterovirus 71 in E.coli and preparation of anti-VP1 monoclonal antibodies

    Institute of Scientific and Technical Information of China (English)

    李晨阳; 王弋; 古艳丽; 古亚楠; 陈海燕; 田璐; 陈丽云; 彭涛

    2012-01-01

    AIM: To express recombinant structural protein VP1 of enterovirus 71 (EV71) in E. coli and prepare anti-VPl monoclonal antibodies (mAbs). METHODS: With PI gene as a template, EV71 VP1 gene was amplified by PCR and cloned into the expression vector pET-32a ( +). After transformation into E. coli TGI, the positive clones were screened and sequenced. The recombinant plasmid was then transformed into BLgold (DE3) and the recombinant protein in inclusion bodies was induced by IPTG and detected by SDS-PAGE. After the inclusion bodies were solubilized with 6 mol/L guanidine hydrochloride, we purified VP1 protein using Ni-NTA affinity chromatography, and then immunized the mice with it to prepare the mAbs against VP1 protein. RESULTS; Recombinant VP1 protein was expressed in E. coli. We obtained totally 24 VP1 monoclonal hybridoma cell strains, of which one was EV71 positive determined by Western blotting and five were positive for I FA. CONCLUSION: These mAbs are valuable reagents for the development of new vaccines and detection kits for EV71.%目的:原核表达EV71结构蛋白VP1并制备其单克隆抗体.方法:以肠道病毒71型( EV71) P1基因为模板,设计引物扩增出目的片段VP1,将其连接至大肠杆菌表达载体pET-32a(+)上,转化大肠杆菌TG1,筛选出阳性克隆后进行测序.将重组表达载体pET-32a (+)-VP1转化大肠杆菌表达菌株BLgold( DE3),对该工程菌进行诱导表达,SDS-PAGE电泳分析,表达产物以包涵体的形式存在.包涵体用6 mol/L盐酸胍溶解,经过Ni-NTA亲和层析法纯化,获得了纯度较高的目的蛋白.用纯化的VP1蛋白免疫小鼠,制备单克隆抗体(mAb).结果:获得了24株mAb,其中1株EV71 Western blot法鉴定呈阳性,5株EV71间接免疫荧光法(IFA)鉴定阳性.结论:成功地制备了VP1的mAb.

  6. Molecular determinants of enterovirus 71 viral entry: cleft around GLN-172 on VP1 protein interacts with variable region on scavenge receptor B 2.

    Science.gov (United States)

    Chen, Pan; Song, Zilin; Qi, Yonghe; Feng, Xiaofeng; Xu, Naiqing; Sun, Yinyan; Wu, Xing; Yao, Xin; Mao, Qunyin; Li, Xiuling; Dong, Wenjuan; Wan, Xiaobo; Huang, Niu; Shen, Xinliang; Liang, Zhenglun; Li, Wenhui

    2012-02-24

    Enterovirus 71 (EV71) is one of the major pathogens that cause hand, foot, and mouth disease outbreaks in young children in the Asia-Pacific region in recent years. Human scavenger receptor class B 2 (SCARB2) is the main cellular receptor for EV71 on target cells. The requirements of the EV71-SCARB2 interaction have not been fully characterized, and it has not been determined whether SCARB2 serves as an uncoating receptor for EV71. Here we compared the efficiency of the receptor from different species including human, horseshoe bat, mouse, and hamster and demonstrated that the residues between 144 and 151 are critical for SCARB2 binding to viral capsid protein VP1 of EV71 and seven residues from the human receptor could convert murine SCARB2, an otherwise inefficient receptor, to an efficient receptor for EV71 viral infection. We also identified that EV71 binds to SCARB2 via a canyon of VP1 around residue Gln-172. Soluble SCARB2 could convert the EV71 virions from 160 S to 135 S particles, indicating that SCARB2 is an uncoating receptor of the virus. The uncoating efficiency of SCARB2 significantly increased in an acidic environment (pH 5.6). These studies elucidated the viral capsid and receptor determinants of enterovirus 71 infection and revealed a possible target for antiviral interventions.

  7. Expression of VP1 protein of enterovirus 71 in E. coli and verification of the expressive product%肠道病毒71型VP1蛋白原核表达及初步鉴定

    Institute of Scientific and Technical Information of China (English)

    李小青; 何威; 段明月; 李丹; 冯媛

    2014-01-01

    目的采用分子克隆技术,构建肠道病毒71型( EV71)VP1全长基因大肠杆菌原核系统表达载体,诱导重组VP1融合蛋白表达。方法自EV71感染患者血清中提取病毒总RNA,进行一步法RT-PCR,扩增编码VP1蛋白的全长基因片段(891 bp),以pET32(a)为表达载体,构建重组表达质粒pET32(a)- VP1,转化E. coli. Rosseta感受态细胞,获得重组工程菌株。经诱导培养,SDS-PAGE电泳,免疫印迹鉴定表达产物。结果获得了含重组表达质粒pET32( a)- VP1的正相阳性工程菌株,经IPTG诱导能高效表达VP1融合蛋白。结论重组工程菌可表达VP1融合蛋白,对研究EV71发病机制及疫苗研制具有重要意义。%Objective The aim of this study are to construct recombinant expression vector containing VP1 whole gene of enterovirus 71 (EV71)by DNA recombinant technology and to induce expression of VP1 fusion protein in E. coli. Methods The interested gene with total extracted from the serum of patients. These patients were infected by EV71 virus. We inserted it into pET32(a)expression vector to construct recombinant expression vector pET32(a)- VP1. The verified recombinants pET32( a)- VP1 were transformed into E. coli. Rossena to produce bacteria strain with positive recombinants,then the strain were analyzed with SDS - PAGE and immunoblotting to verify the expression product -- VP1 fusion protein. Results The positive bacteria containing the recombinant expressive vector pET28(a) - VP1 were constructed successfully, and high level expression of VP1 fusion protein was performed by inducing with IPTG. Conclusion It played an important role in investigating the pathogenesis of EV71 and developing vaccine in the future that the VP1 fusion protein of the positive strain with recombinant were expressed efficiently in E. coli system.

  8. 肠道病毒71型 VP1基因的扩增、克隆、表达及蛋白分析%Amplification,cloning,expression and protein analysis of enterovirus 71 VP1 gene

    Institute of Scientific and Technical Information of China (English)

    刘云锋; 周珍文; 关锐梨; 关启鸿; 骆鸣勇; 周帅

    2014-01-01

    Objective To conduct the amplification,cloning,bioinformatics analysis,prokaryotic expression and purification of enterovirus 71 VP1 gene segment and to initially confirm the biological activity of the recombinant expression product.Methods A pair of specific primers was designed according to GenBank EV71 sequence,viral RNA as a template was extracted from the throat swab specimens in the EV71 patients.EV71 VP1 gene was amplified by RT-PCR.After enzyme digestion,the expression vector pET28a was inserted.The prokaryotic expression vector of pET28a-EV71 VP1 was constructed.Then the E.coli DH5a transforma-tion was performed.IPTG was adopted for induction expression.The expression results were analyzed by using SDS-PAGE and Western blot.The bioinformatics analysis of the sequenced results was performed by the software.Expressed protein was purified and the plates were coated,ELISA was used to test the VP1 specific IgG antibody in serum samples of EV71 positive and COX A16-positive patients.Results The BLAST alignment showed that the homology of the objective gene EV71 VP1 was 99% com-pared with other strains(JQ766207.1)in GenBank.EV71 VP1 protein was about 32×103 ,which mainly existed in the form of in-clusion body.The bioinformatics analysis showed that EV71 VP1 protein was a hydrophilic protein,without transmembrane region and N-terminal signal peptide sequence,the tertiary structure existed.The ELISA results showed that the specific IgG OD value in EV71-positive patients was(2.425±0.521),OD value in COX A16 positive patients was(1.205 ±0.314),the normal control OD value was(0.353±0.128).The sensitivity and specificity of EV71 VP1 protein detection were 84% and 88% respectively.Conclu-sion The pET28a-EV71 VP1 expression vector is successfully constructed;the preliminary analysis on the serum of the infected patients by ELISA shows that the obtained objective protein has higher sensitivity and specificity,which is initially confirmed to have biological activity

  9. Sequence adaptations affecting cleavage of the VP1/2A junction by the 3C protease in foot-and-mouth disease virus-infected cells

    DEFF Research Database (Denmark)

    Gullberg, Maria; Polacek, Charlotta; Belsham, Graham

    2014-01-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor P1-2A is cleaved by the virus-encoded 3C protease to VP0, VP3, VP1 and 2A. It was shown previously that modification of a single amino acid residue (K210E) within the VP1 protein and close to the VP1/2A cleavage site, inhibited clea...

  10. Characterization of the antibody response against EV71 capsid proteins in Chinese individuals by NEIBM-ELISA.

    Science.gov (United States)

    Ding, Yingying; Chen, Xuguang; Qian, Baohua; Wu, Guorong; He, Ting; Feng, Jiaojiao; Gao, Caixia; Wang, Lili; Wang, Jinhong; Li, Xiangyu; Cao, Mingmei; Peng, Heng; Zhao, Chunyan; Pan, Wei

    2015-01-01

    Human enterovirus 71 (EV71) has become the major pathogen of hand, foot, and mouth disease (HFMD) worldwide, while the anti-EV71 antibody responses other than neutralizing epitopes have not been characterized. In this study, EV71 capsid proteins VP1, VP3, VP0 and various VP1 antigens were constructed to analyze anti-EV71 response in severe HFMD cases, non-HFMD outpatient children and normal adults using a novel evolved immunoglobulin-binding molecule (NEIBM)-based ELISA. The high prevalence of antibody responses against all three capsid proteins was demonstrated, and anti-EV71 VP1 showed the main antibody response. Anti-EV71 VP1 antibody response was found to predominantly target to epitopes based on the common enterovirus cross-reactive sequence. Moreover, inhibition pattern against anti-EV71 VP1 reactions in three groups was obviously different. Taken together, these results firstly characterized the anti-EV71 antibody responses which are predominantly against VP1 epitopes based on common enterovirus cross-reactive sequence. This finding could be helpful for the better understanding of anti-EV71 humoral immunity and useful for seroepidemiological surveillance.

  11. Characterization of the antibody response against EV71 capsid proteins in Chinese individuals by NEIBM-ELISA

    OpenAIRE

    Ding, Yingying; Chen, Xuguang; Qian, Baohua; Wu, Guorong; He, Ting; Feng, Jiaojiao; Gao, Caixia; Wang, Lili; Wang, Jinhong; Li, Xiangyu; Cao, Mingmei; Peng, Heng; Zhao, Chunyan; Pan, Wei

    2015-01-01

    Human enterovirus 71 (EV71) has become the major pathogen of hand, foot, and mouth disease (HFMD) worldwide, while the anti-EV71 antibody responses other than neutralizing epitopes have not been characterized. In this study, EV71 capsid proteins VP1, VP3, VP0 and various VP1 antigens were constructed to analyze anti-EV71 response in severe HFMD cases, non-HFMD outpatient children and normal adults using a novel evolved immunoglobulin-binding molecule (NEIBM)-based ELISA. The high prevalence o...

  12. Several recombinant capsid proteins of equine rhinitis a virus show potential as diagnostic antigens.

    Science.gov (United States)

    Li, Fan; Stevenson, Rachel A; Crabb, Brendan S; Studdert, Michael J; Hartley, Carol A

    2005-06-01

    Equine rhinitis A virus (ERAV) is a significant pathogen of horses and is also closely related to Foot-and-mouth disease virus (FMDV). Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA.

  13. Characterization of a nuclear localization signal of canine parvovirus capsid proteins.

    Science.gov (United States)

    Vihinen-Ranta, M; Kakkola, L; Kalela, A; Vilja, P; Vuento, M

    1997-12-01

    We investigated the abilities of synthetic peptides mimicking the potential nuclear localization signal of canine parvovirus (CPV) capsid proteins to translocate a carrier protein to the nucleus following microinjection into the cytoplasm of A72 cells. Possible nuclear localization sequences were chosen for synthesis from CPV capsid protein sequences (VP1, VP2) on the basis of the presence of clustered basic residues, which is a common theme in most of the previously identified targeting peptides. Nuclear targeting activity was found within the N-terminal residues 4-13 (PAKRARRGYK) of the VP1 capsid protein. While replacement of Arg10 with glycine did not affect the activity, replacement of Lys6, Arg7, or Arg9 with glycine abolished it. The targeting activity was found to residue in a cluster of basic residues, Lys5, Arg7, and Arg9. Nuclear import was saturated by excess of unlabelled peptide conjugates (showing that it was a receptor-mediated process). Transport into the nucleus was an energy-dependent and temperature-dependent process actively mediated by the nuclear pores and inhibited by wheat germ agglutinin.

  14. Evolutionary analysis of structural protein gene VP1 of foot-and-mouth disease virus serotype Asia 1.

    Science.gov (United States)

    Zhang, Qingxun; Liu, Xinsheng; Fang, Yuzhen; Pan, Li; Lv, Jianliang; Zhang, Zhongwang; Zhou, Peng; Ding, Yaozhong; Chen, Haotai; Shao, Junjun; Zhao, Furong; Lin, Tong; Chang, Huiyun; Zhang, Jie; Wang, Yonglu; Zhang, Yongguang

    2015-01-01

    Foot-and-mouth disease virus (FMDV) serotype Asia 1 was mostly endemic in Asia and then was responsible for economically important viral disease of cloven-hoofed animals, but the study on its selection and evolutionary process is comparatively rare. In this study, we characterized 377 isolates from Asia collected up until 2012, including four vaccine strains. Maximum likelihood analysis suggested that the strains circulating in Asia were classified into 8 different groups (groups I-VIII) or were unclassified (viruses collected before 2000). On the basis of divergence time analyses, we infer that the TMRCA of Asia 1 virus existed approximately 86.29 years ago. The result suggested that the virus had a high mutation rate (5.745 × 10(-3) substitutions/site/year) in comparison to the other serotypes of FMDV VP1 gene. Furthermore, the structural protein VP1 was under lower selection pressure and the positive selection occurred at many sites, and four codons (positions 141, 146, 151, and 169) were located in known critical antigenic residues. The remaining sites were not located in known functional regions and were moderately conserved, and the reason for supporting all sites under positive selection remains to be elucidated because the power of these analyses was largely unknown.

  15. Comparing Enterovirus 71 with Coxsackievirus A16 by analyzing nucleotide sequences and antigenicity of recombinant proteins of VP1s and VP4s

    Directory of Open Access Journals (Sweden)

    Sun Yu

    2011-11-01

    Full Text Available Abstract Background Enterovirus 71 (EV71 and Coxsackievirus A16 (CA16 are two major etiological agents of Hand, Foot and Mouth Disease (HFMD. EV71 is associated with severe cases but not CA16. The mechanisms contributed to the different pathogenesis of these two viruses are unknown. VP1 and VP4 are two major structural proteins of these viruses, and should be paid close attention to. Results The sequences of vp1s from 14 EV71 and 14 CA16, and vp4s from 10 EV71 and 1 CA16 isolated in this study during 2007 to 2009 HFMD seasons were analyzed together with the corresponding sequences available in GenBank using DNAStar and MEGA 4.0. Phylogenetic analysis of complete vp1s or vp4s showed that EV71 isolated in Beijing belonged to C4 and CA16 belonged to lineage B2 (lineage C. VP1s and VP4s from 4 strains of viruses expressed in E. coli BL21 cells were used to detect IgM and IgG in human sera by Western Blot. The detection of IgM against VP1s of EV71 and CA16 showed consistent results with current infection, while none of the sera were positive against VP4s of EV71 and CA16. There was significant difference in the positive rates between EV71 VP1 and CA16 VP1 (χ2 = 5.02, P 2 = 15.30, P 2 = 26.47, P 2 = 16.78, P Conclusions EV71 and CA16 were highly diverse in the nucleotide sequences of vp1s and vp4s. The sera positive rates of VP1 and VP4 of EV71 were lower than those of CA16 respectively, which suggested a less exposure rate to EV71 than CA16 in Beijing population. Human serum antibodies detected by Western blot using VP1s and VP4s as antigen indicated that the immunological reaction to VP1 and VP4 of both EV71 and CA16 was different.

  16. Display of VP1 on the surface of baculovirus and its immunogenicity against heterologous human enterovirus 71 strains in mice.

    Directory of Open Access Journals (Sweden)

    Tao Meng

    Full Text Available BACKGROUND: Human Enterovirus 71 (EV71 is a common cause of hand, foot and mouth disease (HFMD in young children. It is often associated with severe neurological diseases and has caused high mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no effective vaccine and antiviral agents available against EV71 infections. VP1 is one of the major immunogenic capsid protein of EV71 and plays a crucial role in viral infection. Antibodies against VP1 are important for virus neutralization. METHODOLOGY/PRINCIPAL FINDING: In the present study, infectious EV71 viruses were generated from their synthetic complementary DNA using the human RNA polymerase I reverse genetics system. Secondly, the major immunogenic capsid protein (VP1 of EV71-Fuyang (subgenogroup C4 was displayed on the surface of recombinant baculovirus Bac-Pie1-gp64-VP1 as gp64 fusion protein under a novel White Spot Syndrome Virus (WSSV immediate early ie1 promoter. Baculovirus expressed VP1 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed that VP1 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired VP1 from the insect cell membrane via the budding process. After two immunizations in mice, Bac-Pie1-gp64-VP1 elicited neutralization antibody titer of 1∶64 against EV71 (subgenogroup C4 in an in vitro neutralization assay. Furthermore, the antisera showed high cross-neutralization activities against all 11 subgenogroup EV71 strains. CONCLUSION: Our results illustrated that Bac-Pie1-gp64-VP1 retained native epitopes of VP1 and acted as an effective EV71 vaccine candidate which would enable rapid production without any biosafety concerns.

  17. Expression of Structural Protein VP1 of Poliovirus Sabin2 Strain in Insect Baculovirus Expression System%脊髓灰质炎病毒Sabin2株结构蛋白VP1在昆虫杆状病毒表达系统中的表达

    Institute of Scientific and Technical Information of China (English)

    刘金花; 董关木; 安祺; 曹守春; 孔艳

    2011-01-01

    Objective To express the structural protein VP1 of poliovirus (PV) Sabin2 strain in insect baculovirus expression system. Methods VP1 gene was amplified by RT-PCR from the bulk of PV vaccine prepared with Sabin2 strain, and cloned into vector pFastBac1. The constructed recombinant plasmid pFast-VPl was transformed to E. coli DH10Bac carrying baculovirus shuttle plasmid Bacmid, and the obtained recombinant plasmid Bacmid-VP1 was transfected to sf9 insect cells in mediation of liposome to prepare recombinant baculovirus rBac-VP1. The sf9 cells were infected with rBac-VP1 of passage 2 and determined for expression of VP1 by IFA, based on which the condition for infection was optimized. Results The target gene fragment at a length of 3 203 bp was amplified from the transformants of Bacmid-VP1. The rBac-VP1 of passage 2 roached a titer of 6 × 107 pfu/ml, with which the infected sf9 cells showed green fluorescence under fluorescent microscope. The expression levels of VP1 in sf9 cells infected with rBac-VP1 at various MOIs showed no significant difference, which increased gradually with the increasing time for infection within 96 h.Conclusion The structural protein VP1 of PV Sabin2 strain was successfully expressed in insect baculovirus expression system,which laid a foundation of development of poliovirus subunit vaccine.%目的 在昆虫杆状病毒表达系统中表达脊髓灰质炎病毒(Poliovirus,PV)Sabin2株结构蛋白VP1.方法 从Sabin2疫苗原液中RT-PCR扩增PV结构蛋白VP1基因.克隆入pFastBac1质粒,转化含杆状病毒穿梭载体Bacmid的E coli DH 10Bac,获得重组杆状病毒表达质粒Bacmid-VP1,在脂质体介导下转染sf9昆虫细胞,获得重组杆状病毒rBac-VP1.将第2代rBac-VP1感染sf9细胞,间接免疫荧光法检测VP1蛋白的表达,并优化感染条件.结果 重组杆状病毒表达质粒.Bacmid-VP1转化子可扩增出3 203 bp的目的 片段;第2代rBac-VP1的滴度为6×107 pfu/ml,其感染的sf9细胞在荧光显微

  18. The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

    Science.gov (United States)

    Leisi, Remo; Von Nordheim, Marcus; Ros, Carlos; Kempf, Christoph

    2016-01-01

    Parvovirus B19 (B19V) is a small non-enveloped virus and known as the causative agent for the mild childhood disease erythema infectiosum. B19V has an extraordinary narrow tissue tropism, showing only productive infection in erythroid precursor cells in the bone marrow. We recently found that the viral protein 1 unique region (VP1u) contains an N-terminal receptor-binding domain (RBD), which mediates the uptake of the virus into cells of the erythroid lineage. To further investigate the role of the RBD in connection with a B19V-unrelated capsid, we chemically coupled the VP1u of B19V to the bacteriophage MS2 capsid and tested the internalization capacity of the bioconjugate on permissive cells. In comparison, we studied the cellular uptake and infection of B19V along the erythroid differentiation. The results showed that the MS2-VP1u bioconjugate mimicked the specific internalization of the native B19V into erythroid precursor cells, which further coincides with the restricted infection profile. The successful mimicry of B19V uptake demonstrates that the RBD in the VP1u is sufficient for the endocytosis of the viral capsid. Furthermore, the recombinant VP1u competed with B19V uptake into permissive cells, thus excluding a significant alternative uptake mechanism by other receptors. Strikingly, the VP1u receptor appeared to be expressed only on erythropoietin-dependent erythroid differentiation stages that also provide the necessary intracellular factors for a productive infection. Taken together, these findings suggest that the VP1u binds to a yet-unknown erythroid-specific cellular receptor and thus restricts the virus entry to permissive cells. PMID:27690083

  19. Identification of a conserved neutralizing linear B-cell epitope in the VP1 proteins of duck hepatitis A virus type 1 and 3.

    Science.gov (United States)

    Zhang, Ruihua; Zhou, Guomei; Xin, Yinghao; Chen, Junhao; Lin, Shaoli; Tian, Ye; Xie, Zhijing; Jiang, Shijin

    2015-11-18

    Duck virus hepatitis (DVH), mainly caused by duck hepatitis A virus (DHAV), is a severe disease threaten to duck industry and has worldwide distribution. As the major structural protein, the VP1 protein of DHAV is able to induce neutralizing antibody in ducks. In this study, a monoclonal antibody (mAb) 4F8 against the intact DHAV-1 particles was used to identify the possible epitope in the three serotypes of DHAV. The mAb 4F8 had weak neutralizing activities to both DHAV-1 and DHAV-3, and reacted with the conserved linear B-cell epitopes of (75)GEIILT(80) in DHAV-1 VP1 and (75)GEVILT(80) in DHAV-3 VP1 protein, respectively, while not with DHAV-2 VP1. This was the first report about identification of the common conserved neutralizing linear B-cell epitope of DHAV-1 and DHAV-3, which will facilitate understanding of the antigenic structure of VP1 and the serologic diagnosis of DHAV infection.

  20. Disassociation of the SV40 Genome from Capsid Proteins Prior to Nuclear Entry

    Directory of Open Access Journals (Sweden)

    Kuksin Dmitry

    2012-08-01

    Full Text Available Abstract Background Previously, we demonstrated that input SV40 particles undergo a partial disassembly in the endoplasmic reticulum, which exposes internal capsid proteins VP2 and VP3 to immunostaining. Then, in the cytoplasm, disassembly progresses further to also make the genomic DNA accessible to immune detection, as well as to detection by an ethynyl-2-deoxyuridine (EdU-based chemical reaction. The cytoplasmic partially disassembled SV40 particles retain some of the SV40 capsid proteins, VP1, VP2, and VP3, in addition to the viral genome. Findings In the current study, we asked where in the cell the SV40 genome might disassociate from capsid components. We observed partially disassembled input SV40 particles around the nucleus and, beginning at 12 hours post-infection, 5-Bromo-2-deoxyuridine (BrdU-labeled parental SV40 DNA in the nucleus, as detected using anti-BrdU antibodies. However, among the more than 1500 cells examined, we never detected input VP2/VP3 in the nucleus. Upon translocation of the BrdU-labeled SV40 genomes into nuclei, they were transcribed and, thus, are representative of productive infection. Conclusions Our findings imply that the SV40 genome disassociates from the capsid proteins before or at the point of entry into the nucleus, and then enters the nucleus devoid of VP2/3.

  1. Mechanostability of Proteins and Virus Capsids

    Science.gov (United States)

    Cieplak, Marek

    2013-03-01

    Molecular dynamics of proteins within coarse grained models have become a useful tool in studies of large scale systems. The talk will discuss two applications of such modeling. The first is a theoretical survey of proteins' resistance to constant speed stretching as performed for a set of 17134 simple and 318 multidomain proteins. The survey has uncovered new potent force clamps. They involve formation of cysteine slipknots or dragging of a cystine plug through the cystine ring and lead to characteristic forces that are significantly larger than the common shear-based clamp such as observed in titin. The second application involves studies of nanoindentation processes in virus capsids and elucidates their molecular aspects by showing deviations in behavior compared to the continuum shell model. Across the 35 capsids studied, both the collapse force and the elastic stiffness are observed to vary by a factor of 20. The changes in mechanical properties do not correlate simply with virus size or symmetry. There is a strong connection to the mean coordination number , defined as the mean number of interactions to neighboring amino acids. The Young's modulus for thin shell capsids rises roughly quadratically with - 6, where 6 is the minimum coordination for elastic stability in three dimensions. Supported by European Regional Development Fund, through Innovative Economy grant Nanobiom (POIG.01.01.02-00-008/08)

  2. Engineering bacterial surface displayed human norovirus capsid proteins: A novel system to explore interaction between norovirus and ligands

    Directory of Open Access Journals (Sweden)

    Mengya eNiu

    2015-12-01

    Full Text Available Human noroviruses (HuNoVs are major contributors to acute nonbacterial gastroenteritis outbreaks. Many aspects of HuNoVs are poorly understood due to both the current inability to culture HuNoVs, and the lack of efficient small animal models. Surrogates for HuNoVs, such as recombinant viral like particles (VLPs expressed in eukaryotic system or P particles expressed in prokaryotic system, have been used for studies in immunology and interaction between the virus and its receptors. However, it is difficult to use VLPs or P particles to collect or isolate potential ligands binding to these recombinant capsid proteins. In this study, a new strategy was used to collect HuNoVs binding ligands through the use of ice nucleation protein (INP to display recombinant capsid proteins of HuNoVs on bacterial surfaces. The viral protein-ligand complex could be easily separated by a low speed centrifugation step. This system was also used to explore interaction between recombinant capsid proteins of HuNoVs and their receptors. In this system, the VP1 capsid encoding gene (ORF2 and the protruding domain (P domain encoding gene (3’ terminal fragment of ORF2 of HuNoVs GI.1 and GII.4 were fused with 5’ terminal fragment of ice nucleation protein encoding gene (inaQn. The results demonstrated that the recombinant VP1 and P domains of HuNoVs were expressed and anchored on the surface of Escherichia coli BL21 cells after the bacteria were transformed with the corresponding plasmids. Both cell surface displayed VP1 and P domains could be recognized by HuNoVs specific antibodies and interact with the viral histo-blood group antigens receptors. In both cases, displayed P domains had better binding abilities than VP1. This new strategy of using displayed HuNoVs capsid proteins on the bacterial surface could be utilized to separate HuNoVs binding components from complex samples, to investigate interaction between the virus and its receptors, as well as to develop an

  3. Positive Selection Analysis of VP1 Genes of Worldwide Human Enterovirus 71 Viruses

    Institute of Scientific and Technical Information of China (English)

    Wei-feng SHI; Zhong ZHANG; Ai-she DUN; Yan-zhou ZHANG; Guang-fu YU; Dong-ming ZHUANG; Chao-dong ZHU

    2009-01-01

    Human enterovirus 71 viruses have been long circulating throughout the world. In this study, we performed a positive selection analysis of the VP1 genes of capsid proteins from Enterovirus 71 viruses. Our results showed that although most sites were under negative or neutral evolution, four positions of the VP1 genes were under positive selection pressure. This might account for the spread and frequent outbreaks of the viruses and the enhanced neurovirulence. In particular, position 98 might be involved in neutralizing antibodies, modulating the virus-receptor interaction and enhancing the virulence of the viruses. Moreover, both positions 145 and 241 might correlate to determine the receptor specificity. However, these positions did not display much difference in amino acid polymorphism. In addition, no position in the VP1 genes of viruses isolated from China was under positive selection.

  4. The Oligomerization Domain of VP3, the Scaffolding Protein of Infectious Bursal Disease Virus, Plays a Critical Role in Capsid Assembly

    Science.gov (United States)

    Maraver, Antonio; Oña, Ana; Abaitua, Fernando; González, Dolores; Clemente, Roberto; Ruiz-Díaz, Jose A.; Castón, Jose R.; Pazos, Florencio; Rodriguez, Jose F.

    2003-01-01

    Infectious bursal disease virus (IBDV) capsids are formed by a single protein layer containing three polypeptides, pVP2, VP2, and VP3. Here, we show that the VP3 protein synthesized in insect cells, either after expression of the complete polyprotein or from a VP3 gene construct, is proteolytically degraded, leading to the accumulation of product lacking the 13 C-terminal residues. This finding led to identification of the VP3 oligomerization domain within a 24-amino-acid stretch near the C-terminal end of the polypeptide, partially overlapping the VP1 binding domain. Inactivation of the VP3 oligomerization domain, by either proteolysis or deletion of the polyprotein gene, abolishes viruslike particle formation. Formation of VP3-VP1 complexes in cells infected with a dual recombinant baculovirus simultaneously expressing the polyprotein and VP1 prevented VP3 proteolysis and led to efficient virus-like particle formation in insect cells. PMID:12743301

  5. Imaging of the alphavirus capsid protein during virus replication.

    Science.gov (United States)

    Zheng, Yan; Kielian, Margaret

    2013-09-01

    Alphaviruses are enveloped viruses with highly organized structures. The nucleocapsid (NC) core contains a capsid protein lattice enclosing the plus-sense RNA genome, and it is surrounded by a lipid bilayer containing a lattice of the E1 and E2 envelope glycoproteins. Capsid protein is synthesized in the cytoplasm and particle budding occurs at the plasma membrane (PM), but the traffic and assembly of viral components and the exit of virions from host cells are not well understood. To visualize the dynamics of capsid protein during infection, we developed a Sindbis virus infectious clone tagged with a tetracysteine motif. Tagged capsid protein could be fluorescently labeled with biarsenical dyes in living cells without effects on virus growth, morphology, or protein distribution. Live cell imaging and colocalization experiments defined distinct groups of capsid foci in infected cells. We observed highly motile internal puncta that colocalized with E2 protein, which may represent the transport machinery that capsid protein uses to reach the PM. Capsid was also found in larger nonmotile internal structures that colocalized with cellular G3BP and viral nsP3. Thus, capsid may play an unforeseen role in these previously observed G3BP-positive foci, such as regulation of cellular stress granules. Capsid puncta were also observed at the PM. These puncta colocalized with E2 and recruited newly synthesized capsid protein; thus, they may be sites of virus assembly and egress. Together, our studies provide the first dynamic views of the alphavirus capsid protein in living cells and a system to define detailed mechanisms during alphavirus infection.

  6. Molecular interactions of Epstein-Barr virus capsid proteins.

    Science.gov (United States)

    Wang, Wen-Hung; Chang, Li-Kwan; Liu, Shih-Tung

    2011-02-01

    The capsids of herpesviruses, which comprise major and minor capsid proteins, have a common icosahedral structure with 162 capsomers. An electron microscopic study shows that Epstein-Barr virus (EBV) capsids in the nucleus are immunolabeled by anti-BDLF1 and anti-BORF1 antibodies, indicating that BDLF1 and BORF1 are the minor capsid proteins of EBV. Cross-linking and electrophoresis studies of purified BDLF1 and BORF1 revealed that these two proteins form a triplex that is similar to that formed by the minor capsid proteins, VP19C and VP23, of herpes simplex virus type 1 (HSV-1). Although the interaction between VP23, a homolog of BDLF1, and the major capsid protein VP5 could not be verified biochemically in earlier studies, the interaction between BDLF1 and the EBV major capsid protein, viral capsid antigen (VCA), can be confirmed by glutathione S-transferase (GST) pulldown assay and coimmunoprecipitation. Additionally, in HSV-1, VP5 interacts with only the middle region of VP19C; in EBV, VCA interacts with both the N-terminal and middle regions of BORF1, a homolog of VP19C, revealing that the proteins in the EBV triplex interact with the major capsid protein differently from those in HSV-1. A GST pulldown study also identifies the oligomerization domains in VCA and the dimerization domain in BDLF1. The results presented herein reveal how the EBV capsid proteins interact and thereby improve our understanding of the capsid structure of the virus.

  7. Low levels of foot-and-mouth disease virus 3C protease expression are required to achieve optimal capsid protein expression and processing in mammalian cells

    DEFF Research Database (Denmark)

    Polacek, Charlotta; Gullberg, Maria; Li, Jiong;

    2013-01-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor (P1-2A) is processed by the virus-encoded 3C protease (3Cpro) to produce VP0, VP3, VP1 and 2A. Within the virus-encoded polyprotein, the P1-2A and 3Cpro can be expected to be produced at equivalent concentrations. However, using...... production of diagnostic reagents and improved vaccines against foot-and-mouth disease....

  8. Effects of dendritic cells pulsed with VP1 protein of foot-and-mouth disease virus on IFN-γ release of T lymphocyte%负载口蹄疫病毒VP1蛋白质的树突状细胞对T细胞产生IFN-γ的影响

    Institute of Scientific and Technical Information of China (English)

    李娜; 张雷; 安鹏丽; 高云欢; 张丽; 王家鑫

    2011-01-01

    目的:研究负载口蹄疫病毒VP1蛋白质的树突状细胞对淋巴结T细胞产生IFN-γ的影响.方法:构建pET32a-VP1原核表达载体,经IPTG诱导表达并纯化重组蛋白VP1.制备骨髓源树突状细胞(BMDC)和淋巴结T细胞,将纯化的VP1蛋白质负载BMDC后与淋巴结T细胞共培养,收集不同时间点的共培养上清液,用ELISA检测其IFN-γ的含量.结果:本实验成功构建了pET32a-VP1原核表达载体,并获得了VP1蛋白质.在负载VP1蛋白质的BMDC与T细胞共培养后,实验组各时间点上清液的IFN-γ含量均高于对照组(3小时除外),特别是在共培养后9、24和48小时,差异显著.结论:负载FMDV VP1蛋白质的BMDC可有效激活淋巴结T细胞,从而启动Th1细胞免疫应答,分泌大量IFN-γ.%0bjectivre:To study 1he activation ofT ]ym phocytes by bone m orrow-derired dendritic eels (BMDC ) pulsedwilh VP1 protein of fbot-and-m ou1h disease virus (FMDV ) in vitro .M e1hods:The prokaryotic expressbn vector of pET32a-VPl was constructed and induced to expressVPl by PTG .VP1 protein was purified by common SDSrPAGE and efectroelutfan approach .BMDC pulsedwith FMDV VP1 protein w ere co-cultured w ith lym ph node T eels .Supema1an1s w ere harvested at indicated tin e points and 1he FN-y levels of supematente w ere deter- mined with ELBA .Results :The prokaryotic expression vector was successfully constructed and VP1 protein was expressed. A fier ocrculture of lym ph node T cells w ith BM D C pulsed w ith V PI protein ,the FN-γ levels of test groups w ere higher 1han control groups w ith significant differences at9 h ,24 h ,and 48 h butno significant difference at3 h.Conclusion :The BM DC pulsed with FM DV VP1 protein are able ti activate 1he T eels efHcientiy ,resulting in Thl response w ith vigorous release of FN-r.

  9. A viable simian virus 40 variant with a deletion in the overlapping genes for virion proteins VP1, VP2 and VP3.

    Science.gov (United States)

    Norkin, L C; Piatak, M

    1982-12-01

    Nucleotide sequence analysis was used to determine the exact location of a deletion in the late region of the SP2 mutant of simian virus 40 (SV40), a viable small-plaque variant isolated from a persistent infection of rhesus monkey kidney cells. The results indicate that six base pairs are deleted from that part of the SV40 genome in which the coding regions for the three virion proteins, VP1, VP2 and VP3, overlap. This implies that all three virion proteins are affected by the deletion. This finding is discussed with respect to the viability of SP2.

  10. Assembly and characterization of foot-and-mouth disease virus empty capsid particles expressed within mammalian cells

    DEFF Research Database (Denmark)

    Gullberg, Maria; Muszynski, Bartosz; Organtini, Lindsey J.;

    2013-01-01

    The foot-and-mouth disease virus (FMDV) structural protein precursor, P1-2A, is cleaved by the virus-encoded 3C protease (3Cpro) into the capsid proteins VP0, VP1 and VP3 (and 2A). In some systems, it is difficult to produce large amounts of these processed capsid proteins since 3Cpro can be toxic...

  11. The VP1 S154D mutation of type Asia1 foot-and-mouth disease virus enhances viral replication and pathogenicity.

    Science.gov (United States)

    Lian, Kaiqi; Yang, Fan; Zhu, Zixiang; Cao, Weijun; Jin, Ye; Liu, Huanan; Li, Dan; Zhang, Keshan; Guo, Jianhong; Liu, Xiangtao; Zheng, Haixue

    2016-04-01

    One of the proteins encoded by the foot-and-mouth disease virus (FMDV), the VP1 protein, a capsid protein, plays an important role in integrin receptor attachment and humoral immunity-mediated host responses. The integrin receptor recognition motif and an important antigenic epitope exist within the G-H loop, which is comprised of amino acids 134-160 of the VP1 protein. FMDV strain, Asia1/HN/CHA/06, isolated from a pig, was passaged four times in suckling mice and sequenced. Sequencing analyses showed that there was a mutation of the integrin receptor recognition motif Arg-Gly-Asp/Arg-Asp-Asp (RGD/RDD, VP1 143-145) and a VP1 154 serine/Asp (VP1 S154D) mutation in the G-H loop of the VP1 protein. The influence of the RGD/RDD mutation on Asia1 FMDV disease phenotype has been previously studied. In this study, to determine the influence of the VP1 S154D mutation on FMDV Asia1 replication and pathogenicity, two recombinant FMDVs with different residues only at the VP1 154 site were rescued by reverse genetics techniques and their infectious potential in host cells and pathogenicity in pigs were compared. Our data indicates that the VP1 S154D mutation increases the replication level of FMDV Asia1/HN/CHA/06 in BHK-21, IB-RS-2, and PK-15 cells and enhances pathogenicity in pigs. Through the transient transfection-infection assay to compare integrin receptor usage of two recombinant viruses, the result shows that the VP1 S154D mutation markedly increases the ability of type Asia1 FMDV to use the integrin receptors αυβ6 and αυβ8 from pig. This study identifies a key research target for illuminating the role of residues located at G-H loop in FMDV pathogenicity.

  12. 3D reconstruction and capsid protein characterization of grass carp reovirus

    Institute of Scientific and Technical Information of China (English)

    FANG; Qin; Shah; Sanket; LIANG; Yuyao; Z.; H.; ZHOU

    2005-01-01

    Grass carp reovirus (GCRV) is a relatively new virus first isolated in China and is a member of the Aquareovirus genus of the Reoviridae family. Recent report of genomic sequencing showed that GCRV shared high degree of homology with mammalian reovirus (MRV). As a step of our effort to understand the structural basis of GCRV pathogenesis, we determined the three-dimensional (3D) structure of GCRV capsid at 17 (A) resolution by electron cryomicroscopy. Each GCRV capsid has a multilayered organization, consisting of an RNA core, an inner, middle and outer protein layer. The outer layer is made up of 200 trimers that are arranged on an incomplete T=13 icosahedral lattice. A characteristic feature of this layer is the depression resulting from the absence of trimers around the peripentonal positions, revealing the underlying trimers on the middle layer. There are 120 subunits in the inner layer arranged with T=1 symmetry. These structural features are common to other members of the Reoviridae. Moreover, SDS-PAGE analysis showed that GCRV virions contain seven structural proteins (VP1-VP7). These structural proteins have a high degree of sequence homology to MRV, consistent with the structural similarities observed in our study. The high structural similarities of isolated GCRV and MRV suggest that future structural studies focusing on GCRV entering into and replicating within its host cell are necessary in order to fully understand the structural basis of GCRV pathogenesis.

  13. Recombinant VP1, an Akt inhibitor, suppresses progression of hepatocellular carcinoma by inducing apoptosis and modulation of CCL2 production.

    Directory of Open Access Journals (Sweden)

    Tai-An Chen

    Full Text Available BACKGROUND: The application of viral elements in tumor therapy is one facet of cancer research. Recombinant capsid protein VP1 (rVP1 of foot-and-mouth disease virus has previously been demonstrated to induce apoptosis in cancer cell lines. Here, we aim to further investigate its apoptotic mechanism and possible anti-metastatic effect in murine models of hepatocellular carcinoma (HCC, one of the most common human cancers worldwide. METHODOLOGY/PRINCIPAL FINDINGS: Treatment with rVP1 inhibited cell proliferation in two murine HCC cell lines, BNL and Hepa1-6, with IC₅₀ values in the range of 0.1-0.2 µM. rVP1 also induced apoptosis in these cells, which was mediated by Akt deactivation and dissociation of Ku70-Bax, and resulted in conformational changes and mitochondrial translocation of Bax, leading to the activation of caspases-9, -3 and -7. Treatment with 0.025 µM rVP1, which did not affect the viability of normal hepatocytes, suppressed cell migration and invasion via attenuating CCL2 production. The production of CCL2 was modulated by Akt-dependent NF-κB activation that was decreased after rVP1 treatment. The in vivo antitumor effects of rVP1 were assessed in both subcutaneous and orthotopic mouse models of HCC in immune-competent BALB/c mice. Intratumoral delivery of rVP1 inhibited subcutaneous tumor growth as a result of increased apoptosis. Intravenous administration of rVP1 in an orthotopic HCC model suppressed tumor growth, inhibited intra-hepatic metastasis, and prolonged survival. Furthermore, a decrease in the serum level of CCL2 was observed in rVP1-treated mice. CONCLUSIONS/SIGNIFICANCE: The data presented herein suggest that, via inhibiting Akt phosphorylation, rVP1 suppresses the growth, migration, and invasion of murine HCC cells by inducing apoptosis and attenuating CCL2 production both in vitro and in vivo. Recombinant protein VP1 thus has the potential to be developed as a new therapeutic agent for HCC.

  14. H1PVAT is a novel and potent early-stage inhibitor of poliovirus replication that targets VP1.

    Science.gov (United States)

    Tijsma, Aloys; Thibaut, Hendrik Jan; Spieser, Stéphane A H; De Palma, Armando; Koukni, Mohamed; Rhoden, Eric; Oberste, Steve; Pürstinger, Gerhard; Volny-Luraghi, Antonia; Martin, Javier; Marchand, Arnaud; Chaltin, Patrick; Neyts, Johan; Leyssen, Pieter

    2014-10-01

    A novel small molecule, H1PVAT, was identified as a potent and selective inhibitor of the in vitro replication of all three poliovirus serotypes, whereas no activity was observed against other enteroviruses. Time-of-drug-addition studies revealed that the compound interfered with an early stage of virus replication. Four independently-selected H1PVAT-resistant virus variants uniformly carried the single amino acid substitution I194F in the VP1 capsid protein. Poliovirus type 1 strain Sabin, reverse-engineered to contain this substitution, proved to be completely insensitive to the antiviral effect of H1PVAT and was cross-resistant to the capsid-binding inhibitors V-073 and pirodavir. The VP1 I194F mutant had a smaller plaque phenotype than wild-type virus, and the amino acid substitution rendered the virus more susceptible to heat inactivation. Both for the wild-type and VP1 I194F mutant virus, the presence of H1PVAT increased the temperature at which the virus was inactivated, providing evidence that the compound interacts with the viral capsid, and that capsid stabilization and antiviral activity are not necessarily correlated. Molecular modeling suggested that H1PVAT binds with high affinity in the pocket underneath the floor of the canyon that is involved in receptor binding. Introduction of the I194F substitution in the model of VP1 induced a slight concerted rearrangement of the core β-barrel in this pocket, which disfavors binding of the compound. Taken together, the compound scaffold, to which H1PVAT belongs, may represent another promising class of poliovirus capsid-binding inhibitors next to V-073 and pirodavir. Potent antivirals against poliovirus will be essential in the poliovirus eradication end-game.

  15. Genetic Analysis of the P1 Region of Human Enterovirus 71 Strains and Expression of the 55 F StrainVP1 Protein

    Institute of Scientific and Technical Information of China (English)

    Jian-qiang Li; Jun-jie Yang; Xiu-juan Fan; Zhen-peng Sun; Yan Sun; Huan Li; Zi-xin Meng; Wei Li

    2012-01-01

    Enterovirus 71 (EV71) is a member of the Entero-virus genus of the Picornaviridae family and is the major cause of Hand,foot,and mouth disease (HFMD) in children.Different strains from Gansu were cloned and the P1 protein was sequenced and analysed.Results indicate that there are three kinds of EV71 infections prevalent in Gansu.The VP1 protein from one of these strains,55F,was expressed.The recombinant protein was expressed with high level and reacted specifically with the EV71 patient antibody,the recombinant protein was also applied to raise antiserum in rabbits and after the fourth injection a high titer of antiserum was detected by ELISA assay.These data are useful for further clarification of prevalent EV71 strains in the north of China at the molecular level and provide a basis for EV71 diagnosis.

  16. Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation.

    Science.gov (United States)

    Wittkop, Linda; Schwarz, Alexandra; Cassany, Aurelia; Grün-Bernhard, Stefanie; Delaleau, Mildred; Rabe, Birgit; Cazenave, Christian; Gerlich, Wolfram; Glebe, Dieter; Kann, Michael

    2010-07-01

    Capsids of hepatitis B virus and other hepadnaviruses contain a cellular protein kinase, which phosphorylates the capsid protein. Some phosphorylation sites are shown to be essential for distinct steps of viral replication as pregenome packaging or plus strand DNA synthesis. Although different protein kinases have been reported to phosphorylate the capsid protein, varying experimental approaches do not allow direct comparison. Furthermore, the activity of a specific protein kinase has not yet been correlated to steps in the hepadnaviral life cycle. In this study we show that capsids from various sources encapsidate active protein kinase Calpha, irrespective of hepatitis B virus genotype and host cell. Treatment of a virion expressing cell line with a pseudosubstrate inhibitor showed that inhibition of protein kinase C phosphorylation did not affect genome maturation but resulted in capsid accumulation and inhibited virion release to the medium. Our results imply that different protein kinases have distinct functions within the hepadnaviral life cycle.

  17. Structure of the small outer capsid protein, Soc: a clamp for stabilizing capsids of T4-like phages.

    Science.gov (United States)

    Qin, Li; Fokine, Andrei; O'Donnell, Erin; Rao, Venigalla B; Rossmann, Michael G

    2010-01-29

    Many viruses need to stabilize their capsid structure against DNA pressure and for survival in hostile environments. The 9-kDa outer capsid protein (Soc) of bacteriophage T4, which stabilizes the virus, attaches to the capsid during the final stage of maturation. There are 870 Soc molecules that act as a "glue" between neighboring hexameric capsomers, forming a "cage" that stabilizes the T4 capsid against extremes of pH and temperature. Here we report a 1.9 A resolution crystal structure of Soc from the bacteriophage RB69, a close relative of T4. The RB69 crystal structure and a homology model of T4 Soc were fitted into the cryoelectron microscopy reconstruction of the T4 capsid. This established the region of Soc that interacts with the major capsid protein and suggested a mechanism, verified by extensive mutational and biochemical studies, for stabilization of the capsid in which the Soc trimers act as clamps between neighboring capsomers. The results demonstrate the factors involved in stabilizing not only the capsids of T4-like bacteriophages but also many other virus capsids.

  18. RNA-binding region of Macrobrachium rosenbergii nodavirus capsid protein.

    Science.gov (United States)

    Goh, Zee Hong; Mohd, Nur Azmina Syakirin; Tan, Soon Guan; Bhassu, Subha; Tan, Wen Siang

    2014-09-01

    White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein.

  19. Antigenic properties of avian hepatitis E virus capsid protein.

    Science.gov (United States)

    Zhao, Qin; Syed, Shahid Faraz; Zhou, En-Min

    2015-10-22

    Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease and hepatitis-splenomegaly syndrome in chickens, and is genetically and antigenically related to mammalian HEVs. HEV capsid protein contains immunodominant epitopes and induces a protective humoral immune response. A better understanding of the antigenic composition of this protein is critically important for the development of effective vaccine and sensitive and specific serological assays. To date, six linear antigenic domains (I-VI) have been characterized in avian HEV capsid protein and analyzed for their applications in the serological diagnosis and vaccine design. Domains I and V induce strong immune response in chickens and are common to avian, human, and swine HEVs, indicating that the shared epitopes hampering differential diagnosis of avian HEV infection. Domains III and IV are not immunodominant and elicit a weak immune response. Domain VI, located in the N-terminal region of the capsid protein, can also trigger an intense immune response, but the anti-domain VI antibodies are transient. The protection analysis showed that the truncated capsid protein containing the C-terminal 268 amino acid residues expressed by the bacterial system can provide protective immunity against avian HEV infection in chickens. However, the synthetic peptides incorporating the different linear antigenic domains (I-VI) and epitopes are non-protective. The antigenic composition of avian HEV capsid protein is altogether complex. To develop an effective vaccine and accurate serological diagnostic methods, more conformational antigenic domains or epitopes are to be characterized in detail.

  20. Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells.

    Directory of Open Access Journals (Sweden)

    Cong Haolong

    Full Text Available Enterovirus 71 (EV71 is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human.

  1. A Novel Murine Model of Parvovirus Associated Dilated Cardiomyopathy Induced by Immunization with VP1-Unique Region of Parvovirus B19

    Science.gov (United States)

    Šimoliūnas, Egidijus; Rinkūnaitė, Ieva; Smalinskaitė, Luka; Podkopajev, Andrej; Bironaitė, Daiva; Weis, Cleo-Aron; Marx, Alexander; Bukelskienė, Virginija; Gretz, Norbert; Grabauskienė, Virginija; Labeit, Dittmar; Labeit, Siegfried

    2016-01-01

    Background. Parvovirus B19 (B19V) is a common finding in endomyocardial biopsy specimens from myocarditis and dilated cardiomyopathy patients. However, current understanding of how B19V is contributing to cardiac damage is rather limited due to the lack of appropriate mice models. In this work we demonstrate that immunization of BALB/c mice with the major immunogenic determinant of B19V located in the unique sequence of capsid protein VP1 (VP1u) is an adequate model to study B19V associated heart damage. Methods and Results. We immunized mice in the experimental group with recombinant VP1u; immunization with cardiac myosin derived peptide served as a positive reference and phosphate buffered saline served as negative control. Cardiac function and dimensions were followed echocardiographically 69 days after immunization. Progressive dilatation of left ventricle and decline of ejection fraction were observed in VP1u- and myosin-immunized mice. Histologically, severe cardiac fibrosis and accumulation of heart failure cells in lungs were observed 69 days after immunization. Transcriptomic profiling revealed ongoing cardiac remodeling and immune process in VP1u- and myosin-immunized mice. Conclusions. Immunization of BALB/c mice with VP1u induces dilated cardiomyopathy in BALB/c mice and it could be used as a model to study clinically relevant B19V associated cardiac damage. PMID:27812527

  2. Crystal Structure of the Human Astrovirus Capsid Protein

    Science.gov (United States)

    Toh, Yukimatsu; Harper, Justin; Dryden, Kelly A.; Yeager, Mark; Méndez, Ernesto

    2016-01-01

    ABSTRACT Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. HAstV is a nonenveloped virus with a T=3 capsid and a positive-sense RNA genome. The capsid protein (CP) of HAstV is synthesized as a 90-kDa precursor (VP90) that can be divided into three linear domains: a conserved N-terminal domain, a hypervariable domain, and an acidic C-terminal domain. Maturation of HAstV requires proteolytic processing of the astrovirus CP both inside and outside the host cell, resulting in the removal of the C-terminal domain and the breakdown of the rest of the CP into three predominant protein species with molecular masses of ∼34, 27/29, and 25/26 kDa, respectively. We have now solved the crystal structure of VP9071–415 (amino acids [aa] 71 to 415 of VP90) of human astrovirus serotype 8 at a 2.15-Å resolution. VP9071–415 encompasses the conserved N-terminal domain of VP90 but lacks the hypervariable domain, which forms the capsid surface spikes. The structure of VP9071–415 is comprised of two domains: an S domain, which adopts the typical jelly-roll β-barrel fold, and a P1 domain, which forms a squashed β-barrel consisting of six antiparallel β-strands similar to what was observed in the hepatitis E virus (HEV) capsid structure. Fitting of the VP9071–415 structure into the cryo-electron microscopy (EM) maps of HAstV produced an atomic model for a continuous, T=3 icosahedral capsid shell. Our pseudoatomic model of the human HAstV capsid shell provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation needed for virus infectivity. Such information has potential applications in the development of a virus-like particle (VLP) vaccine as well as small-molecule drugs targeting astrovirus assembly/maturation. IMPORTANCE Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. As a nonenveloped virus

  3. L2, the minor capsid protein of papillomavirus

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Joshua W. [Department of Pathology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Roden, Richard B.S., E-mail: roden@jhmi.edu [Department of Pathology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Department of Oncology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Department of Gynecology and Obstetrics, The Johns Hopkins University, Baltimore, MD 21287 (United States)

    2013-10-15

    The capsid protein L2 plays major roles in both papillomavirus assembly and the infectious process. While L1 forms the majority of the capsid and can self-assemble into empty virus-like particles (VLPs), L2 is a minor capsid component and lacks the capacity to form VLPs. However, L2 co-assembles with L1 into VLPs, enhancing their assembly. L2 also facilitates encapsidation of the ∼8 kbp circular and nucleosome-bound viral genome during assembly of the non-enveloped T=7d virions in the nucleus of terminally differentiated epithelial cells, although, like L1, L2 is not detectably expressed in infected basal cells. With respect to infection, L2 is not required for particles to bind to and enter cells. However L2 must be cleaved by furin for endosome escape. L2 then travels with the viral genome to the nucleus, wherein it accumulates at ND-10 domains. Here, we provide an overview of the biology of L2. - Highlights: • L2 is the minor antigen of the non-enveloped T=7d icosahedral Papillomavirus capsid. • L2 is a nuclear protein that can traffic to ND-10 and facilitate genome encapsidation. • L2 is critical for infection and must be cleaved by furin. • L2 is a broadly protective vaccine antigen recognized by neutralizing antibodies.

  4. CapsidMaps: protein-protein interaction pattern discovery platform for the structural analysis of virus capsids using Google Maps.

    Science.gov (United States)

    Carrillo-Tripp, Mauricio; Montiel-García, Daniel Jorge; Brooks, Charles L; Reddy, Vijay S

    2015-04-01

    Structural analysis and visualization of protein-protein interactions is a challenging task since it is difficult to appreciate easily the extent of all contacts made by the residues forming the interfaces. In the case of viruses, structural analysis becomes even more demanding because several interfaces coexist and, in most cases, these are formed by hundreds of contacting residues that belong to multiple interacting coat proteins. CapsidMaps is an interactive analysis and visualization tool that is designed to benefit the structural virology community. Developed as an improved extension of the φ-ψ Explorer, here we describe the details of its design and implementation. We present results of analysis of a spherical virus to showcase the features and utility of the new tool. CapsidMaps also facilitates the comparison of quaternary interactions between two spherical virus particles by computing a similarity (S)-score. The tool can also be used to identify residues that are solvent exposed and in the process of locating antigenic epitope regions as well as residues forming the inside surface of the capsid that interact with the nucleic acid genome. CapsidMaps is part of the VIPERdb Science Gateway, and is freely available as a web-based and cross-browser compliant application at http://viperdb.scripps.edu.

  5. 利用Asia 1型口蹄疫病毒VP1蛋白的单克隆抗体建立单抗竞争ELISA方法%Establishment of Monoclonal Antibody Competitive ELISA Using Monoclonal Antibody Against VP1 Protein of Asia 1 Type Foot-and-Mouth Disease Virus

    Institute of Scientific and Technical Information of China (English)

    林彤; 邵军军; 丛国正; 独军政; 高闪电; 常惠芸; 谢庆阁

    2009-01-01

    Using the purified VP1 protein of Asia 1 type foot-and-mouth disease virus as the antigen, the purified monoclonal antibody was labeled by the sodium periodate method and the monoclonal antibody competitive ELISA was established in this study. Ten positive porcine foot-and-mouth disease serums and more than two hundreds negative serum were tested, and the results were the same as the background of samples. The sensitivity test and replicate test indicated that this method was stable and sensitive, which was suitable for monitoring Asia 1 type porcine foot-and-mouth disease virus antibody.

  6. Parvovirus capsid disorders cholesterol-rich membranes.

    Science.gov (United States)

    Pakkanen, Kirsi; Kirjavainen, Sanna; Mäkelä, Anna R; Rintanen, Nina; Oker-Blom, Christian; Jalonen, Tuula O; Vuento, Matti

    2009-02-06

    In this study canine parvovirus, CPV, was found to induce disorder in DPPC:cholesterol membranes in acidic conditions. This acidicity-induced fluidizing effect is suggested to originate from the N-terminus of the viral capsid protein VP1. In accordance with the model membrane studies, a fluidizing effect was seen also in the endosomal membranes during CPV infection implying an important functional role of the fluidization in the endocytic entry of the virus.

  7. Impact of capsid conformation and Rep-capsid interactions on adeno-associated virus type 2 genome packaging.

    Science.gov (United States)

    Bleker, Svenja; Pawlita, Michael; Kleinschmidt, Jürgen A

    2006-01-01

    Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefold pore nearly completely abolished Rep-capsid interaction and packaging. This suggests a Rep-binding site at the highly conserved amino acids at or close to the pores formed by the capsid protein pentamers. A different mutant (P. Wu, W. Xiao, T. Conlon, J. Hughes, M. Agbandje-McKenna, T. Ferkol, T. Flotte, and N. Muzyczka, J. Virol. 74:8635-8647, 2000) with an amino acid exchange at the interface of capsid protein pentamers led to a complete block of DNA encapsidation. Analysis of the capsid conformation of this mutant revealed that the pores at the fivefold axes were occupied by VP1/VP2 N termini, thereby preventing DNA introduction into the capsid. Nevertheless, the corresponding capsids had more Rep proteins bound than wild-type AAV, showing that correct Rep interaction with the capsid depends on a defined capsid conformation. Both mutant types together support the conclusion that the pores at the fivefold symmetry axes are involved in genome packaging and that capsid conformation-dependent Rep-capsid interactions play an essential role in the packaging process.

  8. Quantum dot-induced viral capsid assembling in dissociation buffer.

    Science.gov (United States)

    Gao, Ding; Zhang, Zhi-Ping; Li, Feng; Men, Dong; Deng, Jiao-Yu; Wei, Hong-Ping; Zhang, Xian-En; Cui, Zong-Qiang

    2013-01-01

    Viruses encapsulating inorganic nanoparticles are a novel type of nanostructure with applications in biomedicine and biosensors. However, the encapsulation and assembly mechanisms of these hybridized virus-based nanoparticles (VNPs) are still unknown. In this article, it was found that quantum dots (QDs) can induce simian virus 40 (SV40) capsid assembly in dissociation buffer, where viral capsids should be disassembled. The analysis of the transmission electron microscope, dynamic light scattering, sucrose density gradient centrifugation, and cryo-electron microscopy single particle reconstruction experimental results showed that the SV40 major capsid protein 1 (VP1) can be assembled into ≈25 nm capsids in the dissociation buffer when QDs are present and that the QDs are encapsulated in the SV40 capsids. Moreover, it was determined that there is a strong affinity between QDs and the SV40 VP1 proteins (KD=2.19E-10 M), which should play an important role in QD encapsulation in the SV40 viral capsids. This study provides a new understanding of the assembly mechanism of SV40 virus-based nanoparticles with QDs, which may help in the design and construction of other similar virus-based nanoparticles.

  9. Revised Mimivirus major capsid protein sequence reveals intron-containing gene structure and extra domain

    Directory of Open Access Journals (Sweden)

    Suzan-Monti Marie

    2009-05-01

    Full Text Available Abstract Background Acanthamoebae polyphaga Mimivirus (APM is the largest known dsDNA virus. The viral particle has a nearly icosahedral structure with an internal capsid shell surrounded with a dense layer of fibrils. A Capsid protein sequence, D13L, was deduced from the APM L425 coding gene and was shown to be the most abundant protein found within the viral particle. However this protein remained poorly characterised until now. A revised protein sequence deposited in a database suggested an additional N-terminal stretch of 142 amino acids missing from the original deduced sequence. This result led us to investigate the L425 gene structure and the biochemical properties of the complete APM major Capsid protein. Results This study describes the full length 3430 bp Capsid coding gene and characterises the 593 amino acids long corresponding Capsid protein 1. The recombinant full length protein allowed the production of a specific monoclonal antibody able to detect the Capsid protein 1 within the viral particle. This protein appeared to be post-translationnally modified by glycosylation and phosphorylation. We proposed a secondary structure prediction of APM Capsid protein 1 compared to the Capsid protein structure of Paramecium Bursaria Chlorella Virus 1, another member of the Nucleo-Cytoplasmic Large DNA virus family. Conclusion The characterisation of the full length L425 Capsid coding gene of Acanthamoebae polyphaga Mimivirus provides new insights into the structure of the main Capsid protein. The production of a full length recombinant protein will be useful for further structural studies.

  10. Primate TRIM5 proteins form hexagonal nets on HIV-1 capsids

    Science.gov (United States)

    Li, Yen-Li; Chandrasekaran, Viswanathan; Carter, Stephen D; Woodward, Cora L; Christensen, Devin E; Dryden, Kelly A; Pornillos, Owen; Yeager, Mark; Ganser-Pornillos, Barbie K; Jensen, Grant J; Sundquist, Wesley I

    2016-01-01

    TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids. DOI: http://dx.doi.org/10.7554/eLife.16269.001 PMID:27253068

  11. Antigenic structure of the capsid protein of rabbit haemorrhagic disease virus

    DEFF Research Database (Denmark)

    Martinez-Torrecuadrada, Jorge L.; Cortes, Elena; Vela, Carmen;

    1998-01-01

    Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid is composed of a single 60 kDa protein. The capsid protein gene was cloned in Escherichia coli using the pET3 system, and the antigenic structure of RHDV VP60 was dissected using 11 monoclonal...

  12. Anti-VP1 and anti-VP2 antibodies detected by immunofluorescence assays in patients with acute human parvovirus B19 infection

    Directory of Open Access Journals (Sweden)

    Pereira Renata FA

    2001-01-01

    Full Text Available Acute human parvovirus B19 infection is followed by an antibody response to the structural proteins of the viral capsid (VP1 and VP2. We used 80 sera collected from 58 erythema infectiosum and 6 transient aplastic crisis patients to test IgM and IgG antibodies against these two proteins in an immunofluorescence assay (IFA using Sf9 cells infected with recombinant baculovirus expressing either VP1 or VP2 antigen. Although less sensitive than IgM capture enzyme immunoassay using native antigen (MACEIA, we could detect anti-VP1 or anti-VP2 IgM antibodies by IFA in 49 patients with acute infection (76.6%. Detection of IgG anti-VP1 and anti-VP2 by IFA, however, was as sensitive as IgG detection by indirect enzyme immunoassay. By applying IgG avidity IFA to sera of the 15 IgM IFA negative patients we were able to confirm acute infection in further 12 cases by IFA. Overall, acute infection was confirmed by IFA in 61 (95.3% of the 64 patients.

  13. Identification of specific antigenic epitope at N-terminal segment of enterovirus 71 (EV-71) VP1 protein and characterization of its use in recombinant form for early diagnosis of EV-71 infection

    OpenAIRE

    Zhang, Jianhua; Jiang, Bingfu; Xu, Mingjie; Dai, Xing; Purdy, Michael A.; Meng, Jihong

    2014-01-01

    Human enterovirus 71 (EV-71) is the main etiologic agent of hand, foot and mouth disease (HFMD). We sought to identify EV-71 specific antigens and develop serologic assays for acute-phase EV-71 infection. A series of truncated proteins within the N-terminal 100 amino acids (aa) of EV-71 VP1 was expressed in Escherichia coli. Western blot (WB) analysis showed that positions around 11–21 aa contain EV-71-specific antigenic sites, whereas positions 1–5 and 51–100 contain epitopes shared with hum...

  14. The hepatitis B virus core protein intradimer interface modulates capsid assembly and stability.

    Science.gov (United States)

    Selzer, Lisa; Katen, Sarah P; Zlotnick, Adam

    2014-09-02

    During the hepatitis B virus (HBV) life cycle, capsid assembly and disassembly must ensure correct packaging and release of the viral genome. Here we show that changes in the dynamics of the core protein play an important role in regulating these processes. The HBV capsid assembles from 120 copies of the core protein homodimer. Each monomer contains a conserved cysteine at position 61 that can form an intradimer disulfide that we use as a marker for dimer conformational states. We show that dimers in the context of capsids form intradimer disulfides relatively rapidly. Surprisingly, compared to reduced dimers, fully oxidized dimers assembled slower and into capsids that were morphologically similar but less stable. We hypothesize that oxidized protein adopts a geometry (or constellation of geometries) that is unfavorable for capsid assembly, resulting in weaker dimer-dimer interactions as well as slower assembly kinetics. Our results suggest that structural flexibility at the core protein intradimer interface is essential for regulating capsid assembly and stability. We further suggest that capsid destabilization by the C61-C61 disulfide has a regulatory function to support capsid disassembly and release of the viral genome.

  15. The Role of VP1 Amino Acid Residue 145 of Enterovirus 71 in Viral Fitness and Pathogenesis in a Cynomolgus Monkey Model.

    Directory of Open Access Journals (Sweden)

    Chikako Kataoka

    2015-07-01

    Full Text Available Enterovirus 71 (EV71, a major causative agent of hand, foot, and mouth disease, occasionally causes severe neurological symptoms. We identified P-selectin glycoprotein ligand-1 (PSGL-1 as an EV71 receptor and found that an amino acid residue 145 in the capsid protein VP1 (VP1-145 defined PSGL-1-binding (PB and PSGL-1-nonbinding (non-PB phenotypes of EV71. However, the role of PSGL-1-dependent EV71 replication in neuropathogenesis remains poorly understood. In this study, we investigated viral replication, genetic stability, and the pathogenicity of PB and non-PB strains of EV71 in a cynomolgus monkey model. Monkeys were intravenously inoculated with cDNA-derived PB and non-PB strains of EV71, EV71-02363-EG and EV71-02363-KE strains, respectively, with two amino acid differences at VP1-98 and VP1-145. Mild neurological symptoms, transient lymphocytopenia, and inflammatory cytokine responses, were found predominantly in the 02363-KE-inoculated monkeys. During the early stage of infection, viruses were frequently detected in clinical samples from 02363-KE-inoculated monkeys but rarely in samples from 02363-EG-inoculated monkeys. Histopathological analysis of central nervous system (CNS tissues at 10 days postinfection revealed that 02363-KE induced neuropathogenesis more efficiently than that induced by 02363-EG. After inoculation with 02363-EG, almost all EV71 variants detected in clinical samples, CNS, and non-CNS tissues, possessed a G to E amino acid substitution at VP1-145, suggesting a strong in vivo selection of VP1-145E variants and CNS spread presumably in a PSGL-1-independent manner. EV71 variants with VP1-145G were identified only in peripheral blood mononuclear cells in two out of four 02363-EG-inoculated monkeys. Thus, VP1-145E variants are mainly responsible for the development of viremia and neuropathogenesis in a non-human primate model, further suggesting the in vivo involvement of amino acid polymorphism at VP1-145 in cell

  16. A combination vaccine for allergy and rhinovirus infections based on rhinovirus-derived surface protein VP1 and a nonallergenic peptide of the major timothy grass pollen allergen Phl p 1.

    Science.gov (United States)

    Edlmayr, Johanna; Niespodziana, Katarzyna; Linhart, Birgit; Focke-Tejkl, Margarete; Westritschnig, Kerstin; Scheiblhofer, Sandra; Stoecklinger, Angelika; Kneidinger, Michael; Valent, Peter; Campana, Raffaela; Thalhamer, Josef; Popow-Kraupp, Theresia; Valenta, Rudolf

    2009-05-15

    Allergens and rhinovirus infections are among the most common elicitors of respiratory diseases. We report the construction of a recombinant combination vaccine for allergy and rhinovirus infections based on rhinovirus-derived VP1, the surface protein which is critically involved in infection of respiratory cells, and a nonallergenic peptide of the major grass pollen allergen Phl p 1. Recombinant hybrid molecules consisting of VP1 and a Phl p 1-derived peptide of 31 aa were expressed in Escherichia coli. The hybrid molecules did not react with IgE Abs from grass pollen allergic patients and lacked allergenic activity when exposed to basophils from allergic patients. Upon immunization of mice and rabbits, the hybrids did not sensitize against Phl p 1 but induced protective IgG Abs that cross-reacted with group 1 allergens from different grass species and blocked allergic patients' IgE reactivity to Phl p 1 as well as Phl p 1-induced basophil degranulation. Moreover, hybrid-induced IgG Abs inhibited rhinovirus infection of cultured human epithelial cells. The principle of fusing nonallergenic allergen-derived peptides onto viral carrier proteins may be used for the engineering of safe allergy vaccines which also protect against viral infections.

  17. Interactions of the HSV-1 UL25 Capsid Protein with Cellular Microtubule-associated Protein

    Institute of Scientific and Technical Information of China (English)

    Lei GUO; Ying ZHANG; Yan-chun CHE; Wen-juan WU; Wei-zhong LI; Li-chun WANG; Yun LIAO; Long-ding LIU; Qi-han LI

    2008-01-01

    An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β-D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.

  18. Sequence analysis and structural implications of rotavirus capsid proteins.

    Science.gov (United States)

    Parbhoo, N; Dewar, J B; Gildenhuys, S

    Rotavirus is the major cause of severe virus-associated gastroenteritis worldwide in children aged 5 and younger. Many children lose their lives annually due to this infection and the impact is particularly pronounced in developing countries. The mature rotavirus is a non-enveloped triple-layered nucleocapsid containing 11 double stranded RNA segments. Here a global view on the sequence and structure of the three main capsid proteins, VP2, VP6 and VP7 is shown by generating a consensus sequence for each of these rotavirus proteins, for each species obtained from published data of representative rotavirus genotypes from across the world and across species. Degree of conservation between species was represented on homology models for each of the proteins. VP7 shows the highest level of variation with 14-45 amino acids showing conservation of less than 60%. These changes are localised to the outer surface alluding to a possible mechanism in evading the immune system. The middle layer, VP6 shows lower variability with only 14-32 sites having lower than 70% conservation. The inner structural layer made up of VP2 showed the lowest variability with only 1-16 sites having less than 70% conservation across species. The results correlate with each protein's multiple structural roles in the infection cycle. Thus, although the nucleotide sequences vary due to the error-prone nature of replication and lack of proof reading, the corresponding amino acid sequence of VP2, 6 and 7 remain relatively conserved. Benefits of this knowledge about the conservation include the ability to target proteins at sites that cannot undergo mutational changes without influencing viral fitness; as well as possibility to study systems that are highly evolved for structure and function in order to determine how to generate and manipulate such systems for use in various biotechnological applications.

  19. Assembly of the small outer capsid protein, Soc, on bacteriophage T4: a novel system for high density display of multiple large anthrax toxins and foreign proteins on phage capsid.

    Science.gov (United States)

    Li, Qin; Shivachandra, Sathish B; Zhang, Zhihong; Rao, Venigalla B

    2007-07-27

    Bacteriophage T4 capsid is a prolate icosahedron composed of the major capsid protein gp23*, the vertex protein gp24*, and the portal protein gp20. Assembled on its surface are 810 molecules of the non-essential small outer capsid protein, Soc (10 kDa), and 155 molecules of the highly antigenic outer capsid protein, Hoc (39 kDa). In this study Soc, a "triplex" protein that stabilizes T4 capsid, is targeted for molecular engineering of T4 particle surface. Using a defined in vitro assembly system, anthrax toxins, protective antigen, lethal factor and their domains, fused to Soc were efficiently displayed on the capsid. Both the N and C termini of the 80 amino acid Soc polypeptide can be simultaneously used to display antigens. Proteins as large as 93 kDa can be stably anchored on the capsid through Soc-capsid interactions. Using both Soc and Hoc, up to 1662 anthrax toxin molecules are assembled on the phage T4 capsid under controlled conditions. We infer from the binding data that a relatively high affinity capsid binding site is located in the middle of the rod-shaped Soc, with the N and C termini facing the 2- and 3-fold symmetry axes of the capsid, respectively. Soc subunits interact at these interfaces, gluing the adjacent capsid protein hexamers and generating a cage-like outer scaffold. Antigen fusion does interfere with the inter-subunit interactions, but these interactions are not essential for capsid binding and antigen display. These features make the T4-Soc platform the most robust phage display system reported to date. The study offers insights into the architectural design of bacteriophage T4 virion, one of the most stable viruses known, and how its capsid surface can be engineered for novel applications in basic molecular biology and biotechnology.

  20. Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yoon Sik, E-mail: yumshak@naver.com; Seo, Hyun Wook, E-mail: suruk@naver.com; Jung, Guhung, E-mail: drjung@snu.ac.kr

    2015-02-13

    Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study that Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. - Highlights: • We examined H{sub 2}O{sub 2} and GSH modulate HBV capsid assembly. • H{sub 2}O{sub 2} facilitates HBV capsid assembly in the presence of Hsp90. • GSH inhibits function of Hsp90 in facilitating HBV capsid assembly. • H{sub 2}O{sub 2} and GSH induce conformation change of Hsp90.

  1. Immunogenicity of Newcastle disease virus vectors expressing Norwalk virus capsid protein in the presence or absence of VP2 protein.

    Science.gov (United States)

    Kim, Shin-Hee; Chen, Shun; Jiang, Xi; Green, Kim Y; Samal, Siba K

    2015-10-01

    Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirus-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans.

  2. Limited cross-reactivity of mouse monoclonal antibodies against Dengue virus capsid protein among four serotypes

    Directory of Open Access Journals (Sweden)

    Noda M

    2012-11-01

    Full Text Available Megumi Noda,1 Promsin Masrinoul,1 Chaweewan Punkum,1 Chonlatip Pipattanaboon,2,3 Pongrama Ramasoota,2,4 Chayanee Setthapramote,2,3 Tadahiro Sasaki,6 Mikiko Sasayama,1 Akifumi Yamashita,1,5 Takeshi Kurosu,6 Kazuyoshi Ikuta,6 Tamaki Okabayashi11Mahidol-Osaka Center for Infectious Diseases, 2Center of Excellence for Antibody Research, 3Department of Microbiology and Immunology, 4Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, Thailand; 5Graduate School of Life Science, Tohoku University, Sendai, Miyagi, 6Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, JapanBackground: Dengue illness is one of the important mosquito-borne viral diseases in tropical and subtropical regions. Four serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4 are classified in the Flavivirus genus of the family Flaviviridae. We prepared monoclonal antibodies against DENV capsid protein from mice immunized with DENV-2 and determined the cross-reactivity with each serotype of DENV and Japanese encephalitis virus.Methods and results: To clarify the relationship between the cross-reactivity of monoclonal antibodies and the diversity of these viruses, we examined the situations of flaviviruses by analyses of phylogenetic trees. Among a total of 60 prepared monoclonal antibodies specific for DENV, five monoclonal antibodies stained the nuclei of infected cells and were found to be specific to the capsid protein. Three were specific to DENV-2, while the other two were cross-reactive with DENV-2 and DENV-4. No monoclonal antibodies were cross-reactive with all four serotypes. Phylogenetic analysis of DENV amino acid sequences of the capsid protein revealed that DENV-2 and DENV-4 were clustered in the same branch, while DENV-1 and DENV-3 were clustered in the other branch. However, these classifications of the capsid protein were different from those of the

  3. Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus infected cells

    DEFF Research Database (Denmark)

    Kristensen, Thea; Normann, Preben; Gullberg, Maria

    2016-01-01

    The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been des...... have implications for the testing of potential antiviral agents targeting the FMDV 3C protease....

  4. 肠道病毒71型VP1~VP4基因克隆及其表达产物的免疫原性%Gene cloning and immunogenicity analysis of the structural proteins VP1-VP4 of enterovirus 71

    Institute of Scientific and Technical Information of China (English)

    宋远斌; 余楠; 何思杰; 陈欣欣; 王斌; 车小燕; 曾其毅

    2011-01-01

    Objective To investigate the changes of auditory parameters during anesthesia and establish the assessment indicators for anesthesia monitoring in animal experiments. Methods BALB/c mice of 4 to 6 weeks were given a single intraperitoneal dose of urethane, and the auditory evoked potential in the surgically exposed inferior colliculus in response to pure tone stimulation was recorded during urethane metabolism. The latency and amplitude data of the waves were extracted using Matlab software to analyze their variations during urethane metabolism. Results The latency of the auditory evoked potential showed slight variation and was well correlated to time. The latency decreased progressively during urethane metabolism, fast in the initial 2 h and tending to stabilize afterwards. Conclusion The latency of the auditory evoked potential can be more suitable indicators than the amplitude for anesthetic effect monitoring.%目的 克隆并表达肠道病毒71型VP1~VP4蛋白基因,初步鉴定其免疫原性.方法 抽提病毒RNA,经RT-PCR方法分别扩增出VP1~VP4蛋白基因片段,经克隆后,在QIA表达系统中表达,表达产物用8 mol/L尿素洗涤及Ni柱亲和层析纯化后,用肠道病毒71型免疫兔血清和柯萨奇病毒A16型免疫兔血清对重组蛋白进行Western blotting及ELISA鉴定.结果 构建的重组质粒pQE30a/VP1~VP4经IPTG诱导,重组蛋白VP1~VP4高效表达并纯化成功,经Western blotting及ELISA证实重组蛋白VP1~VP4可以被免疫兔血清特异识别.结论 肠道病毒71型VP1~VP4蛋白表达载体在大肠杆菌M15中高效表达.纯化产物具有较强的免疫原性,为今后EV71亚单位疫苗的研究和检测试剂盒提供了参考.

  5. Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo

    Directory of Open Access Journals (Sweden)

    Victoria A. Meliopoulos

    2016-11-01

    Full Text Available The disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1 capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2 capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3 from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction.

  6. Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Juan [China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Microbiology and Immunology, Nanjing Medical University (China); Wang, Shixia [China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Medicine, University of Massachusetts Medical School (United States); Gan, Weihua [Department of Pediatrics, The Second Affiliated Hospital, Nanjing Medical University (China); Zhang, Wenhong [Department of Infectious Diseases, Huashan Hospital, Fudan University (China); Ju, Liwen [School of Public Health, Fudan University (China); Huang, Zuhu [Department of Infectious Diseases, The First Affiliated Hospital, Nanjing Medical University (China); China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Lu, Shan, E-mail: shan.lu@umassmed.edu [Department of Infectious Diseases, The First Affiliated Hospital, Nanjing Medical University (China); China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Medicine, University of Massachusetts Medical School (United States)

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer EV71 is a major emerging infectious disease in many Asian countries. Black-Right-Pointing-Pointer Inactivated EV71 vaccines are in clinical studies but their safety and efficacy are unknown. Black-Right-Pointing-Pointer Developing subunit based EV71 vaccines is significant and novel antigen design is needed. Black-Right-Pointing-Pointer DNA immunization is an efficient tool to test the immunogenicity of VP1 based EV71 vaccines. Black-Right-Pointing-Pointer Multiple VP1 antigens are developed showing immunogenic potential. -- Abstract: Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71 (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines.

  7. Capsid coding sequences of foot-and-mouth disease viruses are determinants of pathogenicity in pigs

    DEFF Research Database (Denmark)

    Lohse, Louise; Jackson, Terry; Bøtner, Anette;

    2012-01-01

    The surface exposed capsid proteins, VP1, VP2 and VP3, of foot-and-mouth disease virus (FMDV) determine its antigenicity and the ability of the virus to interact with host-cell receptors. Hence, modification of these structural proteins may alter the properties of the virus. In the present study we...... B64 virus and the two chimeric viruses are identical to each other except for the capsid coding region. Animals exposed to O1K B64 did not exhibit signs of disease, while pigs exposed to each of the other viruses showed typical clinical signs of foot-and-mouth disease (FMD). All pigs infected...

  8. Cooperative effect of the VP1 amino acids 98E, 145A and 169F in the productive infection of mouse cell lines by enterovirus 71 (BS strain)

    Science.gov (United States)

    Victorio, Carla Bianca Luena; Xu, Yishi; Ng, Qimei; Meng, Tao; Chow, Vincent TK; Chua, Kaw Bing

    2016-01-01

    Enterovirus 71 (EV71) is a neurotrophic virus that causes hand, foot and mouth disease (HFMD) and occasional neurological infection among children. It infects primate cells but not rodent cells, primarily due to the incompatibility between the virus and the expressed form of its receptor, scavenger receptor class B member 2 (SCARB2) protein, on rodent cells (mSCARB2). We previously generated adapted strains (EV71:TLLm and EV71:TLLmv) that were shown to productively infect primate and rodent cell lines and whose genomes exhibited a multitude of non-synonymous mutations compared with the EV71:BS parental virus. In this study, we aimed to identify mutations that are necessary for productive infection of murine cells by EV71:BS. Using reverse genetics and site-directed mutagenesis, we constructed EV71 infectious clones with specific mutations that generated amino acid substitutions in the capsid VP1 and VP2 proteins. We subsequently assessed the infection induced by clone-derived viruses (CDVs) in mouse embryonic fibroblast NIH/3T3 and murine neuroblastoma Neuro-2a cell lines. We found that the CDV:BS-VP1K98E,E145A,L169F with three substitutions in the VP1 protein—K98E, E145A and L169F—productively infected both mouse cell lines for at least three passages of the virus in murine cells. Moreover, the virus gained the ability to utilize the mSCARB2 protein to infect murine cell lines. These results demonstrate that the three VP1 residues cooperate to effectively interact with the mSCARB2 protein on murine cells and permit the virus to infect murine cells. Gain-of-function studies similar to the present work provide valuable insight into the mutational trajectory required for EV71 to infect new host cells previously non-susceptible to infection. PMID:27329847

  9. Quantum dot-induced viral capsid assembling in dissociation buffer

    Directory of Open Access Journals (Sweden)

    Gao D

    2013-06-01

    Full Text Available Ding Gao,1,2 Zhi-Ping Zhang,1 Feng Li,3 Dong Men,1 Jiao-Yu Deng,1 Hong-Ping Wei,1 Xian-En Zhang,1 Zong-Qiang Cui1 1State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 2Graduate University of Chinese Academy of Sciences, Beijing, 3Division of Nanobiomedicine and i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, People's Republic of China Abstract: Viruses encapsulating inorganic nanoparticles are a novel type of nanostructure with applications in biomedicine and biosensors. However, the encapsulation and assembly mechanisms of these hybridized virus-based nanoparticles (VNPs are still unknown. In this article, it was found that quantum dots (QDs can induce simian virus 40 (SV40 capsid assembly in dissociation buffer, where viral capsids should be disassembled. The analysis of the transmission electron microscope, dynamic light scattering, sucrose density gradient centrifugation, and cryo-electron microscopy single particle reconstruction experimental results showed that the SV40 major capsid protein 1 (VP1 can be assembled into ≈25 nm capsids in the dissociation buffer when QDs are present and that the QDs are encapsulated in the SV40 capsids. Moreover, it was determined that there is a strong affinity between QDs and the SV40 VP1 proteins (KD = 2.19E-10 M, which should play an important role in QD encapsulation in the SV40 viral capsids. This study provides a new understanding of the assembly mechanism of SV40 virus-based nanoparticles with QDs, which may help in the design and construction of other similar virus-based nanoparticles. Keywords: quantum dots, simian virus 40, self-assembly, encapsulation, virus-based nanoparticles

  10. Trichodysplasia spinulosa-Associated Polyomavirus Uses a Displaced Binding Site on VP1 to Engage Sialylated Glycolipids.

    Directory of Open Access Journals (Sweden)

    Luisa J Ströh

    2015-08-01

    Full Text Available Trichodysplasia spinulosa-associated Polyomavirus (TSPyV was isolated from a patient suffering from trichodysplasia spinulosa, a skin disease that can appear in severely immunocompromised patients. While TSPyV is one of the five members of the polyomavirus family that are directly linked to a human disease, details about molecular recognition events, the viral entry pathway, and intracellular trafficking events during TSPyV infection remain unknown. Here we have used a structure-function approach to shed light on the first steps of TSPyV infection. We established by cell binding and pseudovirus infection studies that TSPyV interacts with sialic acids during attachment and/or entry. Subsequently, we solved high-resolution X-ray structures of the major capsid protein VP1 of TSPyV in complex with three different glycans, the branched GM1 glycan, and the linear trisaccharides α2,3- and α2,6-sialyllactose. The terminal sialic acid of all three glycans is engaged in a unique binding site on TSPyV VP1, which is positioned about 18 Å from established sialic acid binding sites of other polyomaviruses. Structure-based mutagenesis of sialic acid-binding residues leads to reduction in cell attachment and pseudovirus infection, demonstrating the physiological relevance of the TSPyV VP1-glycan interaction. Furthermore, treatments of cells with inhibitors of N-, O-linked glycosylation, and glycosphingolipid synthesis suggest that glycolipids play an important role during TSPyV infection. Our findings elucidate the first molecular recognition events of cellular infection with TSPyV and demonstrate that receptor recognition by polyomaviruses is highly variable not only in interactions with sialic acid itself, but also in the location of the binding site.

  11. Porcine circovirus-2 capsid protein induces cell death in PK15 cells

    Energy Technology Data Exchange (ETDEWEB)

    Walia, Rupali; Dardari, Rkia, E-mail: rdardari@ucalgary.ca; Chaiyakul, Mark; Czub, Markus

    2014-11-15

    Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathways involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.

  12. Poliovirus-associated protein kinase: Destabilization of the virus capsid and stimulation of the phosphorylation reaction by Zn sup 2+

    Energy Technology Data Exchange (ETDEWEB)

    Ratka, M.; Lackmann, M.; Ueckermann, C.; Karlins, U.; Koch, G. (Univ. of Hamburg (West Germany))

    1989-09-01

    The previously described poliovirus-associated protein kinase activity phosphorylates viral proteins VP0 and VP2 as well as exogenous proteins in the presence of Mg{sup 2+}. In this paper, the effect of Zn{sup 2+} on the phosphorylation reaction and the stability of the poliovirus capsid has been studied in detail and compared to that of Mg{sup 2+}. In the presence of Zn{sup 2+}, phosphorylation of capsid proteins VP2 and VP4 is significantly higher while phosphorylation of VP0 and exogenous phosphate acceptor proteins is not detected. The results indicate the activation of more than one virus-associated protein kinase by Zn{sup 2+}. The ion-dependent behavior of the enzyme activities is observed independently of whether the virus was obtained from HeLa or green monkey kidney cells. The poliovirus capsid is destabilized by Zn{sup 2+}. This alteration of the poliovirus capsid structure is a prerequisite for effective phosphorylation of viral capsid proteins. The increased level of phosphorylation of viral capsid proteins results in further destabilization of the viral capsid. As a result of the conformational changes, poliovirus-associated protein kinase activities dissociate from the virus particle. The authors suggest that the destabilizing effect of phosphorylation on the viral capsid plays a role in uncoating of poliovirus.

  13. Chimeric VLPs with GII.3 P2 domain in a backbone of GII.4 VP1 confers novel HBGA binding ability.

    Science.gov (United States)

    Huo, Yuqi; Wang, Wenhui; Ling, Tong; Wan, Xin; Ding, Li; Shen, Shuo; Huo, Jinling; Zhang, Shanfeng; Wang, Mingchen; Wang, Yumei; Liu, Yubing

    2016-09-15

    Noroviruses (NoVs) are a leading cause of non-bacterial acute gastroenteritis worldwide. The prevalence of Genogroup II, genotype 3 (GII.3) NoVs is secondary to the epidemic GII.4 strains which show broad spectrum binding activities against multiple types of histo-blood group antigens (HBGAs). In our previous study it was found that GII.3 NoV VLPs exhibited no binding activity to all synthetic and salivary HBGAs tested. To determine the compatibility of P2 domains between different genotypes and its effect over the binding specificity to HBGAs, we swapped the P2 domain of a GII.4 strain (Sydney 2012-like variant) with that of a GII.3 strain (GII.4-VP1/GII.3-P2). In vitro VLP-HBGA binding and binding blockade assays were used to characterize the binding patterns of GII.4-VP1/GII.3-P2 chimeric capsid protein. Expression of GII.4-VP1/GII.3-P2 chimeric capsid protein using recombinant bacuolovirus expression system led to assembly of virus like particles (VLPs). In vitro VLP-HBGA binding assay using synthetic and salivary HBGAs indicated binding activities to blood type A (trimer), Le(x) and blood type A, B and O salivary HBGAs. In vitro VLP-HBGA binding blockade assay indicated that the binding could be blocked by rabbit hyperimmune serum against GII.3 VLPs, but not hyperimmune sera against GI.2 and GII.4 VLPs. These results indicate that the observed binding activities may be caused by conformational changes of inserted P2 domain and possibly reflect the actual binding profile of GII.3 VLPs. The currently observed absence of binding of GII.3 NoV VLPs to salivary or synthetic HBGAs might be due to absence of other unknown factors.

  14. Solid-State NMR Studies of HIV-1 Capsid Protein Assemblies

    OpenAIRE

    HAN, YUN; Ahn, Jinwoo; Concel, Jason; Byeon, In-Ja L.; Gronenborn, Angela M.; YANG, Jun; Polenova, Tatyana

    2010-01-01

    In mature HIV-1 virions, a 26.6 kDa CA protein is assembled into a characteristic cone shaped core (capsid) that encloses the RNA viral genome. The assembled capsid structure is best described by a fullerene cone model that is made up from a hexameric lattice containing a variable number of CA pentamers, thus allowing for closure of tubular or conical structures. In this report, we present a solid-state NMR analysis of the wild type HIV-1 CA protein, prepared as conical and spherical assembli...

  15. Topography of the Human Papillomavirus Minor Capsid Protein L2 during Vesicular Trafficking of Infectious Entry

    Science.gov (United States)

    DiGiuseppe, Stephen; Keiffer, Timothy R.; Bienkowska-Haba, Malgorzata; Luszczek, Wioleta; Guion, Lucile G. M.; Müller, Martin

    2015-01-01

    ABSTRACT The human papillomavirus (HPV) capsid is composed of the major capsid protein L1 and the minor capsid protein L2. During entry, the HPV capsid undergoes numerous conformational changes that result in endosomal uptake and subsequent trafficking of the L2 protein in complex with the viral DNA to the trans-Golgi network. To facilitate this transport, the L2 protein harbors a number of putative motifs that, if capable of direct interaction, would interact with cytosolic host cell factors. These data imply that a portion of L2 becomes cytosolic during infection. Using a low concentration of digitonin to selectively permeabilize the plasma membrane of infected cells, we mapped the topography of the L2 protein during infection. We observed that epitopes within amino acid residues 64 to 81 and 163 to 170 and a C-terminal tag of HPV16 L2 are exposed on the cytosolic side of intracellular membranes, whereas an epitope within residues 20 to 38, which are upstream of a putative transmembrane region, is luminal. Corroborating these findings, we also found that L2 protein is sensitive to trypsin digestion during infection. These data demonstrate that the majority of the L2 protein becomes accessible on the cytosolic side of intracellular membranes in order to interact with cytosolic factors to facilitate vesicular trafficking. IMPORTANCE In order to complete infectious entry, nonenveloped viruses have to pass cellular membranes. This is often achieved through the viral capsid protein associating with or integrating into intracellular membrane. Here, we determine the topography of HPV L2 protein in the endocytic vesicular compartment, suggesting that L2 becomes a transmembrane protein with a short luminal portion and with the majority facing the cytosolic side for interaction with host cell transport factors. PMID:26246568

  16. Internal Proteins of the Procapsid and Mature Capsids of Herpes Simplex Virus 1 Mapped by Bubblegram Imaging

    Science.gov (United States)

    Wu, Weimin; Newcomb, William W.; Cheng, Naiqian; Aksyuk, Anastasia; Winkler, Dennis C.

    2016-01-01

    ABSTRACT The herpes simplex virus 1 (HSV-1) capsid is a huge assembly, ∼1,250 Å in diameter, and is composed of thousands of protein subunits with a combined mass of ∼200 MDa, housing a 100-MDa genome. First, a procapsid is formed through coassembly of the surface shell with an inner scaffolding shell; then the procapsid matures via a major structural transformation, triggered by limited proteolysis of the scaffolding proteins. Three mature capsids are found in the nuclei of infected cells. A capsids are empty, B capsids retain a shrunken scaffolding shell, and C capsids—which develop into infectious virions—are filled with DNA and ostensibly have expelled the scaffolding shell. The possible presence of other internal proteins in C capsids has been moot as, in cryo-electron microscopy (cryo-EM), they would be camouflaged by the surrounding DNA. We have used bubblegram imaging to map internal proteins in all four capsids, aided by the discovery that the scaffolding protein is exceptionally prone to radiation-induced bubbling. We confirmed that this protein forms thick-walled inner shells in the procapsid and the B capsid. C capsids generate two classes of bubbles: one occupies positions beneath the vertices of the icosahedral surface shell, and the other is distributed throughout its interior. A likely candidate is the viral protease. A subpopulation of C capsids bubbles particularly profusely and may represent particles in which expulsion of scaffold and DNA packaging are incomplete. Based on the procapsid structure, we propose that the axial channels of hexameric capsomers afford the pathway via which the scaffolding protein is expelled. IMPORTANCE In addition to DNA, capsids of tailed bacteriophages and their distant relatives, herpesviruses, contain internal proteins. These proteins are often essential for infectivity but are difficult to locate within the virion. A novel adaptation of cryo-EM based on detecting gas bubbles generated by radiation

  17. Residues of the UL25 protein of herpes simplex virus that are required for its stable interaction with capsids.

    Science.gov (United States)

    Cockrell, Shelley K; Huffman, Jamie B; Toropova, Katerina; Conway, James F; Homa, Fred L

    2011-05-01

    The herpes simplex virus 1 (HSV-1) UL25 gene product is a minor capsid component that is required for encapsidation, but not cleavage, of replicated viral DNA. UL25 is located on the capsid surface in a proposed heterodimer with UL17, where five copies of the heterodimer are found at each of the capsid vertices. Previously, we demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids. To further define the UL25 capsid binding domain, we generated recombinant viruses with either small truncations or amino acid substitutions in the UL25 N terminus. Studies of these mutants demonstrated that there are two important regions within the capsid binding domain. The first 27 amino acids are essential for capsid binding of UL25, while residues 26 to 39, which are highly conserved in the UL25 homologues of other alphaherpesviruses, were found to be critical for stable capsid binding. Cryo-electron microscopy reconstructions of capsids containing either a small tag on the N terminus of UL25 or the green fluorescent protein (GFP) fused between amino acids 50 and 51 of UL25 demonstrate that residues 1 to 27 of UL25 contact the hexon adjacent to the penton. A second region, most likely centered on amino acids 26 to 39, contacts the triplex that is one removed from the penton. Importantly, both of these UL25 capsid binding regions are essential for the stable packaging of full-length viral genomes.

  18. Liposome entrapment and immunogenic studies of a synthetic lipophilic multiple antigenic peptide bearing VP1 and VP3 domains of the hepatitis A virus: a robust method for vaccine design.

    Science.gov (United States)

    Haro, Isabel; Pérez, Silvia; García, Mónica; Chan, Weng C; Ercilla, Guadalupe

    2003-04-10

    Multiple antigen peptides (MAP) have been demonstrated to be efficient immunological reagents for the induction of immune responses to a variety of infectious agents. Several peptide domains of the hepatitis A virus (HAV) capsid proteins, mainly VP1 and VP3, are the immunodominant targets for a protective antibody response. In the present study we analyse the immunogenic properties of a tetrameric heterogeneous palmitoyl-derivatised MAP containing two defined HAV peptide sequences, VP1(11-25) and VP3(102-121), in rabbits immunised with either Freund's adjuvant or multilamellar liposomes. The immune response was evaluated with a specific enzyme immunoassay using MAP[VP1+VP3], VP1 and VP3 as targets. The avidity of the immune response was measured by a non-competitive enzyme-linked immunosorbent assay and by the surface plasmon resonance technology. Antisera raised against the lipo-MAP peptide entrapped in liposomes demonstrated high avidity of binding with affinity rate constants approximately one order of magnitude greater than those obtained with the Freund's protocol.

  19. Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein.

    Science.gov (United States)

    Wangman, Pradit; Senapin, Saengchan; Chaivisuthangkura, Parin; Longyant, Siwaporn; Rukpratanporn, Sombat; Sithigorngul, Paisarn

    2012-03-20

    The gene encoding the capsid protein of Macrobrachium rosenbergii nodavirus (MrNV) was cloned into pGEX-6P-1 expression vector and then transformed into the Escherichia coli strain BL21. After induction, capsid protein-glutathione-S-transferase (GST-MrNV; 64 kDa) was produced. The recombinant protein was separated using SDS-PAGE, excised from the gel, electro-eluted and then used for immunization for monoclonal antibody (MAb) production. Four MAbs specific to the capsid protein were selected and could be used to detect natural MrNV infections in M. rosenbergii by dot blotting, Western blotting and immunohistochemistry without cross-reaction with uninfected shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was 10 fmol µl-1 of the GST-MrNV, as determined using dot blotting. However, the sensitivity of the MAb on dot blotting with homogenate from naturally infected M. rosenbergii was approximately 200-fold lower than that of 1-step RT-PCR. Immunohistochemical analysis using these MAbs with infected shrimp tissues demonstrated staining in the muscles, nerve cord, gill, heart, loose connective tissue and inter-tubular tissue of the hepatopancreas. Although the positive reactions occurred in small focal areas, the immunoreactivity was clearly demonstrated. The MAbs targeted different epitopes of the capsid protein and will be used to develop a simple immunoassay strip test for rapid detection of MrNV.

  20. Mutation of the N-Terminal Region of Chikungunya Virus Capsid Protein: Implications for Vaccine Design

    Science.gov (United States)

    Liu, Xiang; Zaid, Ali; Goh, Lucas Y. H.; Hobson-Peters, Jody; Hall, Roy A.; Merits, Andres

    2017-01-01

    ABSTRACT Mosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of the Togaviridae family responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP)-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS) in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS) attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT)-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infection in vitro. Additionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design. PMID:28223458

  1. Mutation of the N-Terminal Region of Chikungunya Virus Capsid Protein: Implications for Vaccine Design

    Directory of Open Access Journals (Sweden)

    Adam Taylor

    2017-02-01

    Full Text Available Mosquito-transmitted chikungunya virus (CHIKV is an arthritogenic alphavirus of the Togaviridae family responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infection in vitro. Additionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design.

  2. Electrostatic potential of human immunodeficiency virus type 2 and rhesus macaque simian immunodeficiency virus capsid proteins

    Directory of Open Access Journals (Sweden)

    Katarzyna eBozek

    2012-06-01

    Full Text Available Human immunodeficiency virus type 2 (HIV-2 and simian immunodeficiency virus isolated from a macaque monkey (SIVmac are assumed to have originated from simian immunodeficiency virus isolated from sooty mangabey (SIVsm. Despite their close similarity in genome structure, HIV-2 and SIVmac show different sensitivities to TRIM5α, a host restriction factor against retroviruses. The replication of HIV-2 strains is potently restricted by rhesus (Rh monkey TRIM5α, while that of SIVmac strain 239 (SIVmac239 is not. Viral capsid protein is the determinant of this differential sensitivity to TRIM5α, as the HIV-2 mutant carrying SIVmac239 capsid protein evaded Rh TRIM5α-mediated restriction. However, the molecular determinants of this restriction mechanism are unknown. Electrostatic potential on the protein-binding site is one of the properties regulating protein-protein interactions. In this study, we investigated the electrostatic potential on the interaction surface of capsid protein of HIV-2 strain GH123 and SIVmac239. Although HIV-2 GH123 and SIVmac239 capsid proteins share more than 87% amino acid identity, we observed a large difference between the two molecules with the HIV-2 GH123 molecule having predominantly positive and SIVmac239 predominantly negative electrostatic potential on the surface of the loop between α-helices 4 and 5 (L4/5. As L4/5 is one of the major determinants of Rh TRIM5α sensitivity of these viruses, the present results suggest that the binding site of the Rh TRIM5α may show complementarity to the HIV-2 GH123 capsid surface charge distribution.

  3. Essential C-Terminal region of the baculovirus minor capsid protein VP80 binds DNA

    NARCIS (Netherlands)

    Marek, M.; Merten, O.W.; Francis-Devaraj, F.; Oers, van M.M.

    2012-01-01

    The essential Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) minor capsid protein VP80 has been recently shown to interact with the virus-triggered, nuclear F-actin cytoskeleton. A role for VP80 in virus morphogenesis has been proposed in the maturation of progeny nucleocapsids and

  4. Cloning and Sequence Analysis of Capsid Protein Gene of Iridovirus Indonesian Isolates

    Directory of Open Access Journals (Sweden)

    Murwantoko .

    2015-11-01

    Full Text Available generated by an Adobe application 11.5606 Iridovirus was known as agents that caused serious systemic disease in freshwater and marine fishes. The mortality up to 100% of orange-spotted grouper (Epinephelus coioides due to iridovirus infection has been reported in Indonesia. The gene encoding capsid protein of iridovirus is supposed to be conserved and has the potency for the development of control methods. The objectives of this study are to clone the gene encoding capsid protein iridovirus and to analyze their sequences. The   spleen tissues of orange-spotted grouper were collected and extracted their DNA. The DNA fragment of capsid protein of iridovirus genes were amplified by PCR using designed primers with the extraction DNA as templates. The amplified DNA fragments were cloned in pBSKSII and sequenced.  The genes encoding capsid protein of iridovirus from Jepara and Bali were successfully amplified and cloned. The Jepara clone (IJP03 contained complete open reading frame (ORF of the gene composed by 1362 bp nucleotides which encoded 453 amino acids. Those Jepara and Bali (IGD01 clones shared 99.8% similarity in nucleotide level and 99.4% at amino acid level. Based on those sequences, Indonesian iridovirus was belonged to genus Megalocystivirus and shared 99,6-99,9% similarity on nucleotide level with DGIV, ISKNV, MCIV, and ALIV Normal 0 36 false false false

  5. Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain.

    Science.gov (United States)

    Selzer, Lisa; Kant, Ravi; Wang, Joseph C-Y; Bothner, Brian; Zlotnick, Adam

    2015-11-20

    Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle.

  6. Capsid structure and dynamics of a human rhinovirus probed by hydrogen exchange mass spectrometry.

    Science.gov (United States)

    Wang, Lintao; Smith, David L

    2005-06-01

    Viral capsids are dynamic protein assemblies surrounding viral genomes. Despite the high-resolution structures determined by X-ray crystallography and cryo-electron microscopy, their in-solution structure and dynamics can be probed by hydrogen exchange. We report here using hydrogen exchange combined with protein enzymatic fragmentation and mass spectrometry to determine the capsid structure and dynamics of a human rhinovirus, HRV14. Capsid proteins (VP1-4) were labeled with deuterium by incubating intact virus in D(2)O buffer at neutral pH. The labeled proteins were digested by immobilized pepsin to give peptides analyzed by capillary reverse-phase HPLC coupled with nano-electrospray mass spectrometry. Deuterium levels incorporated at amide linkages in peptic fragments were measured for different exchange times from 12 sec to 30 h to assess the amide hydrogen exchange rates along each of the four protein backbones. Exchange results generally agree with the crystal structure of VP1-4,with extended, flexible terminal and surface-loop regions in fast exchange and folded helical and sheet structures in slow exchange. In addition, three alpha-helices, one from each of VP1-3, exhibited very slow exchange, indicating high stability of the protomeric interface. The beta-strands at VP3 N terminus also had very slow exchange, suggesting stable pentamer contacts. It was noted, however, that the interface around the fivefold axis had fast and intermediate exchange, indicating relatively more flexibility. Even faster exchange rates were found in the N terminus of VP1 and most segments of VP4, suggesting high flexibilities, which may correspond to their potential roles in virus uncoating.

  7. DNA condensates organized by the capsid protein VP15 in White Spot Syndrome Virus.

    Science.gov (United States)

    Liu, Yingjie; Wu, Jinlu; Chen, Hu; Hew, Choy Leong; Yan, Jie

    2010-12-20

    The White Spot Syndrome Virus (WSSV) has a large circular double-stranded DNA genome of around 300kb and it replicates in the nucleus of the host cells. The machinery of how the viral DNA is packaged has been remained unclear. VP15, a highly basic protein, is one of the major capsid proteins found in the virus. Previously, it was shown to be a DNA binding protein and was hypothesized to participate in the viral DNA packaging process. Using Atomic Force Microscopy imaging, we show that the viral DNA is associated with a (or more) capsid proteins. The organized viral DNA qualitatively resembles the conformations of VP15 induced DNA condensates in vitro. Furthermore, single-DNA manipulation experiments revealed that VP15 is able to condense single DNA against forces of a few pico Newtons. Our results suggest that VP15 may aid in the viral DNA packaging process by directly condensing DNA.

  8. Facilitation of Rice Stripe Virus Accumulation in the Insect Vector by Himetobi P Virus VP1

    Directory of Open Access Journals (Sweden)

    Shuo Li

    2015-03-01

    Full Text Available The small brown planthopper (SBPH is the main vector for rice stripe virus (RSV, which causes serious rice stripe disease in East Asia. To characterize the virus-vector interactions, the SBPH cDNA library was screened with RSV ribonucleoprotein (RNP as bait using a GAL4-based yeast two-hybrid system. The interaction between RSV-RNP and the Himetobi P virus (HiPV, an insect picorna-like virus VP1 protein was identified. The relationships between HiPV and RSV in SBPH were further investigated, and the results showed that the titer of RSV was commonly higher in single insect that exhibited more VP1 expression. After the VP1 gene was repressed by RNA silencing, the accumulation of RSV decreased significantly in the insect, whereas the virus acquisition ability of SBPH was unaffected, which suggests that HiPV VP1 potentially facilitates the accumulation of RSV in SBPH.

  9. T lymphocyte activation by dendritic cells pulsed with VP1-VP4 fusion protein of foot-and-mouth disease virus%负载口蹄疫病毒VPl-VP4融合蛋白质的树突状细胞对T细胞的活化效应

    Institute of Scientific and Technical Information of China (English)

    李娜; 李丽敏; 安鹏丽; 高云欢; 董昌海; 王家鑫

    2012-01-01

    To study the activation of T lymphocytes by bone marrow derived dendritic cells (BMDC) pulsed with re- combinant VP1-VP4 fusion protein of foot-and-mouth disease virus (FMDV) in vitro. The prokaryotic expression vector of pET32a-VP1-VP4 was constructed and VP1-VP4 fusion protein was expressed and purified by common SDS-PAGE and electroelution approach. BMDC pulsed with FMDV VP1-VP4 fusion protein were co-cultured with lymph node T cells. Supernatants were harvested at indicated time points and the IFN-γ levels of supernatants were determined with ELISA. The data show that BMDC pulsed with FMDV VP1-VP4 fusion protein are able to degrade VP1-VP4 antigen in lysosome and present the resulting peptides by MHC-II molecule for activating the T cells effi- ciently,leading to Thl-like response characteristic of release of IFN-γ.%为研究负载口蹄疫病毒VPl-VP4融合蛋白质的树突状细胞对淋巴结T细胞的活化效应,通过构建pET32a-VPl-VP4原核表达系统制备VPl-VP4融合蛋白。将纯化VPl-VP4融合蛋白负载骨髓源树突状细胞(BMDC)后与淋巴结T细胞共培养,用ELISA检测不同时间点的共培养上清液中IFN-γ的含量。结果表明,负载FMDVVPl-VP4融合蛋白后的BMDC可通过溶酶体-MHC-Ⅱ类分子途径有效地激活淋巴结T细胞,从而启动Thl细胞免疫应答,分泌大量IFN-γ。

  10. Transient Bluetongue virus serotype 8 capsid protein expression in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Albertha R. van Zyl

    2016-03-01

    Full Text Available Bluetongue virus (BTV causes severe disease in domestic and wild ruminants, and has recently caused several outbreaks in Europe. Current vaccines include live-attenuated and inactivated viruses; while these are effective, there is risk of reversion to virulence by mutation or reassortment with wild type viruses. Subunit or virus-like particle (VLP vaccines are safer options: VLP vaccines produced in insect cells by expression of the four BTV capsid proteins are protective against challenge; however, this is a costly production method. We investigated production of BTV VLPs in plants via Agrobacterium-mediated transient expression, an inexpensive production system very well suited to developing country use. Leaves infiltrated with recombinant pEAQ-HT vectors separately encoding the four BTV-8 capsid proteins produced more proteins than recombinant pTRA vectors. Plant expression using the pEAQ-HT vector resulted in both BTV-8 core-like particles (CLPs and VLPs; differentially controlling the concentration of infiltrated bacteria significantly influenced yield of the VLPs. In situ localisation of assembled particles was investigated by using transmission electron microscopy (TEM and it was shown that a mixed population of core-like particles (CLPs, consisting of VP3 and VP7 and VLPs were present as paracrystalline arrays in the cytoplasm of plant cells co-expressing all four capsid proteins.

  11. Detection of major capsid protein of infectious myonecrosis virus in shrimps using monoclonal antibodies.

    Science.gov (United States)

    Seibert, Caroline H; Borsa, Mariana; Rosa, Rafael D; Cargnin-Ferreira, Eduardo; Pereira, Alitiene M L; Grisard, Edmundo C; Zanetti, Carlos R; Pinto, Aguinaldo R

    2010-10-01

    Infectious myonecrosis virus (IMNV) has been causing a progressive disease in farm-reared shrimps in Brazil and Indonesia. Immunodiagnostic methods for IMNV detection, although reliable, are not employed currently because monoclonal antibodies (MAbs) against this virus are not available. In this study, a fragment of the IMNV major capsid protein gene, comprising amino acids 300-527 (IMNV(300-527)), was cloned and expressed in Escherichia coli. The nucleotide sequence of the recombinant IMNV(300-527) fragment displayed a high degree of identity to the major capsid protein of IMNV isolates from Brazil (99%) and Indonesia (98%). Ten MAbs were generated against the expressed fragment, and eight of these, mostly IgG(2a) or IgG(2b), were able to bind to IMNV in tissue extracts from shrimps infected naturally in immunodot-blot assays. Six of these MAbs recognized a approximately 100 kDa protein in a Western-blot, which is the predicted mass of IMNV major capsid protein, and also bound to viral inclusions present in muscle fibroses and in coagulative myonecrosis, as demonstrated by immunohistochemistry. Among all those MAbs created, four did not cross-react with non-infected shrimp tissues; this observation supports their applicability as a sensitive and specific immunodiagnosis of IMNV infection in shrimps.

  12. Targeting of herpesvirus capsid transport in axons is coupled to association with specific sets of tegument proteins

    OpenAIRE

    Luxton, G.W. Gant; Haverlock, Sarah; Coller, Kelly Elizabeth; Antinone, Sarah Elizabeth; Pincetic, Andrew; Smith, Gregory Allan

    2005-01-01

    The capsids of neurotropic herpesviruses have the remarkable ability to move in specific directions within axons. By modulating bidirectional capsid transport to favor either retrograde (minus-end) or anterograde (plus-end) motion, these viruses travel to sensory ganglia or peripheral tissue at specific stages of infection. By using correlative motion analysis to simultaneously monitor the trafficking of distinct viral proteins in living neurons, we demonstrate that viral “tegument” proteins ...

  13. Foot-and-mouth disease virus capsid proteins; analysis of protein processing, assembly and utility as vaccines

    DEFF Research Database (Denmark)

    Belsham, Graham

    precursor enhances the yield of processed capsid proteins and their assembly into empty capsid particles within mammalian cells. Such particles can potentially form the basis of a vaccine but they may only have the same properties as the current inactivated vaccines. We have expressed the FMDV P1-2A alone...... or with FMDV 3Cpro using a “single cycle” alphavirus vector based on Semliki Forest virus (SFV). Cattle vaccinated with these rSFV-FMDV vectors alone, produced anti-FMDV antibodies but the immune response was insufficient to give protection against FMDV challenge. However, vaccination with these vectors primed...... a much stronger immune response against FMDV post-challenge. In subsequent experiments, cattle were sequentially vaccinated with a rSFV-FMDV followed by recombinant FMDV empty capsid particles, or vice versa, prior to challenge. Animals given a primary vaccination with the rSFV-FMDV vector...

  14. Bacteriophage P23-77 capsid protein structures reveal the archetype of an ancient branch from a major virus lineage.

    Science.gov (United States)

    Rissanen, Ilona; Grimes, Jonathan M; Pawlowski, Alice; Mäntynen, Sari; Harlos, Karl; Bamford, Jaana K H; Stuart, David I

    2013-05-07

    It has proved difficult to classify viruses unless they are closely related since their rapid evolution hinders detection of remote evolutionary relationships in their genetic sequences. However, structure varies more slowly than sequence, allowing deeper evolutionary relationships to be detected. Bacteriophage P23-77 is an example of a newly identified viral lineage, with members inhabiting extreme environments. We have solved multiple crystal structures of the major capsid proteins VP16 and VP17 of bacteriophage P23-77. They fit the 14 Å resolution cryo-electron microscopy reconstruction of the entire virus exquisitely well, allowing us to propose a model for both the capsid architecture and viral assembly, quite different from previously published models. The structures of the capsid proteins and their mode of association to form the viral capsid suggest that the P23-77-like and adeno-PRD1 lineages of viruses share an extremely ancient common ancestor.

  15. Specific interaction of capsid protein and importin-{alpha}/{beta} influences West Nile virus production

    Energy Technology Data Exchange (ETDEWEB)

    Bhuvanakantham, Raghavan; Chong, Mun-Keat [Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597 (Singapore); Ng, Mah-Lee, E-mail: micngml@nus.edu.sg [Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597 (Singapore)

    2009-11-06

    West Nile virus (WNV) capsid (C) protein has been shown to enter the nucleus of infected cells. However, the mechanism by which C protein enters the nucleus is unknown. In this study, we have unveiled for the first time that nuclear transport of WNV and Dengue virus C protein is mediated by their direct association with importin-{alpha}. This interplay is mediated by the consensus sequences of bipartite nuclear localization signal located between amino acid residues 85-101 together with amino acid residues 42 and 43 of C protein. Elucidation of biological significance of importin-{alpha}/C protein interaction demonstrated that the binding efficiency of this association influenced the nuclear entry of C protein and virus production. Collectively, this study illustrated the molecular mechanism by which the C protein of arthropod-borne flavivirus enters the nucleus and showed the importance of importin-{alpha}/C protein interaction in the context of flavivirus life-cycle.

  16. Genetic variation of VP1 region in poliovirus and circulation of vaccine-derived poliovirus%脊髓灰质炎病毒VP1区的基因变化及疫苗衍生脊髓灰质炎病毒的流行情况

    Institute of Scientific and Technical Information of China (English)

    陈志慧; 徐闻青

    2010-01-01

    脊髓灰质炎(脊灰)病毒有3个血清型.VP1是脊灰病毒衣壳蛋白之一,它有4个抗原决定簇,可以诱导中和抗体,具有血清型特异性.脊灰病毒由于其RNA依赖性RNA聚合酶缺乏严密的自我校正功能,在自身复制过程中精确性下降,因此,会产生很多点突变,出现一些VP1区核苷酸突变热点.口服脊灰减毒活疫苗(OPV)的应用,有效地控制了脊灰,但因OPV病毒在人体肠道内复制过程中发生碱基突变,导致其神经毒力回升,引起疫苗相关麻痹型脊灰和出现疫苗衍生脊灰病毒(VDPV),使全球多次发生循环VDPV事件,对消灭脊灰带来新的挑战.为此,有关学者建议停止使用OPV,改用脊灰灭活疫苗或两种疫苗相结合的序贯免疫程序.%Polioviruses are classified into three serotypes.VP1 is one of the capsid proteins and has four epitopes which can stimulate neutralizing antibody to polioviruses.This capsid protein also determines the specificity of serotypes.Because the viral RNA polymerases generally lack self-proofreading mechanisms,polioviruses become one of the most rapidly evolving viruses and have several rapid variation sites in VP1 gene.With the routine immunization with oral poliovirus vaccine(OPV), the incidence of poliomyelitis has decreased dramatically.However, genetic variation may occur when the OPV viruses replicate in host's intestinal mucosa,resulting in reversion to neurovirulence of the polioviruses and leading to vaccine-associated paralytic poliomyelitis and vaccine-derived poliovirus (VDPV).There have been several events of the circulating VDPV worldwide, which makes new challenge to polio eradication.Therefore, experts suggest an immunization programme of inactivated poliovirus vaccine (IPV) in stead of OPV, or adoption of sequential immunization with IPV and OPV.

  17. Location of the Bacteriophage P22 Coat Protein C-terminus Provides Opportunities for the Design of Capsid Based Materials

    OpenAIRE

    Servid, Amy; Jordan, Paul; O’Neil, Alison; Prevelige, Peter; Douglas, Trevor

    2013-01-01

    Rational design of modifications to the interior and exterior surfaces of virus-like particles (VLPs) for future therapeutic and materials applications is based on structural information about the capsid. Existing cryo-electron microscopy based models suggest that the C-terminus of the bacteriophage P22 coat protein (CP) extends towards the capsid exterior. Our biochemical analysis through genetic manipulations of the C-terminus supports the model where the CP C-terminus is exposed on the ext...

  18. Optimization of Substitution Matrix for Sequence Alignment of Major Capsid Proteins of Human Herpes Simplex Virus

    Directory of Open Access Journals (Sweden)

    Vipan Kumar Sohpal

    2011-12-01

    Full Text Available Protein sequence alignment has become an informative tool in modern molecular biology research. A number of substitution matrices have been readily available for sequence alignments, but it is challenging task to compute optimal matrices for alignment accuracy. Here, we used the parameter optimization procedure to select the optimal Q of substitution matrices for major viral capsid protein of human herpes simplex virus. Results predict that Blosum matrix is most accurate on alignment benchmarks, and Blosum 60 provides the optimal Q in all substitution matrices. PAM 200 matrices results slightly below than Blosum 60, while VTML matrices are intermediate of PAM and VT matrices under dynamic programming.

  19. The Herpes Simplex Virus 1 UL17 Protein Is the Second Constituent of the Capsid Vertex-Specific Component Required for DNA Packaging and Retention▿

    OpenAIRE

    Toropova, Katerina; Huffman, Jamie B.; Homa, Fred L.; James F Conway

    2011-01-01

    The herpes simplex virus (HSV) UL17 and UL25 minor capsid proteins are essential for DNA packaging. They are thought to comprise a molecule arrayed in five copies around each of the capsid vertices. This molecule was initially termed the “C-capsid-specific component” (CCSC) (B. L. Trus et al., Mol. Cell 26:479-489, 2007), but as we have subsequently observed this feature on reconstructions of A, B, and C capsids, we now refer to it more generally as the “capsid vertex-specific component” (CVS...

  20. Molecular comparison and evolutionary analyses of VP1 nucleotide sequences of new African human enterovirus 71 isolates reveal a wide genetic diversity.

    Directory of Open Access Journals (Sweden)

    Maël Bessaud

    Full Text Available Most circulating strains of Human enterovirus 71 (EV-A71 have been classified primarily into three genogroups (A to C on the basis of genetic divergence between the 1D gene, which encodes the VP1 capsid protein. The aim of the present study was to provide further insights into the diversity of the EV-A71 genogroups following the recent description of highly divergent isolates, in particular those from African countries, including Madagascar. We classified recent EV-A71 isolates by a large comparison of 3,346 VP1 nucleotidic sequences collected from GenBank. Analysis of genetic distances and phylogenetic investigations indicated that some recently-reported isolates did not fall into the genogroups A-C and clustered into three additional genogroups, including one Indian genogroup (genogroup D and 2 African ones (E and F. Our Bayesian phylogenetic analysis provided consistent data showing that the genogroup D isolates share a recent common ancestor with the members of genogroup E, while the isolates of genogroup F evolved from a recent common ancestor shared with the members of the genogroup B. Our results reveal the wide diversity that exists among EV-A71 isolates and suggest that the number of circulating genogroups is probably underestimated, particularly in developing countries where EV-A71 epidemiology has been poorly studied.

  1. Identification of two functional nuclear localization signals in the capsid protein of duck circovirus

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Sun, Ya-Ni [College of Veterinary Medicine, Northwest A and F University, Shanxi, Yangling 712100 (China); Gao, Ji-Ming; Xie, Zhi-Jing [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Wang, Yu [Department of Basic Medical Sciences, Taishan Medical College, Shandong, Taian 271000 (China); Zhu, Yan-Li [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Jiang, Shi-Jin, E-mail: sjjiang@sdau.edu.cn [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China)

    2013-02-05

    The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

  2. Construction of eukaryotic expression vector system for expression of VP1 gene of encephalomyocarditis virus%脑心肌炎病毒VP1基因真核表达载体的构建及表达

    Institute of Scientific and Technical Information of China (English)

    凡静静; 冯若飞; 杨妍梅; 张海霞; 李向茸; 王丹; 谢晶莹; 马忠仁

    2013-01-01

    The objective of this study was to construct a eukaryotic expression vector harboring VP1 gene of encephalomyocarditis virus(EMCV) ,and to express the VP1 gene in CHO cells. VP1 gene segment of EMCV was amplified by RT-PCR,and then cloned into the pEGFP-Cl vector to construct eukaryotic expression vector pEGFP-C1-VP1. The constructed plasmid was then transfected into the CHO cell via Lipo-fectamineTM 2000 Reagent. Transcription of VP1 gene in the CHO cell was tested via RT-PCR,and localization and reactinogenicity of VP1 protein in the CHO cell were detected by using immunohistochemistry and Western-blot,respectively. Enzyme digestion of PCR products and sequencing showed that the recombinant plasmid pEGFP-Cl-VPl was successfully constructed. RT-PCR result showed that the VP1 gene was tran-scripted in the CHO cell. Immunohistochemistry result showed that the expressed VP1 protein was mainly distributed in the membrane of CHO cells, and a little in cytoplasm. Western-blot showed that the expressed VP1 protein could bind to rabbit anti-EMCV antibody,indicating the immunoreactivity of recombinant VP1 protein.%为构建脑心肌炎病毒(EMCV) VP1基因的真核表达载体,并在CHO细胞中进行表达,通过RT-PCR扩增目的基因,酶切连接后将VP1基因克隆入真核表达载体pEGFP-C1中,通过脂质体法转染重组质粒pEGFP-C1-VP1,提取CHO细胞的总RNA,用RT-PCR检测目的基因的转录情况,并利用免疫组织化学和Western-blot分析检测目的基因表达情况、VP1蛋白在细胞中的定位及其抗原性.PCR、酶切验证及测序结果证实,重组质粒构建成功,绿色荧光蛋白正常表达.提取试验组细胞的总RNA,进行RT-PCR,在900 bp处扩增出了目的条带.免疫组织化学结果显示,VP1主要分布在CHO细胞的细胞膜中,少量存在于胞浆中.Western-blot分析结果证实,VP1能与兔抗EMCV抗体特异性结合.表明VP1蛋白在CHO细胞中表达良好,表达的蛋白具有反应原性.

  3. Sequence analysis and location of capsid proteins within RNA 2 of strawberry latent ringspot virus.

    Science.gov (United States)

    Kreiah, S; Strunk, G; Cooper, J I

    1994-09-01

    The nucleotide sequence of the RNA 2 of a strawberry isolate (H) of strawberry latent ringspot virus (SLRSV) comprised 3824 nucleotides and contained one long open reading frame with a theoretical coding capacity of 890 amino acids equivalent to a protein of 98.8K. The N-terminal amino acid sequences of virion-derived proteins were determined by Edman degradation allowing the capsid coding regions to be located and serine/glycine cleavage sites to be identified within the polyprotein. The amino acid sequence in the capsid coding region of an isolate of SLRSV from flowering cherry in New Zealand was 97% identical to that of SLRSV-H. Except in the 3' and 5' terminal non-coding sequences, computer-based alignment and comparison algorithms did not reveal any substantial homologies between RNA 2 of SLRSV-H and the equivalent genomic segments in the nepoviruses arabis mosaic, cherry leaf roll, grapevine fanleaf, raspberry ringspot, grapevine hungarian chrome mosaic, tomato blackring, tomato ringspot, tobacco ringspot, or in the comoviruses cowpea mosaic and red clover mottle. Despite the similarities in overall genome organization, data from RNA 2 remain insufficient for unambiguous positioning of SLRSV in relation to species/genera in the Comoviridae.

  4. RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins.

    Science.gov (United States)

    Yang, Zhu; Reeves, Michael; Ye, Jun; Trang, Phong; Zhu, Li; Sheng, Jingxue; Wang, Yu; Zen, Ke; Wu, Jianguo; Liu, Fenyong

    2015-06-24

    An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%-99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy.

  5. Analogs of LDL Receptor Ligand Motifs in Dengue Envelope and Capsid Proteins as Potential Codes for Cell Entry

    OpenAIRE

    Juan Guevara; Jaime Romo; Troy McWhorter; Natalia Valentinova Guevara

    2015-01-01

    It is established that cell entry of low density lipoprotein particles (LLPs) containing Apo B100 and Apo E is mediated by receptors and GAGs. Receptor ligand motifs, XBBBXXBX, XBBXBX, and ΨBΨXB, and mono- and bipartite NLS sequences are abundant in Apo E and Apo B100 as well as in envelope and capsid proteins of Dengue viruses 1–4 (DENV1–4). Synthetic, fluorescence-labeled peptides of sequences in DENV2 envelope protein, and DENV3 capsid that include these motifs were used to conduct a quali...

  6. Novel chimeric foot-and-mouth disease virus-like particles harboring serotype O VP1 protect guinea pigs against challenge.

    Science.gov (United States)

    Li, Haitao; Li, Zhiyong; Xie, Yinli; Qin, Xiaodong; Qi, Xingcai; Sun, Peng; Bai, Xingwen; Ma, Youji; Zhang, Zhidong

    2016-02-01

    Foot-and-mouth disease is a highly contagious, acute viral disease of cloven-hoofed animal species causing severe economic losses worldwide. Among the seven serotypes of foot-and-mouth disease virus (FMDV), serotype O is predominant, but its viral capsid is more acid sensitive than other serotypes, making it more difficult to produce empty serotype O VLPs in the low pH insect hemolymph. Therefore, a novel chimeric virus-like particle (VLP)-based candidate vaccine for serotype O FMDV was developed and characterized in the present study. The chimeric VLPs were composed of antigenic VP1 from serotype O and segments of viral capsid proteins from serotype Asia1. These VLPs elicited significantly higher FMDV-specific antibody levels in immunized mice than did the inactivated vaccine. Furthermore, the chimeric VLPs protected guinea pigs from FMDV challenge with an efficacy similar to that of the inactivated vaccine. These results suggest that chimeric VLPs have the potential for use in vaccines against serotype O FMDV infection.

  7. Interaction between Bluetongue virus outer capsid protein VP2 and vimentin is necessary for virus egress

    Directory of Open Access Journals (Sweden)

    Roy Polly

    2007-01-01

    Full Text Available Abstract Background The VP2 outer capsid protein Bluetongue Virus (BTV is responsible for receptor binding, haemagglutination and eliciting host-specific immunity. However, the assembly of this outer capsid protein on the transcriptionally active viral core would block transcription of the virus. Thus assembly of the outer capsid on the core particle must be a tightly controlled process during virus maturation. Earlier studies have detected mature virus particles associated with intermediate filaments in virus infected cells but the viral determinant for this association and the effect of disrupting intermediate filaments on virus assembly and release are unknown. Results In this study it is demonstrated that BTV VP2 associates with vimentin in both virus infected cells and in the absence of other viral proteins. Further, the determinants of vimentin localisation are mapped to the N-terminus of the protein and deletions of aminio acids between residues 65 and 114 are shown to disrupt VP2-vimentin association. Site directed mutation also reveals that amino acid residues Gly 70 and Val 72 are important in the VP2-vimentin association. Mutation of these amino acids resulted in a soluble VP2 capable of forming trimeric structures similar to unmodified protein that no longer associated with vimentin. Furthermore, pharmacological disruption of intermediate filaments, either directly or indirectly through the disruption of the microtubule network, inhibited virus release from BTV infected cells. Conclusion The principal findings of the research are that the association of mature BTV particles with intermediate filaments are driven by the interaction of VP2 with vimentin and that this interaction contributes to virus egress. Furthermore, i the N-terminal 118 amino acids of VP2 are sufficient to confer vimentin interaction. ii Deletion of amino acids 65–114 or mutation of amino acids 70–72 to DVD abrogates vimentin association. iii Finally

  8. Cross-serotype immunity induced by immunization with a conserved rhinovirus capsid protein.

    Directory of Open Access Journals (Sweden)

    Nicholas Glanville

    Full Text Available Human rhinovirus (RV infections are the principle cause of common colds and precipitate asthma and COPD exacerbations. There is currently no RV vaccine, largely due to the existence of ∼150 strains. We aimed to define highly conserved areas of the RV proteome and test their usefulness as candidate antigens for a broadly cross-reactive vaccine, using a mouse infection model. Regions of the VP0 (VP4+VP2 capsid protein were identified as having high homology across RVs. Immunization with a recombinant VP0 combined with a Th1 promoting adjuvant induced systemic, antigen specific, cross-serotype, cellular and humoral immune responses. Similar cross-reactive responses were observed in the lungs of immunized mice after infection with heterologous RV strains. Immunization enhanced the generation of heterosubtypic neutralizing antibodies and lung memory T cells, and caused more rapid virus clearance. Conserved domains of the RV capsid therefore induce cross-reactive immune responses and represent candidates for a subunit RV vaccine.

  9. Modeling of the human rhinovirus C capsid suggests possible causes for antiviral drug resistance.

    Science.gov (United States)

    Basta, Holly A; Ashraf, Shamaila; Sgro, Jean-Yves; Bochkov, Yury A; Gern, James E; Palmenberg, Ann C

    2014-01-05

    Human rhinoviruses of the RV-C species are recently discovered pathogens with greater clinical significance than isolates in the RV-A+B species. The RV-C cannot be propagated in typical culture systems; so much of the virology is necessarily derivative, relying on comparative genomics, relative to the better studied RV-A+B. We developed a bioinformatics-based structural model for a C15 isolate. The model showed the VP1-3 capsid proteins retain their fundamental cores relative to the RV-A+B, but conserved, internal RV-C residues affect the shape and charge of the VP1 hydrophobic pocket that confers antiviral drug susceptibility. When predictions of the model were tested in organ cultures or ALI systems with recombinant C15 virus, there was a resistance to capsid-binding drugs, including pleconaril, BTA-188, WIN56291, WIN52035 and WIN52084. Unique to all RV-C, the model predicts conserved amino acids within the pocket and capsid surface pore leading to the pocket may correlate with this activity.

  10. Nucleotide sequence of maize dwarf mosaic virus capsid protein gene and its expression in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    赛吉庆; 康良仪; 黄忠; 史春霖; 田波; 谢友菊

    1995-01-01

    The 3’-terminal 1 279 nucleotide sequence of maize dwarf mosaic virus (MDMV) genome has been determined. This sequence contains an open reading frame of 1023 nudeotides and a 3’ -non-coding region of 256 nucleotides. The open reading frame includes all of the coding regions for the viral capsid protein (CP) and part of the viral nuclear inclusion protein (Nib). The predicted viral CP consists of 313 amino acid residues with a calculated molecular weight of 35400. The amino acid sequence of the viral CP derived from MDMV cDNA shows about 47%-54% homology to that of 4 other potyviruses. The viral CP gene was constructed in frame with the lacZ gene in pUC19 plasmid and expressed in E. coli cells. The fusion polypeptide positively reacted in Western blot with an antiserum prepared against the native viral CP.

  11. Protection of chickens against avian hepatitis E virus (avian HEV) infection by immunization with recombinant avian HEV capsid protein.

    Science.gov (United States)

    Guo, H; Zhou, E M; Sun, Z F; Meng, X J

    2007-04-12

    Avian hepatitis E virus (avian HEV) is an emerging virus associated with hepatitis-splenomegaly syndrome in chickens in North America. Avian HEV is genetically and antigenically related to human HEV, the causative agent of hepatitis E in humans. In the lack of a practical animal model, avian HEV infection in chickens has been used as a model to study human HEV replication and pathogenesis. A 32 kDa recombinant ORF2 capsid protein of avian HEV expressed in Escherichia coli was found having similar antigenic structure as that of human HEV containing major neutralizing epitopes. To determine if the capsid protein of avian HEV can be used as a vaccine, 20 chickens were immunized with purified avian HEV recombinant protein with aluminum as adjuvant and another 20 chickens were mock immunized with KLH precipitated in aluminum as controls. Both groups of chickens were subsequently challenged with avian HEV. All the tested mock-immunized control chickens developed typical avian HEV infection characterized by viremia, fecal virus shedding and seroconversion to avian HEV antibodies. Gross hepatic lesions were also found in portion of these chickens. In contrast, none of the tested chickens immunized with avian HEV capsid protein had detectable viremia, fecal virus shedding or observable gross hepatitis lesions. The results from this study suggested that immunization of chickens with avian HEV recombinant ORF2 capsid protein with aluminum as adjuvant can induce protective immunity against avian HEV infection. Chickens are a useful small animal model to study anti-HEV immunity and pathogenesis.

  12. Structural Basis for the Development of Avian Virus Capsids That Display Influenza Virus Proteins and Induce Protective Immunity

    OpenAIRE

    Pascual, Elena; Mata, Carlos P.; Gómez-Blanco, Josué; Moreno, Noelia; Bárcena, Juan; Blanco, Esther; Rodríguez-Frandsen, Ariel; Nieto, Amelia; Carrascosa, José L.; Castón, José R.

    2014-01-01

    Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ∼70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an am...

  13. Production and Application of Polyclonal Antibodies Against Recombinant Capsid Protein of Extra Small Virus of Macrobrachium rosenbergii.

    Science.gov (United States)

    Neethi, V; Sivakumar, N; Kumar, Kundan; Rajendran, K V; Makesh, M

    2012-12-01

    Macrobrachium rosenbergii nodavirus along with a satellite virus, extra small virus (XSV) causes white tail disease (WTD) in the giant freshwater prawn M. rosenbergii. Infected M. rosenbergii postlarvae were collected from a hatchery in Kakinada, Andhra Pradesh. The gene coding the capsid protein of XSV was cloned in a bacterial expression vector pRSET A and the recombinant protein was expressed in Escherichia coli BL21(DE3)pLysS cells. The recombinant protein was purified by Nickel affinity chromatography. Polyclonal antibodies were produced in mice against the recombinant protein and the antibodies reacted specifically with the recombinant protein and XSV in WTD-infected tissues. This is the first report of detection of XSV using antibodies against recombinant capsid protein.

  14. Genetic Variation of the VP1 Gene of the Virulent Duck Hepatitis A Virus Type 1 (DHAV-1) Isolates in Shandong Province of China

    Institute of Scientific and Technical Information of China (English)

    Jiming Gao; Junhao Chen; Xingkui Si; Zhijing Xie; Yanli Zhu; Xingxiao Zhang; Shujing Wang; Shijin Jiang

    2012-01-01

    To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1(DHAV-1) isolates,the virulence,cross neutralization assays and the complete sequence of the virion protein 1(VP1) gene of nine virulent DHAV-1 strains,which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008,were tested.The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses(ELD50s) and the median lethal doses(LD50s),respectively.The results showed that the ELD5s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 106/mL to 1.44 × 107/mL,while the LD50s were 2.39 × 105/mL to 6.15 × 106/mL.Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates,respectively.Compared with other virulent,moderate virulent,attenuated vaccine and mild strains,the VP1 genes of the 9 strains shared 89.8%-99.7% similarity at the nucleotide level and 92.4%-99.6% at amino acid level with other DHAV-1 strains.There were three hypervariable regions at the C-terminus(as 158-160,180-193 and 205-219) and other variable points in VPI protein,but which didn't cause virulence of DHAV-1 change.

  15. Mechanism for Coordinated RNA Packaging and Genome Replication by Rotavirus Polymerase VP1

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Xiaohui; McDonald, Sarah M.; Tortorici, M. Alejandra; Tao, Yizhi Jane; Vasquez-Del Carpio, Rodrigo; Nibert, Max L.; Patton, John T.; Harrison, Stephen C. (Harvard-Med); (NIH); (CH-Boston)

    2009-04-08

    Rotavirus RNA-dependent RNA polymerase VP1 catalyzes RNA synthesis within a subviral particle. This activity depends on core shell protein VP2. A conserved sequence at the 3' end of plus-strand RNA templates is important for polymerase association and genome replication. We have determined the structure of VP1 at 2.9 {angstrom} resolution, as apoenzyme and in complex with RNA. The cage-like enzyme is similar to reovirus {lambda}3, with four tunnels leading to or from a central, catalytic cavity. A distinguishing characteristic of VP1 is specific recognition, by conserved features of the template-entry channel, of four bases, UGUG, in the conserved 3' sequence. Well-defined interactions with these bases position the RNA so that its 3' end overshoots the initiating register, producing a stable but catalytically inactive complex. We propose that specific 3' end recognition selects rotavirus RNA for packaging and that VP2 activates the autoinhibited VP1/RNA complex to coordinate packaging and genome replication.

  16. Multiple functions of capsid proteins in (+) stranded RNA viruses during plant-virus interactions.

    Science.gov (United States)

    Weber, Philipp H; Bujarski, Jozef J

    2015-01-22

    In addition to providing a protective shell for genomic RNA(s), the coat (capsid) proteins (CPs) of plus-stranded RNA viruses play a variety of other functions that condition the plant-virus relationship. In this review we outline the extensive research progress that has been made within the last decade on those CP characteristics that relate to virus infectivity, pathogenicity, symptom expression, interactions with host factors, virus movement, vector transmission, host range, as well as those used to study virus evolution. By discussing the examples among a variety of plant RNA viruses we show that in addition to general features and pathways, the involvement of CPs may assume very distinct tasks that depend on the particular virus life style. Research perspectives and potential applications are discussed at the end.

  17. Analysis of epitopes in the capsid protein of avian hepatitis E virus by using monoclonal antibodies.

    Science.gov (United States)

    Dong, Shiwei; Zhao, Qin; Lu, Mingzhe; Sun, Peiming; Qiu, Hongkai; Zhang, Lu; Lv, Junhua; Zhou, En-Min

    2011-02-01

    Avian hepatitis E virus (HEV) is related genetically and antigenically to human and swine HEVs and capsid protein of avian HEV shares approximately 48-49% amino acid sequence identities with those of human and swine HEVs. Six monoclonal antibodies (MAbs) were produced and used to locate different epitopes in the ORF2 region of aa 339-570 of avian HEV Chinese isolate. The results showed that five epitopes were located in the aa 339-414 region and one in the aa 510-515 region. Two epitopes located in aa 339-355 and aa 384-414 regions are the immunodominant epitopes on the surface of the avian HEV particles as demonstrated by immune capture of viral particles and immunohistochemical detection of the ORF2 antigens with two MAbs.

  18. Genetic diversity of hepatitis A virus in China: VP3-VP1-2A genes and evidence of quasispecies distribution in the isolates.

    Science.gov (United States)

    Wang, Hao; Zheng, Huihui; Cao, Jingyuan; Zhou, Wenting; Yi, Yao; Jia, Zhiyuan; Bi, Shengli

    2013-01-01

    Hepatitis A virus (HAV) is the most common cause of infectious hepatitis throughout the world, spread largely by the fecal-oral route. To characterize the genetic diversity of the virus circulating in China where HAV in endemic, we selected the outbreak cases with identical sequences in VP1-2A junction region and compiled a panel of 42 isolates. The VP3-VP1-2A regions of the HAV capsid-coding genes were further sequenced and analyzed. The quasispecies distribution was evaluated by cloning the VP3 and VP1-2A genes in three clinical samples. Phylogenetic analysis demonstrated that the same genotyping results could be obtained whether using the complete VP3, VP1, or partial VP1-2A genes for analysis in this study, although some differences did exist. Most isolates clustered in sub-genotype IA, and fewer in sub-genotype IB. No amino acid mutations were found at the published neutralizing epitope sites, however, several unique amino acid substitutions in the VP3 or VP1 region were identified, with two amino acid variants closely located to the immunodominant site. Quasispecies analysis showed the mutation frequencies were in the range of 7.22 x 10(-4) -2.33 x 10(-3) substitutions per nucleotide for VP3, VP1, or VP1-2A. When compared with the consensus sequences, mutated nucleotide sites represented the minority of all the analyzed sequences sites. HAV replicated as a complex distribution of closely genetically related variants referred to as quasispecies, and were under negative selection. The results indicate that diverse HAV strains and quasispecies inside the viral populations are presented in China, with unique amino acid substitutions detected close to the immunodominant site, and that the possibility of antigenic escaping mutants cannot be ruled out and needs to be further analyzed.

  19. Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Zi-Zheng; Sun, Yuan-Yuan; Zhao, Min; Huang, Hui [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhang, Jun; Xia, Ning-Shao [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China); Miao, Ji, E-mail: jmiao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhao, Qinjian, E-mail: qinjian_zhao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China)

    2013-01-18

    Highlights: ► The RNA-binding hnRNP H regulates late viral gene expression. ► hnRNP H activity was inhibited by a late viral protein. ► Specific interaction between HPV L1 and hnRNP H was demonstrated. ► Co-localization of HPV L1 and hnRNP H inside cells was observed. ► Viral capsid protein production, enabling rapid capsid assembly, was implicated. -- Abstract: Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.

  20. The Astrovirus Capsid: A Review

    Science.gov (United States)

    Arias, Carlos F.; DuBois, Rebecca M.

    2017-01-01

    Astroviruses are enterically transmitted viruses that cause infections in mammalian and avian species. Astroviruses are nonenveloped, icosahedral viruses comprised of a capsid protein shell and a positive-sense, single-stranded RNA genome. The capsid protein undergoes dramatic proteolytic processing both inside and outside of the host cell, resulting in a coordinated maturation process that affects cellular localization, virus structure, and infectivity. After maturation, the capsid protein controls the initial phases of virus infection, including virus attachment, endocytosis, and genome release into the host cell. The astrovirus capsid is the target of host antibodies including virus-neutralizing antibodies. The capsid protein also mediates the binding of host complement proteins and inhibits complement activation. Here, we will review our knowledge on the astrovirus capsid protein (CP), with particular attention to the recent structural, biochemical, and virological studies that have advanced our understanding of the astrovirus life cycle. PMID:28106836

  1. P22 coat protein structures reveal a novel mechanism for capsid maturation: stability without auxiliary proteins or chemical crosslinks.

    Science.gov (United States)

    Parent, Kristin N; Khayat, Reza; Tu, Long H; Suhanovsky, Margaret M; Cortines, Juliana R; Teschke, Carolyn M; Johnson, John E; Baker, Timothy S

    2010-03-10

    Viral capsid assembly and stability in tailed, dsDNA phage and Herpesviridae are achieved by various means including chemical crosslinks (unique to HK97), or auxiliary proteins (lambda, T4, phi29, and herpesviruses). All these viruses have coat proteins (CP) with a conserved, HK97-like core structure. We used a combination of trypsin digestion, gold labeling, cryo-electron microscopy, 3D image reconstruction, and comparative modeling to derive two independent, pseudoatomic models of bacteriophage P22 CP: before and after maturation. P22 capsid stabilization results from intersubunit interactions among N-terminal helices and an extensive "P loop," which obviate the need for crosslinks or auxiliary proteins. P22 CP also has a telokin-like Ig domain that likely stabilizes the monomer fold so that assembly may proceed via individual subunit addition rather than via preformed capsomers as occurs in HK97. Hence, the P22 CP structure may be a paradigm for understanding how monomers assemble in viruses like phi29 and HSV-1.

  2. Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis (Florida)

    2012-09-17

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  3. Structural studies of adeno-associated virus serotype 8 capsid transitions associated with endosomal trafficking.

    Science.gov (United States)

    Nam, Hyun-Joo; Gurda, Brittney L; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis

    2011-11-01

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  4. Multiple capsid-stabilizing interactions revealed in a high-resolution structure of an emerging picornavirus causing neonatal sepsis

    Science.gov (United States)

    Shakeel, Shabih; Westerhuis, Brenda M.; Domanska, Ausra; Koning, Roman I.; Matadeen, Rishi; Koster, Abraham J.; Bakker, Arjen Q.; Beaumont, Tim; Wolthers, Katja C.; Butcher, Sarah J.

    2016-07-01

    The poorly studied picornavirus, human parechovirus 3 (HPeV3) causes neonatal sepsis with no therapies available. Our 4.3-Å resolution structure of HPeV3 on its own and at 15 Å resolution in complex with human monoclonal antibody Fabs demonstrates the expected picornavirus capsid structure with three distinct features. First, 25% of the HPeV3 RNA genome in 60 sites is highly ordered as confirmed by asymmetric reconstruction, and interacts with conserved regions of the capsid proteins VP1 and VP3. Second, the VP0 N terminus stabilizes the capsid inner surface, in contrast to other picornaviruses where on expulsion as VP4, it forms an RNA translocation channel. Last, VP1's hydrophobic pocket, the binding site for the antipicornaviral drug, pleconaril, is blocked and thus inappropriate for antiviral development. Together, these results suggest a direction for development of neutralizing antibodies, antiviral drugs based on targeting the RNA-protein interactions and dissection of virus assembly on the basis of RNA nucleation.

  5. Radioimmunoassay for detection of VP1 specific neutralizing antibodies of foot and mouse disease virus

    Energy Technology Data Exchange (ETDEWEB)

    Patzer, E.J.; Jackson, M.L. (Genentech, Inc., South San Francisco CA (USA)); Moore, D.M. (U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, NY (USA))

    1985-01-01

    A solid-phase radioimmunoassay was developed for the detection of antibodies against a specific region of the VP1 protein of the A24 and O1 serotypes of foot and mouth disease virus. The antibody titers from the radioimmunoassay showed a positive correlation with neutralizing antibody titers determined by a mouse protection assay. The specificity of the assay resides in the peptide used as antigen. The assay is rapid, reproducible and does not require the use of whole virions.

  6. Molecular variability analyses of Apple chlorotic leaf spot virus capsid protein

    Indian Academy of Sciences (India)

    T Rana; V Chandel; Y Kumar; R Ram; V Hallan; A A Zaidi

    2010-12-01

    The complete sequences of the coat protein (CP) gene of 26 isolates of Apple chlorotic leaf spot virus (ACLSV) from India were determined. The isolates were obtained from various pome (apple, pear and quince) and stone (plum, peach, apricot, almond and wild Himalayan cherry) fruit trees. Other previously characterized ACLSV isolates and Trichoviruses were used for comparative analysis. Indian ACLSV isolates among themselves and with isolates from elsewhere in the world shared 91–100% and 70–98% sequence identities at the amino acid and nucleotide levels, respectively. The highest degree of variability was observed in the middle portion with 9 amino acid substitutions in contrast to the N-terminal and C-terminal ends, which were maximally conserved with only 4 amino acid substitutions. In phylogenetic analysis no reasonable correlation between host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other Trichoviruses revealed the TaTao ACLSV peach isolate to be phylogenetically closest to Peach mosaic virus, Apricot pseudo chlorotic leaf spot virus and Cherry mottle leaf virus. Recombination analysis (RDP3 ver.2.6) done for all the available ACLSV complete CP sequences of the world and Indian isolates indicate no significant evidence of recombination. However, one recombination event among Indian ACLSV-CP isolates was detected. To the best of our knowledge, this is the first report of complete CP sequence variability study from India and also the first evidence of homologous recombination in ACLSV.

  7. Maize rayado fino virus capsid proteins assemble into virus-like particles in Escherichia coli.

    Science.gov (United States)

    Hammond, Rosemarie W; Hammond, John

    2010-02-01

    Maize rayado fino virus (MRFV; genus Marafivirus; family Tymoviridae) is an isometric plant virus of 30 nm containing two components: empty shells and complete virus particles (encapsidating the 6.3 kb genomic RNA). Both particles are composed of two serologically related, carboxy co-terminal, coat proteins (CP) of apparent molecular mass 21-22 kDa (CP2) and 24-28 kDa (CP1) in a molar ratio of 3:1, respectively; CP1 contains a 37 amino acid amino terminal extension of CP2. In our study, expression of CP1 or CP2 in Escherichia coli resulted in assembly of each capsid protein into virus-like particles (VLPs), appearing in electron microscopy as stain-permeable (CP2) or stain-impermeable particles (CP1). CP1 VLPs encapsidated bacterial 16S ribosomal RNA, but not CP mRNA, while CP2 VLPs encapsidated neither CP mRNA nor 16S ribosomal RNA. Expression of CP1 and CP2 in E. coli using a co-expression vector resulted in the assembly of VLPs which were stain-impermeable and encapsidated CP mRNA. These results suggest that the N-terminal 37 amino acid residues of CP1, although not required for particle formation, may be involved in the assembly of complete virions and that the presence of both CP1 and CP2 in the particle is required for specific encapsidation of MRFV CP mRNA.

  8. Subcellular localization and rearrangement of endoplasmic reticulum by Brome mosaic virus capsid protein.

    Science.gov (United States)

    Bamunusinghe, Devinka; Seo, Jang-Kyun; Rao, A L N

    2011-03-01

    Genome packaging in the plant-infecting Brome mosaic virus (BMV), a member of the alphavirus-like superfamily, as well as in other positive-strand RNA viruses pathogenic to humans (e.g., poliovirus) and animals (e.g., Flock House virus), is functionally coupled to replication. Although the subcellular localization site of BMV replication has been identified, that of the capsid protein (CP) has remained elusive. In this study, the application of immunofluorescence confocal microscopy to Nicotiana benthamiana leaves expressing replication-derived BMV CP as a green fluorescent protein (GFP) fusion, in conjunction with antibodies to the CP and double-stranded RNA, a presumed marker of RNA replication, revealed that the subcellular localization sites of replication and CP overlap. Our temporal analysis by transmission electron microscopy of ultrastructural modifications induced in BMV-infected N. benthamiana leaves revealed a reticulovesicular network of modified endoplasmic reticulum (ER) incorporating large assemblies of vesicles derived from ER accumulated in the cytoplasm during BMV infection. Additionally, for the first time, we have found by ectopic expression experiments that BMV CP itself has the intrinsic property of modifying ER to induce vesicles similar to those present in BMV infections. The significance of CP-induced vesicles in relation to CP-organized viral functions that are linked to replication-coupled packaging is discussed.

  9. Generation of rabbit anti-truncated VP1 of Enterovirus 71 antibody%兔抗肠道病毒71型截短VP1抗体的制备及鉴定

    Institute of Scientific and Technical Information of China (English)

    任富利; 周辉; 孟胜利; 王泽鋆; 申硕

    2016-01-01

    目的:制备兔抗肠道病毒71型(EV71)截短VP1多克隆抗体,为EV71基础研究奠定基础。方法 RT-PCR扩增EV71VP1-N160基因(480 bp),以pGEX-6p-1为表达载体,构建重组表达质粒pGEX-6p-1-VP1-N160,转化大肠埃希菌BL21(DE3),IPTG诱导表达GST-VP1-N160融合蛋白,并对其进行纯化。以纯化的GST-VP1-N160融合蛋白作为免疫原,经背部皮下免疫新西兰大耳白兔,制备多克隆抗体,ELISA检测多克隆抗体效价,间接免疫荧光法和免疫印迹检测抗体特异性。结果经双酶切鉴定,表明重组表达质粒pGEX-6p-1-VP1-N160构建正确;GST-VP1-N160融合蛋白相对分子质量为35000~55000,主要为不可溶性包涵体形式,其在大肠杆菌中高效表达;纯化后目的蛋白纯度约为95%,蛋白质量浓度1.9 mg/mL;免疫家兔后,免疫血清效价可达1012,抗VP1-N160多克隆抗体可以识别EV71原核表达的重组蛋白及天然病毒抗原中的VP1,特异性好。结论成功制备了抗EV71截短VP1多克隆抗体。%Objective To generate rabbit polyclonal antibody against truncated VP1 of enterovirus 71 ( EV71 ) and to pro-vide a new approach for the fundamental research on EV71. Methods N-terminal fragment (480 bp) of EV71 VP1, des-ignated as VP1-N160, was amplified by RT-PCR and inserted into E. coli BL21(DE3) to construct a recombinant expres-sion vector named as pGEX-6p-1-VP1-N160. After expression induced by IPTG and purification, the fusion protein GST-VP1-N160 was used to immunize New Zealand white rabbit by hypodermic injection in preparation of polyclonal antibody. The titer of the polyclonal antibody was measured by ELISA and specificity was determined by IFA and WB. Results The result of enzymatic double digestion showed that the recombinant plasmid pGEX-6p-1-VP1-N160 was correctly constructed. GST-VP1-N160 fusion protein with a molecular mass of 35 000~55 000 was expressed at high level in E. coli and it was mainly in a form as insoluble inclusion bodies. The

  10. Identification of an antigenic domain in the N-terminal region of avian hepatitis E virus (HEV) capsid protein that is not common to swine and human HEVs.

    Science.gov (United States)

    Wang, Lizhen; Sun, Yani; Du, Taofeng; Wang, Chengbao; Xiao, Shuqi; Mu, Yang; Zhang, Gaiping; Liu, Lihong; Widén, Frederik; Hsu, Walter H; Zhao, Qin; Zhou, En-Min

    2014-12-01

    The antigenic domains located in the C-terminal 268 amino acid residues of avian hepatitis E virus (HEV) capsid protein have been characterized. This region shares common epitopes with swine and human HEVs. However, epitopes in the N-terminal 338 amino acid residues have never been reported. In this study, an antigenic domain located between amino acids 23 and 85 was identified by indirect ELISA using the truncated recombinant capsid proteins as coating antigens and anti-avian HEV chicken sera as primary antibodies. In addition, this domain did not react with anti-swine and human HEV sera. These results indicated that the N-terminal 338 amino acid residues of avian HEV capsid protein do not share common epitopes with swine and human HEVs. This finding is important for our understanding of the antigenicity of the avian HEV capsid protein. Furthermore, it has important implications in the selection of viral antigens for serological diagnosis.

  11. The herpes simplex virus 1 UL17 protein is the second constituent of the capsid vertex-specific component required for DNA packaging and retention.

    Science.gov (United States)

    Toropova, Katerina; Huffman, Jamie B; Homa, Fred L; Conway, James F

    2011-08-01

    The herpes simplex virus (HSV) UL17 and UL25 minor capsid proteins are essential for DNA packaging. They are thought to comprise a molecule arrayed in five copies around each of the capsid vertices. This molecule was initially termed the "C-capsid-specific component" (CCSC) (B. L. Trus et al., Mol. Cell 26:479-489, 2007), but as we have subsequently observed this feature on reconstructions of A, B, and C capsids, we now refer to it more generally as the "capsid vertex-specific component" (CVSC) (S. K. Cockrell et al., J. Virol. 85:4875-4887, 2011). We previously confirmed that UL25 occupies the vertex-distal region of the CVSC density by visualizing a large UL25-specific tag in reconstructions calculated from cryo-electron microscopy (cryo-EM) images. We have pursued the same strategy to determine the capsid location of the UL17 protein. Recombinant viruses were generated that contained either a small tandem affinity purification (TAP) tag or the green fluorescent protein (GFP) attached to the C terminus of UL17. Purification of the TAP-tagged UL17 or a similarly TAP-tagged UL25 protein clearly demonstrated that the two proteins interact. A cryo-EM reconstruction of capsids containing the UL17-GFP protein reveals that UL17 is the second component of the CVSC and suggests that UL17 interfaces with the other CVSC component, UL25, through its C terminus. The portion of UL17 nearest the vertex appears to be poorly constrained, which may provide flexibility in interacting with tegument proteins or the DNA-packaging machinery at the portal vertex. The exposed locations of the UL17 and UL25 proteins on the HSV-1 capsid exterior suggest that they may be attractive targets for highly specific antivirals.

  12. Detention of HPV L1 Capsid Protein and hTERC Gene in Screening of Cervical Cancer

    Directory of Open Access Journals (Sweden)

    Huang Bin

    2013-06-01

    Full Text Available   Objective(s: To investigate the expression of human papilloma virus (HPV L1 capsid protein, and human telomerase RNA component (hTERC in cervical cancer and the role of detection of both genes in screening of cervical cancer.   Materials and Methods: A total of 309 patients were recruited and cervical exfoliated cells were collected. Immunocytochemistry was employed to detect HPV L1 capsid protein, and fluorescent in situ hybridization (FISH was performed to detect the hTERC. Results: The expression of HPV L1 capsid protein reduced with the increase of the histological grade of cervical cells and was negatively related to the grade of cervical lesions. However, the expression of hTERC increased with the increase of the histological grade and positively associated with the grade of cervical lesions. The proportion of patients with L1(-/hTERC(+ was higher in patients with histological grade of CIN2 or higher than that in those with histological grade of CIN1. The L1(+/hTERC(- and L1(-/hTERC(- were negatively related to the grade of cervical lesions. L1(-/hTERC(+ was positively associated with the grade of cervical lesions. The L1/hTERC ratio increased. The negative predictive value of both HPV L1 and hTERC was higher than that of HPV L1 or hTERC, but there was no marked difference in the screening efficacy of cervical cancer among HPV L1, hTERC and HPV L1+hTERC. Conclusion: HPV L1 capsid protein and hTERC gene may serve as markers for the early diagnosis and prediction of cervical lesions. The increase in L1/hTERC ratio reflects the progression of cervical lesions to a certain extent.

  13. Characterization of virus-like particles and identification of capsid proteins in Xanthophyllomyces dendrorhous.

    Science.gov (United States)

    Flores, Oriana; Alcaíno, Jennifer; Fernandez-Lobato, María; Cifuentes, Víctor; Baeza, Marcelo

    2015-04-01

    Two dsRNAs of estimated lengths of 5 (L1) and 3.7 (L2) kpb are commonly found in strains of the basidiomycetous yeast Xanthophyllomyces dendrorhous, and the presence of virus-like particles (VLPs) have been described in some strains. Recently, two putative totiviruses (XdV-L1A and XdV-L1B) were identified from L1 dsRNA and one (XdV-L2) from L2 dsRNA in the strain UCD 67-385. In some strains, there are smaller dsRNAs (0.9-1.4 kb) that probable are satellite elements. In this work, the VLPs from several strains of X. dendrorhous, which differ in their dsRNAs content, were separated by sucrose gradient and characterized in relation to the dsRNAs and proteins that compose them. It was found that all types of dsRNAs were encapsidated into VLPs, supporting the hypothesis that the smaller dsRNAs are satellite molecules. A main protein of approx. 76 or 37 kDa composed the virions that only have the L1-dsRNA or L2-dsRNA, respectively. In the strain UCD 67-385, these both proteins were identified as viral capsid protein (CP), allow to confirm the gag predicted ORFs in XdV-L1A, XdV-L1B, and XdV-L2, with CPs of 76.6, 76.2, and 38.8 kDa, respectively. Analysis of predicted structures of CPs of XdV-L1A and XdV-L1B, showed high similitudes with the CPs of ScV-L-A and other totiviruses.

  14. The evolution of Vp1 gene in enterovirus C species sub-group that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99.

    Directory of Open Access Journals (Sweden)

    Teemu Smura

    Full Text Available Genus Enterovirus (Family Picornaviridae, consists of twelve species divided into genetically diverse types by their capsid protein VP1 coding sequences. Each enterovirus type can further be divided into intra-typic sub-clusters (genotypes. The aim of this study was to elucidate what leads to the emergence of novel enterovirus clades (types and genotypes. An evolutionary analysis was conducted for a sub-group of Enterovirus C species that contains types Coxsackievirus A21 (CVA-21, CVA-24, Enterovirus C95 (EV-C95, EV-C96 and EV-C99. VP1 gene datasets were collected and analysed to infer the phylogeny, rate of evolution, nucleotide and amino acid substitution patterns and signs of selection. In VP1 coding gene, high intra-typic sequence diversities and robust grouping into distinct genotypes within each type were detected. Within each type the majority of nucleotide substitutions were synonymous and the non-synonymous substitutions tended to cluster in distinct highly polymorphic sites. Signs of positive selection were detected in some of these highly polymorphic sites, while strong negative selection was indicated in most of the codons. Despite robust clustering to intra-typic genotypes, only few genotype-specific 'signature' amino acids were detected. In contrast, when different enterovirus types were compared, there was a clear tendency towards fixation of type-specific 'signature' amino acids. The results suggest that permanent fixation of type-specific amino acids is a hallmark associated with evolution of different enterovirus types, whereas neutral evolution and/or (frequency-dependent positive selection in few highly polymorphic amino acid sites are the dominant forms of evolution when strains within an enterovirus type are compared.

  15. A Novel Pharmacophore Model Derived from a Class of Capsid Protein Enterovirus 71 Inhibitors

    Institute of Scientific and Technical Information of China (English)

    DUAN Hong-Xia; YANG Xin-Ling; WANG Dao-Quan; NING Jun; MEI Xiang-Dong; ZHANG Jian

    2012-01-01

    Capsid protein enterovirus 71 (EV71) is one of the major viruses that cause the severe encephalitis and thus result in a high mortality in children less than 5 years of age.In an effort to discover new potent inhibitors against EV71,a novel three-dimensional pharmacophore model was developed on 24 inhibitors with different molecular structures and bioactivities.The best hypothesis (Hypo1) has a high predictive power and consists of four features,namely,one hydrophobic point (HY) and three hydrogen-bond acceptors (HA).Two key features of the best Hypo1,HY1 and HA3 match well with an important narrow hydrophobic canyon and with the surface of LYS274 in the target EV71 active site,respectively.The more versatile feature,HA1,is firstly found to be very influential on these compounds’ bioactivities,which may interact with the other side of the active site in the EV71 receptor.The application of the model is successful in predicting the activities of 30 known EV71 inhibitors with a correlation coefficient of 0.831.Furthermore,Hypo1 demonstrates a superior screening capability for retrieving inhibitors from the database with a high enrichment factor of 70.This study provides some important clues in search for more potent inhibitors against EV71 infection.

  16. Structural polymorphism of the major capsid protein of a double-stranded RNA virus: an amphipathic alpha helix as a molecular switch.

    Science.gov (United States)

    Saugar, Irene; Luque, Daniel; Oña, Ana; Rodríguez, José F; Carrascosa, José L; Trus, Benes L; Castón, José R

    2005-07-01

    The infectious bursal disease virus T=13 viral particle is composed of two major proteins, VP2 and VP3. Here, we show that the molecular basis of the conformational flexibility of the major capsid protein precursor, pVP2, is an amphipatic alpha helix formed by the sequence GFKDIIRAIR. VP2 containing this alpha helix is able to assemble into the T=13 capsid only when expressed as a chimeric protein with an N-terminal His tag. An amphiphilic alpha helix, which acts as a conformational switch, is thus responsible for the inherent structural polymorphism of VP2. The His tag mimics the VP3 C-terminal region closely and acts as a molecular triggering factor. Using cryo-electron microscopy difference imaging, both polypeptide elements were detected on the capsid inner surface. We propose that electrostatic interactions between these two morphogenic elements are transmitted to VP2 to acquire the competent conformations for capsid assembly.

  17. Simulations of HIV capsid protein dimerization reveal the effect of chemistry and topography on the mechanism of hydrophobic protein association

    CERN Document Server

    Yu, Naiyin

    2015-01-01

    Recent work has shown that the hydrophobic protein surfaces in aqueous solution sit near a drying transition. The tendency for these surfaces to expel water from their vicinity leads to self assembly of macromolecular complexes. In this article we show with a realistic model for a biologically pertinent system how this phenomenon appears at the molecular level. We focus on the association of the C-terminal domain (CA-C) of the human immunodeficiency virus (HIV) capsid protein. By combining all-atom simulations with specialized sampling techniques we measure the water density distribution during the approach of two CA-C proteins as a function of separation and amino acid sequence in the interfacial region. The simulations demonstrate that CA-C protein-protein interactions sit at the edge of a dewetting transition and that this mesoscopic manifestation of the underlying liquid-vapor phase transition can be readily manipulated by biology or protein engineering to significantly affect association behavior. While ...

  18. Identification of two neutralization epitopes on the capsid protein of avian hepatitis E virus.

    Science.gov (United States)

    Zhou, E-M; Guo, H; Huang, F F; Sun, Z F; Meng, X J

    2008-02-01

    Avian hepatitis E virus (avian HEV) is genetically and antigenically related to human HEV, the causative agent of hepatitis E. To identify the neutralizing epitopes on the capsid (ORF2) protein of avian HEV, four mAbs (7B2, 1E11, 10A2 and 5G10) against recombinant avian HEV ORF2 protein were generated. mAbs 7B2, 1E11 and 10A2 blocked each other for binding to avian HEV ORF2 protein in a competitive ELISA, whereas 5G10 did not block the other mAbs, suggesting that 7B2, 1E11 and 10A2 recognize the same or overlapping epitopes and 5G10 recognizes a different one. The epitopes recognized by 7B2, 1E11 and 10A2, and by 5G10 were mapped by Western blotting between aa 513 and 570, and between aa 476 and 513, respectively. mAbs 1E11, 10A2 and 5G10 were shown to bind to avian HEV particles in vitro, although only 5G10 reacted to viral antigens in transfected LMH cells. To assess the neutralizing activities of the mAbs, avian HEV was incubated in vitro with each mAb before inoculation into specific-pathogen-free chickens. Both viraemia and faecal virus shedding were delayed in chickens inoculated with the mixtures of avian HEV and 1E11, 10A2 or 5G10, suggesting that these three mAbs partially neutralize avian HEV.

  19. Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein

    Directory of Open Access Journals (Sweden)

    Bugli F

    2014-05-01

    Full Text Available Francesca Bugli,1 Valeria Caprettini,2 Margherita Cacaci,1 Cecilia Martini,1 Francesco Paroni Sterbini,1 Riccardo Torelli,1 Stefano Della Longa,3 Massimiliano Papi,4 Valentina Palmieri,4 Bruno Giardina,5 Brunella Posteraro,1 Maurizio Sanguinetti,1 Alessandro Arcovito5 1Istituto di Microbiologia, Università Cattolica del Sacro Cuore, 2Dipartimento di Fisica, Sapienza Università di Roma, Rome, 3Dipartimento di Medicina Clinica, Sanità Pubblica, Scienze della Vita e dell’Ambiente, Università dell’Aquila, L’Aquila, 4Istituto di Fisica, 5Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy Abstract: In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few micron long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here – providing a large amount of the viral capsid protein in the native

  20. Location of the bacteriophage P22 coat protein C-terminus provides opportunities for the design of capsid-based materials.

    Science.gov (United States)

    Servid, Amy; Jordan, Paul; O'Neil, Alison; Prevelige, Peter; Douglas, Trevor

    2013-09-01

    Rational design of modifications to the interior and exterior surfaces of virus-like particles (VLPs) for future therapeutic and materials applications is based on structural information about the capsid. Existing cryo-electron microscopy-based models suggest that the C-terminus of the bacteriophage P22 coat protein (CP) extends toward the capsid exterior. Our biochemical analysis through genetic manipulations of the C-terminus supports the model where the CP C-terminus is exposed on the exterior of the P22 capsid. Capsids displaying a 6xHis tag appended to the CP C-terminus bind to a Ni affinity column, and the addition of positively or negatively charged coiled coil peptides to the capsid results in association of these capsids upon mixing. Additionally, a single cysteine appended to the CP C-terminus results in the formation of intercapsid disulfide bonds and can serve as a site for chemical modifications. Thus, the C-terminus is a powerful location for multivalent display of peptides that facilitate nanoscale assembly and capsid modification.

  1. 密码子优化型鸭甲肝病毒VP1基因在昆虫细胞中的表达%ENHANCED EXPRESSION OF CODON-OPTIMIZED VP1 GENE OF DUCK HEPATITIS A VIRUS IN INSECT CELLS

    Institute of Scientific and Technical Information of China (English)

    李传峰; 陈宗艳; 孟春春; 梁瑞英; 胡文; 刘光清

    2014-01-01

    为了提高基因A型鸭甲肝病毒(Duck hepatitis A virus,DHAV)VP1基因在昆虫细胞中的表达水平,本研究根据昆虫细胞密码子偏爱性对野生型DHAV VP1(wtVP1)基因进行改造,合成了optiVP1基因,并利用Bac-to-Bac表达系统构建了重组杆状病毒rBacmid-wtVP1和rBacmid-optiVP1,分别转染对数生长期的sf9昆虫细胞表达VP1蛋白。转染72 h后,sf9细胞出现典型的细胞病变(cytopathic effect,CPE), Western-blot和间接免疫荧光法(indirect immunofluorescence assay,IFA)检测结果表明VP1蛋白在重组杆状病毒感染的sf9昆虫细胞中获得了良好表达。用Image J软件对Western-blot扫描的图片进行灰度分析发现, optiVP1基因在昆虫细胞中的表达水平明显高于wtVP1。本研究为进一步研制诊断抗原和新型基因工程疫苗的开发奠定了基础。%The objective of the present study was to enhance expression level of VP1 gene of Duck hepatitis A virus (DHAV) genotype A in insect cells by manipulating the codon usage bias. The codon usage of wild-type DHAV VP1 (wtVP1) gene was optimized and designated as optiVP1. The recombinant rBacmid-optiVP1 and rBacmid-wtVP1 plasmids were then constructed using the Bac-to-Bac baculovirus expression system (BEVS) and then transfected to sf9 insect cells at logarithmic phase for expression of VP1 protein. The typical cytopathic effect was observed in sf9 cells at 72 h post transfection. The expression of VP1 protein in sf9 cells was confirmed in Western blotting and indirect immunofluorescence assay (IFA). The VP1 amounts on Western blotting were measured using the software Image J. The expression level of optiVP1 gene was significantly increased as compared with wtVP1 gene. This study provided a basis for development of diagnostic reagents and genetically engineered novel vaccines for DHAV.

  2. Biophysical and Structural Studies on the Capsid Protein of the Human Immunodeficiency Virus Type 1: A New Drug Target?

    Directory of Open Access Journals (Sweden)

    José L. Neira

    2009-01-01

    Full Text Available AIDS affects 30 million people worldwide and is one of the deadliest epidemics in human history. It is caused by a retrovirus, HIV, whose mature capsid (enclosing the RNA with other proteins is formed by the assembly of several hundred copies of a protein, CA*. The C-terminal domain of such protein, CAC, is a driving force in virus assembly and the connections in the mature capsid lattice indicate that CAC joins through homodimerization of the CA hexamers. In the first part of this work, I shall review the biophysical studies carried out with the dimeric wild-type CAC protein and a mutant monomeric variant. The results open new venues for the development of drugs able to interact either with the dimeric species, hampering its assembly, or with the monomeric species, obstructing its folding. In the second part of this review, I shall describe the structures of complexes of CAC with small molecules able to weaken its dimerization. Furthermore, interactions with other proteins and lipids are also described. The whole set of results suggests that much of the surface of CAC does not accommodate binding per se, but rather binding sites in the protein are predefined, i.e., there are “hot” spots for binding in CAC (whatever be the molecule to bind. These “hot” residues involve most of the dimerization interface (an α-helix of the CAC wild-type protein, but also polypeptide patches at the other helices.

  3. Specific recognition of the major capsid protein of Acanthamoeba polyphaga mimivirus by sera of patients infected by Francisella tularensis.

    Science.gov (United States)

    Pelletier, Nicolas; Raoult, Didier; La Scola, Bernard

    2009-08-01

    Francisella tularensis, a Gram-negative cocobacillus responsible for tularemia, especially severe pneumonia, is a facultative intracellular bacterium classified as a biological agent of category A. Acanthamoeba polyphaga mimivirus (APM) is a recently discovered giant virus suspected to be an agent of both community- and hospital-acquired pneumonia. During specificity testing of antibody to APM detection, it was observed that nearly all patients infected by F. tularensis had elevated antibody titers to APM. In the present study, we investigated this cross-reactivity by immunoproteomics. Apart from the detection of antibodies reactive to new immunoreactive proteins in patients infected by F. tularensis, we showed that the sera of those patients recognize specifically two proteins of APM: the capsid protein and another protein of unknown function. No common protein motif can be detected in silico based on genome analysis of the involved protein. Furthermore, this cross-reactivity was confirmed with the recombinant capsid protein expressed in Escherichia coli. This emphasizes the pitfalls of a serological diagnosis of pneumonia.

  4. Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus.

    Science.gov (United States)

    Deroubaix, Aurélie; Osseman, Quentin; Cassany, Aurélia; Bégu, Dominique; Ragues, Jessica; Kassab, Somar; Lainé, Sébastien; Kann, Michael

    2015-01-01

    Biopsies from patients show that hepadnaviral core proteins and capsids - collectively called core - are found in the nucleus and cytoplasm of infected hepatocytes. In the majority of studies, cytoplasmic core localization is related to low viraemia while nuclear core localization is associated with high viral loads. In order to better understand the molecular interactions leading to core localization, we analysed transfected hepatoma cells using immune fluorescence microscopy. We observed that expression of core protein in the absence of other viral proteins led to nuclear localization of core protein and capsids, while expression of core in the context of the other viral proteins resulted in a predominantly cytoplasmic localization. Analysis of which viral partner was responsible for cytoplasmic retention indicated that the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that ϵ, an RNA structure to which the viral polymerase binds, was essential for cytoplasmic retention. Furthermore, we showed that core protein phosphorylation at Ser 164 was essential for the cytoplasmic core localization phenotype, which is likely to explain differences observed between individual cells.

  5. Aquareovirus NS80 Initiates Efficient Viral Replication by Retaining Core Proteins within Replication-Associated Viral Inclusion Bodies

    OpenAIRE

    Liming Yan; Jie Zhang; Hong Guo; Shicui Yan; Qingxiu Chen; Fuxian Zhang; Qin Fang

    2015-01-01

    Viral inclusion bodies (VIBs) are specific intracellular compartments for reoviruses replication and assembly. Aquareovirus nonstructural protein NS80 has been identified to be the major constituent for forming globular VIBs in our previous study. In this study, we investigated the role of NS80 in viral structural proteins expression and viral replication. Immunofluorescence assays showed that NS80 could retain five core proteins or inner-capsid proteins (VP1-VP4 and VP6), but not outer-capsi...

  6. Structure of the Triatoma virus capsid

    Energy Technology Data Exchange (ETDEWEB)

    Squires, Gaëlle; Pous, Joan [Laboratoire de Virologie Moléculaire et Structurale, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette CEDEX (France); Agirre, Jon [Fundación Biofísica Bizkaia, Barrio Sarriena S/N, 48940 Leioa, Bizkaia (FBB) (Spain); Unidad de Biofísica (UBF, CSIC, UPV/EHU), PO Box 644, 48080 Bilbao (Spain); Rozas-Dennis, Gabriela S. [U.N.S., San Juan 670 (8000) Bahía Blanca (Argentina); U.N.S., Avenida Alem 1253 (8000) Bahía Blanca (Argentina); Costabel, Marcelo D. [U.N.S., Avenida Alem 1253 (8000) Bahía Blanca (Argentina); Marti, Gerardo A. [Centro de Estudios Parasitológicos y de Vectores (CEPAVE-CCT, La Plata, CONICET-UNLP), Calle 2 No. 584 (1900) La Plata (Argentina); Navaza, Jorge; Bressanelli, Stéphane [Laboratoire de Virologie Moléculaire et Structurale, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette CEDEX (France); Guérin, Diego M. A., E-mail: diego.guerin@ehu.es [Fundación Biofísica Bizkaia, Barrio Sarriena S/N, 48940 Leioa, Bizkaia (FBB) (Spain); Unidad de Biofísica (UBF, CSIC, UPV/EHU), PO Box 644, 48080 Bilbao (Spain); Rey, Felix A., E-mail: diego.guerin@ehu.es [Laboratoire de Virologie Moléculaire et Structurale, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette CEDEX (France)

    2013-06-01

    The crystallographic structure of TrV shows specific morphological and functional features that clearly distinguish it from the type species of the Cripavirus genus, CrPV. The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others. Here, the crystal structure of the TrV capsid at 2.5 Å resolution is reported, showing that as expected it is very similar to that of Cricket paralysis virus (CrPV). Nevertheless, a number of distinguishing structural features support the introduction of a new genus (Triatovirus; type species TrV) under the Dicistroviridae family. The most striking differences are the absence of icosahedrally ordered VP4 within the infectious particle and the presence of prominent projections that surround the fivefold axis. Furthermore, the structure identifies a second putative autoproteolytic DDF motif in protein VP3, in addition to the conserved one in VP1 which is believed to be responsible for VP0 cleavage during capsid maturation. The potential meaning of these new findings is discussed.

  7. Genome sequence, structural proteins, and capsid organization of the cyanophage Syn5: a "horned" bacteriophage of marine synechococcus.

    Science.gov (United States)

    Pope, Welkin H; Weigele, Peter R; Chang, Juan; Pedulla, Marisa L; Ford, Michael E; Houtz, Jennifer M; Jiang, Wen; Chiu, Wah; Hatfull, Graham F; Hendrix, Roger W; King, Jonathan

    2007-05-11

    Marine Synechococcus spp and marine Prochlorococcus spp are numerically dominant photoautotrophs in the open oceans and contributors to the global carbon cycle. Syn5 is a short-tailed cyanophage isolated from the Sargasso Sea on Synechococcus strain WH8109. Syn5 has been grown in WH8109 to high titer in the laboratory and purified and concentrated retaining infectivity. Genome sequencing and annotation of Syn5 revealed that the linear genome is 46,214 bp with a 237 bp terminal direct repeat. Sixty-one open reading frames (ORFs) were identified. Based on genomic organization and sequence similarity to known protein sequences within GenBank, Syn5 shares features with T7-like phages. The presence of a putative integrase suggests access to a temperate life cycle. Assignment of 11 ORFs to structural proteins found within the phage virion was confirmed by mass-spectrometry and N-terminal sequencing. Eight of these identified structural proteins exhibited amino acid sequence similarity to enteric phage proteins. The remaining three virion proteins did not resemble any known phage sequences in GenBank as of August 2006. Cryo-electron micrographs of purified Syn5 virions revealed that the capsid has a single "horn", a novel fibrous structure protruding from the opposing end of the capsid from the tail of the virion. The tail appendage displayed an apparent 3-fold rather than 6-fold symmetry. An 18 A resolution icosahedral reconstruction of the capsid revealed a T=7 lattice, but with an unusual pattern of surface knobs. This phage/host system should allow detailed investigation of the physiology and biochemistry of phage propagation in marine photosynthetic bacteria.

  8. Nucleolin interacts with the dengue virus capsid protein and plays a role in formation of infectious virus particles.

    Science.gov (United States)

    Balinsky, Corey A; Schmeisser, Hana; Ganesan, Sundar; Singh, Kavita; Pierson, Theodore C; Zoon, Kathryn C

    2013-12-01

    Dengue virus (DENV) is a mosquito-transmitted flavivirus that can cause severe disease in humans and is considered a reemerging pathogen of significant importance to public health. The DENV capsid (C) protein functions as a structural component of the infectious virion; however, it may have additional functions in the virus replicative cycle. Here, we show that the DENV C protein interacts and colocalizes with the multifunctional host protein nucleolin (NCL). Furthermore, we demonstrate that this interaction can be disrupted by the addition of an NCL binding aptamer (AS1411). Knockdown of NCL with small interfering RNA (siRNA) or treatment of cells with AS1411 results in a significant reduction of viral titers after DENV infection. Western blotting and quantitative RT-PCR (qRT-PCR) analysis revealed no differences in viral RNA or protein levels at early time points postinfection, suggesting a role for NCL in viral morphogenesis. We support this hypothesis by showing that treatment with AS1411 alters the migration characteristics of the viral capsid, as visualized by native electrophoresis. Here, we identify a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics.

  9. Characterization of a protein kinase activity associated with purified capsids of the granulosis virus infecting Plodia interpunctella.

    Science.gov (United States)

    Wilson, M E; Consigli, R A

    1985-06-01

    A cyclic-nucleotide independent protein kinase activity has been demonstrated in highly purified preparations of the granulosis virus infecting the Indian meal moth, Plodia interpunctella. A divalent cation was required for activity. Manganese was the preferred cation and a pH of 8.0 resulted in optimal incorporation of 32P radiolabel into acid-precipitable protein. Although both ATP and GTP could serve as phosphate donors, ATP was utilized more efficiently by the enzyme. The kinase activity was localized to purified capsids; and the basic, internal core protein, VP12, was found to be the predominant viral acceptor. Histones and protamine sulfate could also serve as acceptors for the capsid-associated kinase activity. Using acid hydrolysis and phosphoamino acid analysis of phosphorylated nucleocapsid protein and nuclear magnetic resonance of phosphorylated VP12, it was determined that the enzyme catalyzes the transfer of phosphate to both serine and arginine residues of acceptor proteins. We believe this kinase activity may play a significant role in the viral replication cycle.

  10. Conserved Tryptophan Motifs in the Large Tegument Protein pUL36 Are Required for Efficient Secondary Envelopment of Herpes Simplex Virus Capsids

    Science.gov (United States)

    Ivanova, Lyudmila; Buch, Anna; Döhner, Katinka; Pohlmann, Anja; Binz, Anne; Prank, Ute; Sandbaumhüter, Malte

    2016-01-01

    ABSTRACT Herpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs 1766WD1767 and 1862WE1863 are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17+)Lox-pUL36-WD/AA-WE/AA and HSV-1(17+)Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly. IMPORTANCE Herpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several

  11. EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR AND HUMAN PAPILLOMAVIRUS (HPV L1 CAPSID PROTEIN IN CERVICAL SQUAMOUS INTRAEPITHELIAL LESIONS

    Directory of Open Access Journals (Sweden)

    Balan Raluca

    2010-09-01

    Full Text Available We analyzed the immunohistochemical pattern of epidermal growth factor receptor (EGFR in cervical squamous intraepithelial lesions (SILs in correlation with L1 HPV capsid protein, in order to determine the relationship between EGFR expression and the infection status of human papillomavirus (HPV. The study included 40 cases, 24 LSIL (low grade SIL (CIN1, cervical intraepithelial neoplasia and 16 HSIL (high grade SIL (6 cases of CIN2 and 10 cases of CIN3. The immunoexpression of L1 HPV protein was assessed on conventional cervico-vaginal smears and EGFR was immunohistochemically evaluated on the corresponding cervical biopsies. The HPV L1 capsid protein was expressed in 45.83% of LSIL and 25% of HSIL. EGFR was overexpressed in 62,4% of HSIL (58,4% CIN2 and 41,6% CIN3 and 37,6% LSIL. The immunoexpression of L1 HPV has clinical application in the progression assessment of the cervical precancerous lesions without a correlation to the grade of the cervical SIL. EGFR is expressed by all proliferating squamous epithelial cells, thus corresponding with the grade of SIL. The evaluation of EGFR status, correlated with L1 HPV protein expression, can provide useful data of progression risk of cervical squamous intraepithelial lesions

  12. Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2.

    OpenAIRE

    1994-01-01

    The three-dimensional structure of the complex between the Fab fragment of an anti-human rhinovirus neutralizing antibody (8F5) and a cross-reactive synthetic peptide from the viral capsid protein VP2 has been determined at 2.5 A resolution by crystallographic methods. The refinement is presently at an R factor of 0.18 and the antigen-binding site and viral peptide are well defined. The peptide antigen adopts a compact fold by two tight turns and interacts through hydrogen bonds, some with io...

  13. Molecular evolution of the VP1, VP2, and VP3 genes in human rhinovirus species C.

    Science.gov (United States)

    Kuroda, Makoto; Niwa, Shoichi; Sekizuka, Tsuyoshi; Tsukagoshi, Hiroyuki; Yokoyama, Masaru; Ryo, Akihide; Sato, Hironori; Kiyota, Naoko; Noda, Masahiro; Kozawa, Kunihisa; Shirabe, Komei; Kusaka, Takashi; Shimojo, Naoki; Hasegawa, Shunji; Sugai, Kazuko; Obuchi, Masatsugu; Tashiro, Masato; Oishi, Kazunori; Ishii, Haruyuki; Kimura, Hirokazu

    2015-02-02

    Human rhinovirus species C (HRV-C) was recently discovered, and this virus has been associated with various acute respiratory illnesses (ARI). However, the molecular evolution of the major antigens of this virus, including VP1, VP2, and VP3, is unknown. Thus, we performed complete VP1, VP2, and VP3 gene analyses of 139 clinical HRV-C strains using RT-PCR with newly designed primer sets and next-generation sequencing. We assessed the time-scale evolution and evolutionary rate of these genes using the Bayesian Markov chain Monte Carlo method. In addition, we calculated the pairwise distance and confirmed the positive/negative selection sites in these genes. The phylogenetic trees showed that the HRV-C strains analyzed using these genes could be dated back approximately 400 to 900 years, and these strains exhibited high evolutionary rates (1.35 to 3.74 × 10(-3) substitutions/site/year). Many genotypes (>40) were confirmed in the phylogenetic trees. Furthermore, no positively selected site was found in the VP1, VP2, and VP3 protein. Molecular modeling analysis combined with variation analysis suggested that the exterior surfaces of the VP1, VP2 and VP3 proteins are rich in loops and are highly variable. These results suggested that HRV-C may have an old history and unique antigenicity as an agent of various ARI.

  14. Development of Cell Lines Stably Expressing Staphylococcal Nuclease Fused to Dengue 2 Virus Capsid Protein for CTVI

    Institute of Scientific and Technical Information of China (English)

    Cheng-Feng QIN; E-De QIN

    2004-01-01

    To explore the potential application of capsid-targeted viral inactivation(CTVI)strategy in prophylactic model against dengue virus(DV)infection,here we fused a Ca2+-dependent nuclease,staphylococcal nuclease(SN),to the capsid protein of dengue 2 virus(D2C)at the carboxyl terminal,and constructed the desired expression plasmid pc/D2C-SN and control plasmids pc/D2C-SN* and pc/D2C.A mammalian cell line BHK-21 was transfected by electroporation with those plasmids and thereafter selected by 5 μg/ml blasticidin.The resistant cell clones were then expanding cultured and screened by RT-PCR and Western Blot assays.The nuclease activity of the expressed fusion protein D2C-SN was analyzed by in vitro DNA digestion assay.It was confirmed cell lines stably expressing D2C-SN and control constructs were obtained.The intracellular expressed fusion protein D2C-SN had ideal nuclease activity and no cytotoxicity on mammalian cells.Those engineered cell lines provided the experimental system for CTVI application in prophylactic model and paved the new road for combating DV infection with CTVI.

  15. Development and evaluation of an immunochromatographic strip for rapid detection of capsid protein antigen p27 of avian leukosis virus.

    Science.gov (United States)

    Qian, Kun; Liang, You-zhi; Yin, Li-ping; Shao, Hong-xia; Ye, Jian-qiang; Qin, Ai-jian

    2015-09-01

    A rapid immunochromatographic strip for detecting capsid protein antigen p27 of avian leukosis virus was successfully developed based on two high-affinity monoclonal antibodies. The test strip could detect not only 600pg purified recombinant p27 protein but also quantified avian leukosis virus as low as 70 TCID50, which has comparative sensitivity to the commercial enzyme-linked immunosorbent assay (ELISA) kit. For the evaluation of this test strip, 1100 samples consisting of cloacal swabs, meconium collected from the earliest stool of one day old chicken and virus isolates were assessed both by the strip and by the commercial ELISA kit. The agreement between these two tests was 93.91%, 93.42% and 100%, respectively. The sensitivity and specificity of the strip were also calculated by using the ELISA kit as the standard. This immunochromatographic strip provides advantages of rapid and simple detection of capsid protein antigen p27 of avian leukosis virus, which could be applied as an on-site testing assay and used for control and eradication programs of avian leukosis disease.

  16. Structure of the Three N-Terminal Immunoglobulin Domains of the Highly Immunogenic Outer Capsid Protein from a T4-Like Bacteriophage

    Energy Technology Data Exchange (ETDEWEB)

    Fokine, Andrei; Islam, Mohammad Z.; Zhang, Zhihong; Bowman, Valorie D.; Rao, Venigalla B.; Rossmann, Michael G. (CUA); (Purdue)

    2011-09-16

    The head of bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein (Hoc). One Hoc molecule binds near the center of each hexameric capsomer. Hoc is dispensable for capsid assembly and has been used to display pathogenic antigens on the surface of T4. Here we report the crystal structure of a protein containing the first three of four domains of Hoc from bacteriophage RB49, a close relative of T4. The structure shows an approximately linear arrangement of the protein domains. Each of these domains has an immunoglobulin-like fold, frequently found in cell attachment molecules. In addition, we report biochemical data suggesting that Hoc can bind to Escherichia coli, supporting the hypothesis that Hoc could attach the phage capsids to bacterial surfaces and perhaps also to other organisms. The capacity for such reversible adhesion probably provides survival advantages to the bacteriophage.

  17. Prognostic relevance of human papillomavirus L1 capsid protein detection within mild and moderate dysplastic lesions of the cervix uteri in combination with p16 biomarker

    DEFF Research Database (Denmark)

    Hilfrich, Ralf; Hariri, Jalil

    2008-01-01

    OBJECTIVE: To proof the prognostic relevance of HPV L1 capsid protein detection on colposcopically-guided punch biopsies in combination with p16. STUDY DESIGN: Sections of colposcopically-guided punch biopsies from 191 consecutive cases with at least 5 years of follow-up were stained with HPV L1...... capsid protein antibodies (Cytoactiv screening antibody) and a monoclonal anti-p16 antibody. Fifty sections were derived from a benign group, 91 from low-grade (cervical intraepithelial neoplasia [CIN 1]) lesions and 50 from high-grade (CIN 2 and 3) lesions. RESULTS: Overall only 16.1% of the 87 L1......-negative, p16-positive CIN lesions showed remission of the lesion compared to 72.4% of the double positive cases. None of the L1/p16 double negative CIN lesions progressed. CONCLUSION: HPV L1 capsid protein detection with Cytoactiv screening antibody seems to be a promising new tool to predict the behavior...

  18. 人乳头瘤病毒衣壳蛋白与宫颈病变%Human Papillomavirus′ Capsid Proteins and Cervical Lesions

    Institute of Scientific and Technical Information of China (English)

    黄成琳; 张淑兰

    2014-01-01

    Cervical cancer seriously endangers women′s health,and human papillomavirus (HPV) is considered to be the primary cause. Doctors have been striving to find an effective diagnostic method for judging cervical lesions level and predicting its prognosis. HPV capsid proteins comprise the major capsid protein (L1 capsid protein) and the minor capsid protein (L2 capsid protein),and these two proteins play an important role in assembling into virus particles,trafficking HPV to the cell,and causing the host′s immune reactions. In recent years,studies have shown that the L1 capsid protein can be used to predict the progress and subsidence of cervical lesions. HPV prophylactic vaccines ,which are exploited on the basis of the L1 and L2 capsid protein,are proved to get a good preventive effect in clinical trials. This paper reviews the biological characteristics of HPV and researches progress on HPV capsid protein in cervical lesions in recent years.%宫颈癌严重危害妇女健康,人乳头瘤病毒(HPV)感染是其首要病因。临床医师一直致力于寻找一种能有效判断宫颈病变级别及预测预后的诊断方法。 HPV衣壳蛋白包括主要衣壳蛋白(L1壳蛋白)和次要衣壳蛋白(L2壳蛋白),这两种蛋白在组装成病毒颗粒、协助病毒入胞及引起机体免疫反应等多个方面发挥重要作用。近年研究表明, L1壳蛋白可用于预测宫颈病变的进展与消退。以L1及L2壳蛋白为基础研发的HPV预防性疫苗在临床试验中得到了很好的预防效果。综述HPV生物学特点及近年来有关HPV衣壳蛋白在宫颈病变的研究进展。

  19. Bacterial surface-displayed GII.4 human norovirus capsid proteins bound to surface of Romaine lettuce through HBGA-like molecules

    Science.gov (United States)

    Human Noroviruses (HuNoVs) are the main cause of nonbacterial gastroenteritis. Contaminated produce is a main vehicle for dissemination of HuNoVs. In this study, we used an ice nucleation protein (INP) mediated surface display system to present the protruding domain of GII.4 HuNoV capsid protein (G...

  20. Nucleotide sequence of the capsid protein gene and 3' non-coding region of papaya mosaic virus RNA.

    Science.gov (United States)

    Abouhaidar, M G

    1988-01-01

    The nucleotide sequences of cDNA clones corresponding to the 3' OH end of papaya mosaic virus RNA have been determined. The 3'-terminal sequence obtained was 900 nucleotides in length, excluding the poly(A) tail, and contained an open reading frame capable of giving rise to a protein of 214 amino acid residues with an Mr of 22930. This protein was identified as the viral capsid protein. The 3' non-coding region of PMV genome RNA was about 121 nucleotides long [excluding the poly(A) tail] and homologous to the complementary sequence of the non-coding region at the 5' end of PMV RNA. A long open reading frame was also found in the predicted 5' end region of the negative strand.

  1. Residues of the UL25 Protein of Herpes Simplex Virus That Are Required for Its Stable Interaction with Capsids

    OpenAIRE

    Cockrell, Shelley K.; Huffman, Jamie B.; Toropova, Katerina; James F Conway; Homa, Fred L.

    2011-01-01

    The herpes simplex virus 1 (HSV-1) UL25 gene product is a minor capsid component that is required for encapsidation, but not cleavage, of replicated viral DNA. UL25 is located on the capsid surface in a proposed heterodimer with UL17, where five copies of the heterodimer are found at each of the capsid vertices. Previously, we demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids. To further define the UL25 capsid binding domain, we generated reco...

  2. Radioiodination of advenovirus-associated virus external structural proteins. [/sup 125/I

    Energy Technology Data Exchange (ETDEWEB)

    Lubeck, M.D.; Johnson, F.B.

    1977-12-01

    The three structural polypeptides of adenovirus-associated virus type 3 (AAV-3) were examined to determine their orientation in the viral capsid. Sepharose-bound lactoperoxidase was used to label both dense-band and major-band AAV-3 virions. All three capsid proteins (VP1, VP2, and VP3) were found to be radioiodinated by solid-state lactoperoxidase in both dense-band and major-band virus particles. These findings indicate that the three polypeptides possess sequences that are externally oriented in the virion and that, therefore, no one of the three polypeptides can be considered an exclusively internally core protein.

  3. Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with Altered Tropism

    Science.gov (United States)

    Wu, Pei; Xiao, Wu; Conlon, Thomas; Hughes, Jeffrey; Agbandje-McKenna, Mavis; Ferkol, Thomas; Flotte, Terence; Muzyczka, Nicholas

    2000-01-01

    insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues. PMID:10954565

  4. Interaction study of a novel Macrobrachium rosenbergii effector caspase with B2 and capsid proteins of M. rosenbergii nodavirus reveals their roles in apoptosis.

    Science.gov (United States)

    Youngcharoen, Supak; Senapin, Saengchan; Lertwimol, Tareerat; Longyant, Siwaporn; Sithigorngul, Paisarn; Flegel, Timothy W; Chaivisuthangkura, Parin

    2015-08-01

    Apoptosis is an essential immune response to protect invertebrates from virus infected cells. In shrimp, virus infection has been reported to induce apoptosis. Macrobrachium rosenbergii (Mr) was considered to be a disease-resistant host when compared to penaeid shrimps. Caspase-3 was classified as an executioner caspase which played a key role in virus-induced apoptosis. In this study, an effector caspase gene of M. rosenbergii (Mrcasp) was cloned and characterized. The open reading frame (ORF) of Mrcasp was 957 nucleotide encoding 318 amino acid with a deduced molecular mass of 35.87 kDa. RT-PCR analysis showed the presence of Mrcasp in all examined tissues. The phylogenetic tree indicated that Mrcasp was closely related with caspase 3 of shrimp. The functions of the Mrcasp, B2 and capsid proteins of M. rosenbergii nodavirus (MrNV) were assayed in Sf-9 cells. The results showed that Mrcasp induce apoptotic morphology cells; however, capsid protein of MrNV could inhibit apoptotic cells whereas B2 could neither induce nor inhibit apoptotic cells by DAPI staining. The protein interaction between Mrcasp and viral MrNV structure revealed that Mrcasp did not bind to B2 or capsid protein whereas B2 and capsid proteins could bind directly to each other. This study reported a novel sequence of a full-length Mrcasp and its functional studies indicated that Mrcasp could induce apoptotic cells. Our data is the first report demonstrating the direct protein-protein interaction between capsid protein and B2 protein of MrNV.

  5. Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

    Energy Technology Data Exchange (ETDEWEB)

    Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor; Afonine, Pavel V.; Motwani, Tina; Teschke, Carolyn M.; Parent, Kristin N.; Cingolani, Gino (Rutgers); (LBNL); (Connecticut); (TJU); (MSU)

    2017-01-30

    Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid’) built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging’ is a DNA-dependent symmetrization of portal protein.

  6. Role of a nuclear localization signal on the minor capsid Proteins VP2 and VP3 in BKPyV nuclear entry

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, Shauna M. [Cellular and Molecular Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Zhao, Linbo [Doctoral Program in Cancer Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Bosard, Catherine [Department of Microbiology and Immunology University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Imperiale, Michael J., E-mail: imperial@umich.edu [Cellular and Molecular Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Doctoral Program in Cancer Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Department of Microbiology and Immunology University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States)

    2015-01-01

    BK Polyomavirus (BKPyV) is a ubiquitous nonenveloped human virus that can cause severe disease in immunocompromised populations. After internalization into renal proximal tubule epithelial cells, BKPyV traffics through the ER and enters the cytosol. However, it is unclear how the virus enters the nucleus. In this study, we elucidate a role for the nuclear localization signal located on the minor capsid proteins VP2 and VP3 during infection. Site-directed mutagenesis of a single lysine in the basic region of the C-terminus of the minor capsid proteins abrogated their nuclear localization, and the analogous genomic mutation reduced infectivity. Additionally, through use of the inhibitor ivermectin and knockdown of importin β1, we found that the importin α/β pathway is involved during infection. Overall these data are the first to show the significance of the NLS of the BKPyV minor capsid proteins during infection in a natural host cell. - Highlights: • Polyomaviruses must deliver their genome to the nucleus to replicate. • The minor capsid proteins have a well-conserved nuclear localization signal. • Mutation of this NLS diminishes, but does not completely inhibit, infection.

  7. Vaccination of mice with plasmids expressing processed capsid protein of foot-and-mouth disease virus - Importance of dominant and subdominant epitopes for antigenicity and protection

    DEFF Research Database (Denmark)

    Frimann, Tine; Barfoed, Annette Malene; Aasted, Bent

    2007-01-01

    example of a dominant and variable site. This variability is a problem when designing vaccines against this disease, because it necessitates a close match between vaccine strain and virus in an outbreak. We have introduced a series of mutations into viral capsid proteins with the aim of selectively...

  8. EV71 virus-like particles produced by co-expression of capsid proteins in yeast cells elicit humoral protective response against EV71 lethal challenge

    OpenAIRE

    Wang, Xiaowen; Xiao, Xiangqian; Zhao, Miao; Wei LIU; Pang, Lin; Sun, Xin; Cen, Shan; Burton B Yang; Huang, Yuming; Sheng, Wang; Zeng, Yi

    2016-01-01

    Background Enterovirus 71 (EV71) is the most common causative pathogens of hand, foot and mouth disease (HFMD) associated with severe neurological complications. There is a great need to develop prophylactic vaccine against EV71 infection. Results EV71 virus-like particle (VLP) was produced in yeast expression system by the co-expression of four EV71 structural proteins VP1–VP4. Immunization with the recombinant VLPs elicited potent anti-EV71 antibody responses in adult mice and anti-VLP sera...

  9. C-Terminal Amino Acids 471-507 of Avian Hepatitis E Virus Capsid Protein Are Crucial for Binding to Avian and Human Cells.

    Science.gov (United States)

    Zhang, Xinquan; Bilic, Ivana; Marek, Ana; Glösmann, Martin; Hess, Michael

    2016-01-01

    The infection of chickens with avian Hepatitis E virus (avian HEV) can be asymptomatic or induces clinical signs characterized by increased mortality and decreased egg production in adult birds. Due to the lack of an efficient cell culture system for avian HEV, the interaction between virus and host cells is still barely understood. In this study, four truncated avian HEV capsid proteins (ORF2-1 - ORF2-4) with an identical 338aa deletion at the N-terminus and gradual deletions from 0, 42, 99 and 136aa at the C-terminus, respectively, were expressed and used to map the possible binding site within avian HEV capsid protein. Results from the binding assay showed that three truncated capsid proteins attached to avian LMH cells, but did not penetrate into cells. However, the shortest construct, ORF2-4, lost the capability of binding to cells suggesting that the presence of amino acids 471 to 507 of the capsid protein is crucial for the attachment. The construct ORF2-3 (aa339-507) was used to study the potential binding of avian HEV capsid protein to human and other avian species. It could be demonstrated that ORF2-3 was capable of binding to QT-35 cells from Japanese quail and human HepG2 cells but failed to bind to P815 cells. Additionally, chicken serum raised against ORF2-3 successfully blocked the binding to LMH cells. Treatment with heparin sodium salt or sodium chlorate significantly reduced binding of ORF2-3 to LMH cells. However, heparinase II treatment of LMH cells had no effect on binding of the ORF2-3 construct, suggesting a possible distinct attachment mechanism of avian as compared to human HEV. For the first time, interactions between avian HEV capsid protein and host cells were investigated demonstrating that aa471 to 507 of the capsid protein are needed to facilitate interaction with different kind of cells from different species.

  10. Sphingomyelin induces structural alteration in canine parvovirus capsid

    OpenAIRE

    Pakkanen, Kirsi; Karttunen, Jenni; Virtanen, Salla; Vuento, Matti

    2008-01-01

    One of the essential steps in canine parvovirus (CPV) infection, the release from endosomal vesicles, is dominated by interactions between the virus capsid and the endosomal membranes. In this study, the effect of sphingomyelin and phosphatidyl serine on canine parvovirus capsid and on the phospholipase A2 (PLA2) activity of CPV VP1 unique N-terminus was analyzed. Accordingly, a significant (P ≤ 0.05) shift of tryptophan fluorescence emission peak was detected at pH 5.5 in the presen...

  11. Bacterial Surface-Displayed GII.4 Human Norovirus Capsid Proteins Bound to HBGA-Like Molecules in Romaine Lettuce.

    Science.gov (United States)

    Wang, Ming; Rong, Shaofeng; Tian, Peng; Zhou, Yue; Guan, Shimin; Li, Qianqian; Wang, Dapeng

    2017-01-01

    Human Noroviruses (HuNoVs) are the main cause of non-bacterial gastroenteritis. Contaminated produce is a main vehicle for dissemination of HuNoVs. In this study, we used an ice nucleation protein mediated surface display system to present the protruding domain of GII.4 HuNoV capsid protein on bacterial surface and used it as a new strategy to explore interaction between HuNoV protein and receptor candidates from romaine lettuce. The surface-displayed HuNoV proteins were confirmed on the surface of the transformed bacteria by an immunofluorescence assay. The distribution patterns of the surface-displayed HuNoV proteins in romaine lettuce were identified through a confocal immunofluorescence assay. The surface-displayed HuNoV proteins could be found in the stomata, and the surfaces of vein and leaf of romaine lettuce. The surface-displayed HuNoV proteins could be captured by an ELISA assay utilizing extract from leaf (LE) or vein (VE). The binding of the surface-displayed HuNoV proteins to LE or VE could be competitively blocked by histo-blood group antigens from human saliva. In addition, the binding of the surface-displayed HuNoV proteins to LE or VE could also be attenuated by heat denaturation of lettuce proteins, and abolished by oxidation of lettuce carbohydrates. The results indicated that histo-blood group antigen-like molecules in LE or VE were involved in the binding of the surface-displayed HuNoV proteins to romaine lettuce. All data demonstrated that the surface-displayed HuNoV proteins could be utilized in a new and simple system for investigation of the interaction between the HuNoVs and their candidate ligands.

  12. Capsid protein genetic analysis and viral spread to the spinal cord in cats experimentally infected with feline calicivirus (FCV).

    Science.gov (United States)

    Fujita, Y; Sato, Y; Ohe, K; Sakai, S; Fukuyama, M; Furuhata, K; Kishikawa, S; Yamamoto, S; Kiuchi, A; Hara, M; Ishikawa, Y; Taneno, A

    2005-08-01

    We investigated primitively the molecular basis of the neural spread of a feline calcivirus isolate (FCV-S) from the spinal cord of a cat that died after manifesting excitation. Experimental infections of cats with three clones from parent virus isolate FCV-S, isolated based on plaque size, were performed, and virus recovery from the spinal cord and the nucleotide and predicted amino acid sequences of the viral capsid protein region (ORF2) were compared. In the experimental infection with the one-time cloned virus (C1L1) isolated from a large plaque, the C1L1 was recovered from the spinal cord. In contrast, seven-times cloned C6L7 (from large plaque) and five-times cloned C5S2 (isolated from small plaque) were not recovered from the spinal cord. Genetic analysis of the capsid protein gene of the three viral clones revealed that four bases were different and two amino acids were different at positions 34 (Val in C6L7 and Ala in C1L1 and C5S2) and 46 (Leu in C6L7 and Pro in C1L1 and C5S2) between C6L7 (with large plaque) and C5S2 (with small plaque). The amino acid at position 434 of C1L1 was different from those of C6L7 and C5S2 (Gly in C1L1, D (Asp) in C6L7 and C5S2). From these results, the plaque size seemed not to be related to the spread of virus to the spinal cord. Clone C1L1, which spread to the spinal cord, had a difference of one amino acid from the other two clones, which may be related to the ability to spread to the spinal cord.

  13. Analysis of the termination/reinitiation-mechanism of translation of the minor capsid protein VP2 from the feline calcivirus

    OpenAIRE

    Luttermann, Christine

    2009-01-01

    Die Caliciviren umfassen eine Gruppe tier- und humanpathogener Viren mit einem Genom positiver Polarität. Die caliciviralen Strukturproteine werden ausgehend von einer subgenomischen RNA translatiert, die zwei überlappende Leseraster enthält. Das erste Leseraster kodiert für das virale Hauptkapsidprotein VP1 und das zweite für das minore Kapsidprotein VP2. In transienten Expressionsstudien war die VP2-Translationseffizienz ca. 20fach geringer als die des VP1. Für das Feline Calicivirus (FC...

  14. Direct binding of retromer to human papillomavirus type 16 minor capsid protein L2 mediates endosome exit during viral infection.

    Directory of Open Access Journals (Sweden)

    Andreea Popa

    2015-02-01

    Full Text Available Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.

  15. [Influence of Japanese enciphalitis virus capsid protein on the self-replicate ability of JEV replicon vectors].

    Science.gov (United States)

    Huang, Ying; Liu, Shan; Yang, Peng; Wang, Chao; Du, Yun; Sun, Zhiwei; Yu, Weiyuan

    2010-08-01

    To optimize a self-replicate Japanese enciphalitis virus (JEV) replicon, and to make it as an efficient vector to express the heterologous protein, we constructed three JEV replicons by PCR-based shortening the length of capsid genes. The vectors remained full or part of C gene, based on the JEV replicon pCTCJEV. Lac Z was selected as the reporter gene to verify the self-replicate ability of these DNA-based replicons. While transfected into the cell lines CME-4, which continuously expressing the JEV structure proteins C-prM-E, the JEV replicons pCMW-2M-1LACZ, pCMW-2M-3LACZ, which remained the first 23aa and 68aa of C protein, can express the reporter protein as the same level as pCMW-2M-LACZ with the full-length C protein. These results illustrated that the JEV replicon vector with 69-nt of the C gene can retain the self-replicate ability, and provide valuable tools to construct a possible vector for a long-lasting JEV RNA virus expression system.

  16. Characterization of the invariable residue 51 mutations of human immunodeficiency virus type 1 capsid protein on in vitro CA assembly and infectivity

    Directory of Open Access Journals (Sweden)

    Höglund Stefan

    2007-09-01

    Full Text Available Abstract Background The mature HIV-1 conical core formation proceeds through highly regulated protease cleavage of the Gag precursor, which ultimately leads to substantial rearrangements of the capsid (CAp24 molecule involving both inter- and intra-molecular contacts of the CAp24 molecules. In this aspect, Asp51 which is located in the N-terminal domain of HIV-1 CAp24 plays an important role by forming a salt-bridge with the free imino terminus Pro1 following proteolytic cleavage and liberation of the CAp24 protein from the Pr55Gag precursor. Thus, previous substitution mutation of Asp51 to alanine (D51A has shown to be lethal and that this invariable residue was found essential for tube formation in vitro, virus replication and virus capsid formation. Results We extended the above investigation by introducing three different D51 substitution mutations (D51N, D51E, and D51Q into both prokaryotic and eukaryotic expression systems and studied their effects on in vitro capsid assembly and virus infectivity. Two substitution mutations (D51E and D51N had no substantial effect on in vitro capsid assembly, yet they impaired viral infectivity and particle production. In contrast, the D51Q mutant was defective both for in vitro capsid assembly and for virus replication in cell culture. Conclusion These results show that substitutions of D51 with glutamate, glutamine, or asparagine, three amino acid residues that are structurally related to aspartate, could partially rescue both in vitro capsid assembly and intra-cellular CAp24 production but not replication of the virus in cultured cells.

  17. A tetravalent dengue vaccine containing a mix of domain III-P64k and domain III-capsid proteins induces a protective response in mice.

    Science.gov (United States)

    Izquierdo, Alienys; García, Angélica; Lazo, Laura; Gil, Lázaro; Marcos, Ernesto; Alvarez, Mayling; Valdés, Iris; Hermida, Lisset; Guillén, Gerardo; Guzmán, María G

    2014-10-01

    Recombinant fusion proteins containing domain III of the dengue virus envelope protein fused to the P64k protein from Neisseria meningitidis and domain III of dengue virus type 2 (D2) fused to the capsid protein of this serotype were immunogenic and conferred protection in mice against lethal challenge, as reported previously. Combining the domain III-P64k recombinant proteins of dengue virus types 1, 3 and 4 (D1, D3, and D4) with the domain III-capsid protein from D2, we obtained a novel tetravalent formulation containing different antigens. Here, the IgG and neutralizing antibody response, the cellular immune response, and the protective capacity against lethal challenge in mice immunized with this tetravalent formulation were evaluated. The neutralizing antibody response obtained against D1, D2 and D3, together with the high levels of IFNγ secretion induced after stimulation with the four dengue serotypes, supports the strategy of using a new tetravalent formulation containing domain III of the envelope protein fused to the capsid protein of each dengue virus serotype.

  18. Quantification and modification of the equilibrium dynamics and mechanics of a viral capsid lattice self-assembled as a protein nanocoating

    Science.gov (United States)

    Valbuena, Alejandro; Mateu, Mauricio G.

    2015-09-01

    Self-assembling, protein-based bidimensional lattices are being developed as functionalizable, highly ordered biocoatings for multiple applications in nanotechnology and nanomedicine. Unfortunately, protein assemblies are soft materials that may be too sensitive to mechanical disruption, and their intrinsic conformational dynamism may also influence their applicability. Thus, it may be critically important to characterize, understand and manipulate the mechanical features and dynamic behavior of protein assemblies in order to improve their suitability as nanomaterials. In this study, the capsid protein of the human immunodeficiency virus was induced to self-assemble as a continuous, single layered, ordered nanocoating onto an inorganic substrate. Atomic force microscopy (AFM) was used to quantify the mechanical behavior and the equilibrium dynamics (``breathing'') of this virus-based, self-assembled protein lattice in close to physiological conditions. The results uniquely provided: (i) evidence that AFM can be used to directly visualize in real time and quantify slow breathing motions leading to dynamic disorder in protein nanocoatings and viral capsid lattices; (ii) characterization of the dynamics and mechanics of a viral capsid lattice and protein-based nanocoating, including flexibility, mechanical strength and remarkable self-repair capacity after mechanical damage; (iii) proof of principle that chemical additives can modify the dynamics and mechanics of a viral capsid lattice or protein-based nanocoating, and improve their applied potential by increasing their mechanical strength and elasticity. We discuss the implications for the development of mechanically resistant and compliant biocoatings precisely organized at the nanoscale, and of novel antiviral agents acting on fundamental physical properties of viruses.Self-assembling, protein-based bidimensional lattices are being developed as functionalizable, highly ordered biocoatings for multiple applications

  19. Quantification and modification of the equilibrium dynamics and mechanics of a viral capsid lattice self-assembled as a protein nanocoating.

    Science.gov (United States)

    Valbuena, Alejandro; Mateu, Mauricio G

    2015-09-28

    Self-assembling, protein-based bidimensional lattices are being developed as functionalizable, highly ordered biocoatings for multiple applications in nanotechnology and nanomedicine. Unfortunately, protein assemblies are soft materials that may be too sensitive to mechanical disruption, and their intrinsic conformational dynamism may also influence their applicability. Thus, it may be critically important to characterize, understand and manipulate the mechanical features and dynamic behavior of protein assemblies in order to improve their suitability as nanomaterials. In this study, the capsid protein of the human immunodeficiency virus was induced to self-assemble as a continuous, single layered, ordered nanocoating onto an inorganic substrate. Atomic force microscopy (AFM) was used to quantify the mechanical behavior and the equilibrium dynamics ("breathing") of this virus-based, self-assembled protein lattice in close to physiological conditions. The results uniquely provided: (i) evidence that AFM can be used to directly visualize in real time and quantify slow breathing motions leading to dynamic disorder in protein nanocoatings and viral capsid lattices; (ii) characterization of the dynamics and mechanics of a viral capsid lattice and protein-based nanocoating, including flexibility, mechanical strength and remarkable self-repair capacity after mechanical damage; (iii) proof of principle that chemical additives can modify the dynamics and mechanics of a viral capsid lattice or protein-based nanocoating, and improve their applied potential by increasing their mechanical strength and elasticity. We discuss the implications for the development of mechanically resistant and compliant biocoatings precisely organized at the nanoscale, and of novel antiviral agents acting on fundamental physical properties of viruses.

  20. Purification of recombinant virus-like particles of porcine circovirus type 2 capsid protein using ion-exchange monolith chromatography.

    Science.gov (United States)

    Zaveckas, Mindaugas; Snipaitis, Simas; Pesliakas, Henrikas; Nainys, Juozas; Gedvilaite, Alma

    2015-06-01

    Diseases associated with porcine circovirus type 2 (PCV2) infection are having a severe economic impact on swine-producing countries. The PCV2 capsid (Cap) protein expressed in eukaryotic systems self-assemble into virus-like particles (VLPs) which can serve as antigens for diagnostics or/and as vaccine candidates. In this work, conventional adsorbents as well as a monolithic support with large pore sizes were examined for the chromatographic purification of PCV2 Cap VLPs from clarified yeast lysate. Q Sepharose XL was used for the initial separation of VLPs from residual host nucleic acids and some host cell proteins. For the further purification of PCV2 Cap VLPs, SP Sepharose XL, Heparin Sepharose CL-6B and CIMmultus SO3 monolith were tested. VLPs were not retained on SP Sepharose XL. The purity of VLPs after chromatography on Heparin Sepharose CL-6B was only 4-7% and the recovery of VLPs was 5-7%. Using ion-exchange chromatography on the CIMmultus SO3 monolith, PCV2 Cap VLPs with the purity of about 40% were obtained. The recovery of VLPs after chromatography on the CIMmultus SO3 monolith was 15-18%. The self-assembly of purified PCV2 Cap protein into VLPs was confirmed by electron microscopy. Two-step chromatographic purification procedure of PCV2 Cap VLPs from yeast lysate was developed using Q Sepharose XL and cation-exchange CIMmultus SO3 monolith.

  1. A Medicago truncatula H+-pyrophosphatase gene, MtVP1, improves sucrose accumulation and anthocyanin biosynthesis in potato (Solanum tuberosum L.).

    Science.gov (United States)

    Wang, J W; Wang, H Q; Xiang, W W; Chai, T Y

    2014-05-09

    We recently cloned MtVP1, a type I vacuolar-type H(+)-translocating inorganic pyrophosphatase from Medicago truncatula. In the present study, we investigated the cellular location and the function of this H(+)-PPase in Arabidopsis and potato (Solanum tuberosum L.). An MtVP1::enhanced green fluorescent protein fusion was constructed, which localized to the plasma membrane of onion epidermal cells. Transgenic Arabidopsis thaliana overexpressing MtVP1 had more robust root systems and redder shoots than wild-type (WT) plants under conditions of cold stress. Furthermore, overexpression of MtVP1 in potato accelerated the formation and growth of vegetative organs. The tuber buds and stem base of transgenic potatoes became redder than those of WT plants, but flowering was delayed by approximately half a month. Interestingly, anthocyanin biosynthesis was promoted in transgenic Arabidopsis seedlings and potato tuber buds. The sucrose concentration of transgenic potato tubers and tuber buds was enhanced compared with that of WT plants. Furthermore, sucrose concentration in tubers was higher than that in tuber buds. Although there was no direct evidence to support Fuglsang's hypothetical model regarding the effects of H(+)-PPase on sucrose phloem loading, we speculated that sucrose concentration was increased in tuber buds owing to the increased concentration in tubers. Therefore, overexpressed MtVP1 enhanced sucrose accumulation of source organs, which might enhance sucrose transport to sink organs, thus affecting anthocyanin biosynthesis.

  2. Cyclin-dependent kinase 2 phosphorylates s/t-p sites in the hepadnavirus core protein C-terminal domain and is incorporated into viral capsids.

    Science.gov (United States)

    Ludgate, Laurie; Ning, Xiaojun; Nguyen, David H; Adams, Christina; Mentzer, Laura; Hu, Jianming

    2012-11-01

    Phosphorylation of the hepadnavirus core protein C-terminal domain (CTD) is important for viral RNA packaging, reverse transcription, and subcellular localization. Hepadnavirus capsids also package a cellular kinase. The identity of the host kinase that phosphorylates the core CTD or gets packaged remains to be resolved. In particular, both the human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) core CTDs harbor several conserved serine/threonine-proline (S/T-P) sites whose phosphorylation state is known to regulate CTD functions. We report here that the endogenous kinase in the HBV capsids was blocked by chemical inhibitors of the cyclin-dependent kinases (CDKs), in particular, CDK2 inhibitors. The kinase phosphorylated the HBV CTD at the serine-proline (S-P) sites. Furthermore, we were able to detect CDK2 in purified HBV capsids by immunoblotting. Purified CDK2 phosphorylated the S/T-P sites of the HBV and DHBV CTD in vitro. Inhibitors of CDKs, of CDK2 in particular, decreased both HBV and DHBV CTD phosphorylation in vivo. Moreover, CDK2 inhibitors blocked DHBV CTD phosphorylation, specifically at the S/T-P sites, in a mammalian cell lysate. These results indicate that cellular CDK2 phosphorylates the functionally critical S/T-P sites of the hepadnavirus core CTD and is incorporated into viral capsids.

  3. Functional and Structural Characterization of Novel Type of Linker Connecting Capsid and Nucleocapsid Protein Domains in Murine Leukemia Virus.

    Science.gov (United States)

    Doležal, Michal; Hadravová, Romana; Kožíšek, Milan; Bednárová, Lucie; Langerová, Hana; Ruml, Tomáš; Rumlová, Michaela

    2016-09-23

    The assembly of immature retroviral particles is initiated in the cytoplasm by the binding of the structural polyprotein precursor Gag with viral genomic RNA. The protein interactions necessary for assembly are mediated predominantly by the capsid (CA) and nucleocapsid (NC) domains, which have conserved structures. In contrast, the structural arrangement of the CA-NC connecting region differs between retroviral species. In HIV-1 and Rous sarcoma virus, this region forms a rod-like structure that separates the CA and NC domains, whereas in Mason-Pfizer monkey virus, this region is densely packed, thus holding the CA and NC domains in close proximity. Interestingly, the sequence connecting the CA and NC domains in gammaretroviruses, such as murine leukemia virus (MLV), is unique. The sequence is called a charged assembly helix (CAH) due to a high number of positively and negatively charged residues. Although both computational and deletion analyses suggested that the MLV CAH forms a helical conformation, no structural or biochemical data supporting this hypothesis have been published. Using an in vitro assembly assay, alanine scanning mutagenesis, and biophysical techniques (circular dichroism, NMR, microcalorimetry, and electrophoretic mobility shift assay), we have characterized the structure and function of the MLV CAH. We provide experimental evidence that the MLV CAH belongs to a group of charged, E(R/K)-rich, single α-helices. This is the first single α-helix motif identified in viral proteins.

  4. Sphingomyelin induces structural alteration in canine parvovirus capsid.

    Science.gov (United States)

    Pakkanen, Kirsi; Karttunen, Jenni; Virtanen, Salla; Vuento, Matti

    2008-03-01

    One of the essential steps in canine parvovirus (CPV) infection, the release from endosomal vesicles, is dominated by interactions between the virus capsid and the endosomal membranes. In this study, the effect of sphingomyelin and phosphatidyl serine on canine parvovirus capsid and on the phospholipase A(2) (PLA(2)) activity of CPV VP1 unique N-terminus was analyzed. Accordingly, a significant (P< or =0.05) shift of tryptophan fluorescence emission peak was detected at pH 5.5 in the presence of sphingomyelin, whereas at pH 7.4 a similar but minor shift was observed. This effect may relate to the exposure of VP1 N-terminus in acidic pH as well as to interactions between sphingomyelin and CPV. When the phenomenon was further characterized using circular dichroism spectroscopy, differences in CPV capsid CD spectra with and without sphingomyelin and phosphatidyl serine were detected, corresponding to data obtained with tryptophan fluorescence. However, when the enzymatic activity of CPV PLA(2) was tested in the presence of sphingomyelin, no significant effect in the function of the enzyme was detected. Thus, the structural changes observed with spectroscopic techniques appear not to manipulate the activity of CPV PLA(2), and may therefore implicate alternative interactions between CPV capsid and sphingomyelin.

  5. Phylogenetic Diversity of Marine Cyanophage Isolates and Natural Virus Communities as Revealed by Sequences of Viral Capsid Assembly Protein Gene g20†

    OpenAIRE

    Zhong, Yan; Chen, Feng; Wilhelm, Steven W.; Poorvin, Leo; Hodson, Robert E.

    2002-01-01

    In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to specifically amplify ca. 592-bp fragments of the gene for viral capsid assembly protein g20. Phylogenetic analysis of isolated cyanophages revealed that the marine cyanophages were highly diverse yet more closely related to each other than to enteric coliphage T4. Genetically related marine cyanoph...

  6. Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore.

    Directory of Open Access Journals (Sweden)

    Anusha Panjwani

    2014-08-01

    Full Text Available Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

  7. Phylogenetic distribution of the capsid assembly protein gene (g20 of cyanophages in paddy floodwaters in Northeast China.

    Directory of Open Access Journals (Sweden)

    Ruiyong Jing

    Full Text Available Numerous studies have revealed the high diversity of cyanophages in marine and freshwater environments, but little is currently known about the diversity of cyanophages in paddy fields, particularly in Northeast (NE China. To elucidate the genetic diversity of cyanophages in paddy floodwaters in NE China, viral capsid assembly protein gene (g20 sequences from five floodwater samples were amplified with the primers CPS1 and CPS8. Denaturing gradient gel electrophoresis (DGGE was applied to distinguish different g20 clones. In total, 54 clones differing in g20 nucleotide sequences were obtained in this study. Phylogenetic analysis showed that the distribution of g20 sequences in this study was different from that in Japanese paddy fields, and all the sequences were grouped into Clusters α, β, γ and ε. Within Clusters α and β, three new small clusters (PFW-VII∼-IX were identified. UniFrac analysis of g20 clone assemblages demonstrated that the community compositions of cyanophage varied among marine, lake and paddy field environments. In paddy floodwater, community compositions of cyanophage were also different between NE China and Japan.

  8. [Genetic diversity of capsid assembly protein genes (g20) of cyanophage in different natural environment--a review].

    Science.gov (United States)

    Jing, Ruiyong; Kimura, Makoto; Wang, Guanghua

    2013-11-04

    With the development of molecular biological techniques and progress of sequencing virus genome, scientists pay great attentions to the genetic diversity of viruses, which are ubiquitous and abundant in natural environments. So far, no universal genetic marker, analogous to 16S rDNA and 18S rDNA used for microbial communities exists throughout all viruses. However, some family-specific genes encoding conserved amino acids have been proposed for the evaluation of phage diversity and a series of breakthrough achievements were obtained. In this paper, we targeted the capsid assembly protein genes (g20) of cyanophages and reviewed the recent progress on their genetic diversity in natural environments of marines, lakes and paddy fields and discussed the relationship between distribution of g20 gene of cyanophages and its environments. Those studies showed that the distribution of g20 gene varied with environments and many unique clusters were found in different natural environment. In final, several research issues and the future research tendencies for the study of environmental g20 gene were also addressed in this paper.

  9. Induction of mucosal immunity by intranasal immunization with recombinant adenovirus expressing major epitopes of Porcine circovirus-2 capsid protein.

    Science.gov (United States)

    Liu, Yu-feng; Guo, Quan-hai; Chen, Lu; Zhao, Jun; Chang, Hong-tao; Wang, Xin-wei; Yang, Xia; Wang, Chuan-qing

    2013-07-15

    Porcine circovirus-2 (PCV-2) is primarily transmitted through mucosa, thus the mucosal immunity may constitute an essential feature of vaccination strategies against PCV-2 infection. Mucosal immunity elicited by recombinant replication-deficient adenovirus expressing the major epitopes of PCV-2 capsid protein (rAd/Cap/518) via intranasal (i.n.), intramuscular (i.m.) or oral routes in mice were evaluated. Immunization with rAd/Cap/518 via i.n. route induced higher titers of IgA in saliva, bronchoalveolar and intestinal lavage fluid compared with those immunized via i.m. route. The proportions of CD3+, CD3+CD4+ and CD3+CD8+ T cells were significantly increased in mice immunized with rAd/Cap/518 via i.n. route compared with the control group. Higher levels of IFN-γ were detected in the spleen and mesenteric lymph nodes of mice immunized with rAd/Cap/518 via i.n. route compared with other groups, yet IL-4 was not detected in any group. Real-time PCR analysis confirmed viral DNA loads in the i.m. or i.n. immunization group was lower than that seen in the rAd immunization. These results indicate that i.n. administration of rAd/Cap/518 can elicit humoral and Th1-type cellular protective immunity in both systemic and mucosal immune compartments in mice, representing a promising mucosal vaccine candidate against PCV-2.

  10. High-Resolution X-Ray Structure and Functional Analysis of the Murine Norovirus 1 Capsid Protein Protruding Domain

    Energy Technology Data Exchange (ETDEWEB)

    Taube, Stefan; Rubin, John R.; Katpally, Umesh; Smith, Thomas J.; Kendall, Ann; Stuckey, Jeanne A.; Wobus, Christiane E. (Michigan); (Danforth)

    2010-07-23

    Murine noroviruses (MNV) are closely related to the human noroviruses (HuNoV), which cause the majority of nonbacterial gastroenteritis. Unlike HuNoV, MNV grow in culture and in a small-animal model that represents a tractable model to study norovirus biology. To begin a detailed investigation of molecular events that occur during norovirus binding to cells, the crystallographic structure of the murine norovirus 1 (MNV-1) capsid protein protruding (P) domain has been determined. Crystallization of the bacterially expressed protein yielded two different crystal forms (Protein Data Bank identifiers [PDB ID], 3LQ6 and 3LQE). Comparison of the structures indicated a large degree of structural mobility in loops on the surface of the P2 subdomain. Specifically, the A{prime}-B{prime} and E{prime}-F{prime} loops were found in open and closed conformations. These regions of high mobility include the known escape mutation site for the neutralizing antibody A6.2 and an attenuation mutation site, which arose after serial passaging in culture and led to a loss in lethality in STAT1{sup -/-} mice, respectively. Modeling of a Fab fragment and crystal structures of the P dimer into the cryoelectron microscopy three-dimensional (3D) image reconstruction of the A6.2/MNV-1 complex indicated that the closed conformation is most likely bound to the Fab fragment and that the antibody contact is localized to the A{prime}-B{prime} and E{prime}-F{prime} loops. Therefore, we hypothesize that these loop regions and the flexibility of the P domains play important roles during MNV-1 binding to the cell surface.

  11. Cloning and expression of a truncated pigeon circovirus capsid protein suitable for antibody detection in infected pigeons.

    Science.gov (United States)

    Daum, Iris; Finsterbusch, Tim; Härtle, Stefan; Göbel, Thomas W; Mankertz, Annette; Korbel, Rüdiger; Grund, Christian

    2009-04-01

    Infections with pigeon circovirus (PiCV) (also termed columbid circovirus) occur in meat and racing pigeons (Columba livia) of all ages and have been reported worldwide. A PiCV infection is associated with immunosuppression and the development of young pigeon disease syndrome. An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of virus-specific serum antibody was developed for research purposes. In the absence of a method to propagate PiCV in cell culture, the assay was based on a recombinant truncated capsid protein (rCapPiCV) produced by overexpression in Escherichia coli. A 6xHis-Tag was fused to the N-terminus of the protein to facilitate purification by metal affinity chromatography and detection by anti-His antibody. PiCV-negative and PiCV-positive control sera were generated by inoculation of pigeons with tissue homogenate containing PiCV, followed by five weekly blood sample collections. Western blotting of the immune serum revealed a specific protein band of approximately 32 kDa, which was absent in the pre-immune sera. Using rCapPiCV as antigen in an indirect ELISA, PiCV-specific antibody was detected in sera of the experimentally PiCV-infected pigeons collected at 1 to 5 weeks post infection. By testing 118 field sera collected in the years 1989, 1991, 1994 and 2008 in the rCapPiCV ELISA, virus-specific antibody was detected in 89 (75%) of the sera. The results obtained demonstrate that the rCapPiCV-based indirect ELISA is able to detect PiCV-specific antibodies in pigeon sera and may be a useful tool for PiCV serodiagnosis.

  12. Cloning and Prokaryotic Expression of VP1 Gene of Foot-and-Mouth Disease Virus (FMDV) Type O%O型口蹄疫病毒结构蛋白基因VP1的克隆与原核表达

    Institute of Scientific and Technical Information of China (English)

    付薇; 陈磊; 熊毅; 潘琼; 王常伟; 陈进喜; 胡晓静; 刘棋

    2008-01-01

    According to the complete genome of foot-and-mouth disease virus (FMDV) type O, a pair of special primers was designed to amplify VP1 gene. The VP1 gene was amplified by RT-PCR and subsequently inserted into the expression vector pGEX-6p-1 and induced by IPTG. Then SDS-PAGE showed the expressed protein was 51 kD in molecular weight. Then the product was purified by GSTrap FF columns. The product was detected through Western-blot that showed the protein has antigenicity. It provided fundamental data and materials for further investigation on diagnosis method of FMDV.

  13. Cyclophilin A associates with enterovirus-71 virus capsid and plays an essential role in viral infection as an uncoating regulator.

    Directory of Open Access Journals (Sweden)

    Jie Qing

    2014-10-01

    Full Text Available Viruses utilize host factors for their efficient proliferation. By evaluating the inhibitory effects of compounds in our library, we identified inhibitors of cyclophilin A (CypA, a known immunosuppressor with peptidyl-prolyl cis-trans isomerase activity, can significantly attenuate EV71 proliferation. We demonstrated that CypA played an essential role in EV71 entry and that the RNA interference-mediated reduction of endogenous CypA expression led to decreased EV71 multiplication. We further revealed that CypA directly interacted with and modified the conformation of H-I loop of the VP1 protein in EV71 capsid, and thus regulated the uncoating process of EV71 entry step in a pH-dependent manner. Our results aid in the understanding of how host factors influence EV71 life cycle and provide new potential targets for developing antiviral agents against EV71 infection.

  14. Selective human enterovirus and rhinovirus inhibitors: An overview of capsid-binding and protease-inhibiting molecules.

    Science.gov (United States)

    Shih, Shin-Ru; Chen, Shu-Jen; Hakimelahi, Gholam Hossein; Liu, Hsing-Jang; Tseng, Chen-Tso; Shia, Kak-Shan

    2004-07-01

    The absence of effective vaccines for most viral infections highlights an urgent necessity for the design and development of effective antiviral drugs. Due to the advancement in virology since the late 1980s, several key events in the viral life cycle have been well delineated and a number of molecular targets have been validated, culminating in the emergence of many new antiviral drugs in recent years. Inhibitors against enteroviruses and rhinoviruses, responsible for about half of the human common colds, are currently under active investigation. Agents targeted at either viral protein 1 (VP1), a relatively conserved capsid structure mediating viral adsorption/uncoating process, or 3C protease, which is highly conserved among different serotypes and essential for viral replication, are of great potential to become antipicornavirus drugs.

  15. A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

    Science.gov (United States)

    Kang, Sung-Hwan; Qu, Feng; Morris, T Jack

    2015-12-01

    The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV.

  16. Production of recombinant capsid protein of Macrobrachium rosenbergii nodavirus (r-MCP43) of giant freshwater prawn, M. rosenbergii (de Man) for immunological diagnostic methods.

    Science.gov (United States)

    Farook, M A; Madan, N; Taju, G; Majeed, S Abdul; Nambi, K S N; Raj, N Sundar; Vimal, S; Hameed, A S Sahul

    2014-08-01

    White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) is a serious problem in prawn hatcheries. The gene for capsid protein of MrNV (MCP43) was cloned into pRSET B expression vector. The MCP43 protein was expressed as a protein with a 6-histidine tag in Escherichia coli GJ1158 with NaCl induction. This recombinant protein, which was used to raise the antiserum in rabbits, recognized capsid protein in different WTD-infected post-larvae and adult prawn. Various immunological methods such as Western blot, dot blot and ELISA techniques were employed to detect MrNV in infected samples using the antiserum raised against recombinant MCP43 of MrNV. The dot blot assay using anti-rMCP43 was found to be capable of detecting MrNV in WTD-infected post-larvae as early as at 24 h post-infection. The antiserum raised against r-MCP43 could detect the MrNV in the infected samples at the level of 100 pg of total protein. The capsid protein of MrNV estimated by ELISA using anti-rMCP43 and pure r-MCP43 as a standard was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. The results of immunological diagnostic methods employed in this study were compared with that of RT-PCR to test the efficiency of antiserum raised against r-MCP43 for the detection of MrNV. The Western blot, dot blot and ELISA detected all MrNV-positive coded samples as detected by RT-PCR.

  17. High level expression of the capsid protein of hepatitis E virus in diverse eukaryotic cells using the Semliki Forest virus replicon.

    Science.gov (United States)

    Torresi, J; Meanger, J; Lambert, P; Li, F; Locarnini, S A; Anderson, D A

    1997-12-01

    The capsid protein of hepatitis E virus (HEV) is encoded by open reading frame 2 (ORF 2) and exhibits variable processing when expressed in insect and COS cells, but nothing is known of its processing in cells relevant to its replication. The full-length ORF 2 protein was expressed at high levels in mammalian cells by insertion of ORF 2 in the Semliki Forest virus (SFV) replicon to generate rSFV/HEV ORF 2K. Expression of the capsid protein was detected readily by metabolic labelling and indirect immunofluorescence in BHK-21 cells transfected with RNA transcripts derived from rSFV/HEV ORF 2K. ORF 2 protein was also expressed at high levels in cells of diverse origin, including liver-derived cell lines Huh7 and HepG2, following infection with recombinant virus derived from cotransfection of BHK-21 cells with the rSFV/HEV ORF 2K and helper SFV replicon RNAs. The addition of hypertonic KCl during metabolic labelling reduced the level of host cell protein synthesis and enhanced the detection of intermediates in ORF 2 protein processing. The wide host range and high level expression directed by SFV replicon particles has particular utility in the analysis of cell-specific factors in the protein processing and assembly of non-cultivable viruses such as HEV.

  18. Absolute quantification of norovirus capsid protein in food, water, and soil using synthetic peptides with electrospray and MALDI mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Hartmann, Erica M. [Center for Environmental Security and Security Defense Systems Initiative, The Biodesign Institute, Arizona State University, 781 E. Terrace Mall, Tempe, AZ 85287-5904 (United States); Colquhoun, David R.; Schwab, Kellogg J. [Department of Environmental Health Sciences, The Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205 (United States); Halden, Rolf U., E-mail: halden@asu.edu [Center for Environmental Security and Security Defense Systems Initiative, The Biodesign Institute, Arizona State University, 781 E. Terrace Mall, Tempe, AZ 85287-5904 (United States); Department of Environmental Health Sciences, The Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205 (United States)

    2015-04-09

    Highlights: • Mass spectrometry-based methods for norovirus quantification are developed. • Absolute quantification is achieved using internal heavy isotope-labeled standards. • A single labeled peptide serves in two distinct detection strategies. • These methods are validated for food, water, and soil analysis. • MS-based detection limits are lowered by two orders of magnitude. - Abstract: Norovirus infections are one of the most prominent public health problems of microbial origin in the U.S. and other industrialized countries. Surveillance is necessary to prevent secondary infection, confirm successful cleanup after outbreaks, and track the causative agent. Quantitative mass spectrometry, based on absolute quantitation with stable-isotope labeled peptides, is a promising tool for norovirus monitoring because of its speed, sensitivity, and robustness in the face of environmental inhibitors. In the current study, we present two new methods for the detection of the norovirus genogroup I capsid protein using electrospray and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The peptide TLDPIEVPLEDVR was used to quantify norovirus-like particles down to 500 attomoles with electrospray and 100 attomoles with MALDI. With MALDI, we also demonstrate a detection limit of 1 femtomole and a quantitative dynamic range of 5 orders of magnitude in the presence of an environmental matrix effect. Due to the rapid processing time and applicability to a wide range of environmental sample types (bacterial lysate, produce, milk, soil, and groundwater), mass spectrometry-based absolute quantitation has a strong potential for use in public health and environmental sciences.

  19. Capsid protein oxidation in feline calicivirus using an electrochemical inactivation treatment

    Energy Technology Data Exchange (ETDEWEB)

    Shionoiri, Nozomi; Nogariya, Osamu; Tanaka, Masayoshi; Matsunaga, Tadashi; Tanaka, Tsuyoshi, E-mail: tsuyo@cc.tuat.ac.jp

    2015-02-11

    Highlights: • Feline calicivirus was inactivated electrochemically by a factor of >5 log. • The electrochemical treatment was performed at 0.9 V (vs. Ag/AgCl) for 15 min. • Electrochemical treatment caused oxidation of viral proteins. • Oxidation of viral proteins can lead to loss of viral structural integrity. - Abstract: Pathogenic viral infections are an international public health concern, and viral disinfection has received increasing attention. Electrochemical treatment has been used for treatment of water contaminated by bacteria for several decades, and although in recent years several reports have investigated viral inactivation kinetics, the mode of action of viral inactivation by electrochemical treatment remains unclear. Here, we demonstrated the inactivation of feline calicivirus (FCV), a surrogate for human noroviruses, by electrochemical treatment in a developed flow-cell equipped with a screen-printed electrode. The viral infectivity titer was reduced by over 5 orders of magnitude after 15 min of treatment at 0.9 V vs. Ag/AgCl. Proteomic study of electrochemically inactivated virus revealed oxidation of peptides located in the viral particles; oxidation was not observed in the non-treated sample. Furthermore, transmission electron microscopy revealed that viral particles in the treated sample had irregular structures. These results suggest that electrochemical treatment inactivates FCV via oxidation of peptides in the structural region, causing structural deformation of virus particles. This first report of viral protein damage through electrochemical treatment will contribute to broadening the understanding of viral inactivation mechanisms.

  20. Nuclear export and import of human hepatitis B virus capsid protein and particles.

    Directory of Open Access Journals (Sweden)

    Hung-Cheng Li

    Full Text Available It remains unclear what determines the subcellular localization of hepatitis B virus (HBV core protein (HBc and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS, while ARD-II and ARD-IV behave like two independent nuclear export signals (NES. This conclusion is based on five independent lines of experimental evidence: i Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT. iii By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1, which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel

  1. Magnetic Resonance Imaging Revealed Splenic Targeting of Canine Parvovirus Capsid Protein VP2

    Science.gov (United States)

    Ma, Yufei; Wang, Haiming; Yan, Dan; Wei, Yanquan; Cao, Yuhua; Yi, Peiwei; Zhang, Hailu; Deng, Zongwu; Dai, Jianwu; Liu, Xiangtao; Luo, Jianxun; Zhang, Zhijun; Sun, Shiqi; Guo, Huichen

    2016-03-01

    Canine parvovirus (CPV) is a highly contagious infectious virus, whose infectious mechanism remains unclear because of acute gastroenteritis and the lack of an efficient tool to visualize the virus in real time during virology research. In this study, we developed an iron oxide nanoparticle supported by graphene quantum dots (GQD), namely, FeGQD. In this composite material, GQD acts as a stabilizer; thus, vacancies are retained on the surface for further physical adsorption of the CPV VP2 protein. The FeGQD@VP2 nanocomposite product showed largely enhanced colloidal stability in comparison with bare FeGQD, as well as negligible toxicity both in vitro and in vivo. The composite displayed high uptake into transferrin receptor (TfR) positive cells, which are distinguishable from FeGQD or TfR negative cells. In addition, the composite developed a significant accumulation in spleen rather than in liver, where bare FeGQD or most iron oxide nanoparticles gather. As these evident targeting abilities of FeGQD@VP2 strongly suggested, the biological activity of CPV VP2 was retained in our study, and its biological functions might correspond to CPV when the rare splenic targeting ability is considered. This approach can be applied to numerous other biomedical studies that require a simple yet efficient approach to track proteins in vivo while retaining biological function and may facilitate virus-related research.

  2. Exploiting the yeast L-A viral capsid for the in vivo assembly of chimeric VLPs as platform in vaccine development and foreign protein expression.

    Directory of Open Access Journals (Sweden)

    Frank Powilleit

    Full Text Available A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8(+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i a heterologous model protein (GFP, (ii a per se toxic protein (K28 alpha-subunit, and (iii a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A. Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.

  3. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice.

    Science.gov (United States)

    Valdés, Iris; Bernardo, Lidice; Gil, Lázaro; Pavón, Alekis; Lazo, Laura; López, Carlos; Romero, Yaremis; Menendez, Ivón; Falcón, Viviana; Betancourt, Lázaro; Martín, Jorge; Chinea, Glay; Silva, Ricardo; Guzmán, María G; Guillén, Gerardo; Hermida, Lisset

    2009-11-25

    Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-gamma secretion and protection experiments, mediated by CD4(+) and CD8(+) cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

  4. Cell culture adaptation mutations in foot-and-mouth disease virus serotype A capsid proteins: implications for receptor interactions

    Science.gov (United States)

    In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2), consistently gained several positively charged amino acids...

  5. Essential role of the unordered VP2 n-terminal domain of the parvovirus MVM capsid in nuclear assembly and endosomal enlargement of the virion fivefold channel for cell entry

    Energy Technology Data Exchange (ETDEWEB)

    Sanchez-Martinez, Cristina; Grueso, Esther [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Universidad Autonoma de Madrid, 28049 Cantoblanco, Madrid (Spain); Carroll, Miles [Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury SP4 OJG, Wilts (United Kingdom); Rommelaere, Jean [Deutsches Krebsforschungszentrum Division F010, Im Neuenheimer Feld 242, D-69120 Heidelberg (Germany); Almendral, Jose M., E-mail: jmalmendral@cbm.uam.es [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Universidad Autonoma de Madrid, 28049 Cantoblanco, Madrid (Spain)

    2012-10-10

    The unordered N-termini of parvovirus capsid proteins (Nt) are translocated through a channel at the icosahedral five-fold axis to serve for virus traffick. Heterologous peptides were genetically inserted at the Nt of MVM to study their functional tolerance to manipulations. Insertion of a 5T4-single-chain antibody at VP2-Nt (2Nt) yielded chimeric capsid subunits failing to enter the nucleus. The VEGFR2-binding peptide (V1) inserted at both 2Nt and VP1-Nt efficiently assembled in virions, but V1 disrupted VP1 and VP2 entry functions. The VP2 defect correlated with restricted externalization of V1-2Nt out of the coat. The specific infectivity of MVM and wtVP-pseudotyped mosaic MVM-V1 virions, upon heating and/or partial 2Nt cleavage, demonstrated that some 2Nt domains become intracellularly translocated out of the virus shell and cleaved to initiate entry. The V1 insertion defines a VP2-driven endosomal enlargement of the channel as an essential structural rearrangement performed by the MVM virion to infect.

  6. Genetic analysis of foot-and-mouth disease virus serotype A of Indian origin and detection of positive selection and recombination in leader protease- and capsid-coding regions

    Indian Academy of Sciences (India)

    S B Nagendrakumar; M Madhanmohan; P N Rangarajan; V A Srinivasan

    2009-03-01

    The leader protease (Lpro) and capsid-coding sequences (P1) constitute approximately 3 kb of the foot-and-mouth disease virus (FMDV). We studied the phylogenetic relationship of 46 FMDV serotype A isolates of Indian origin collected during the period 1968–2005 and also eight vaccine strains using the neighbour-joining tree and Bayesian tree methods. The viruses were categorized under three major groups – Asian, Euro-South American and European. The Indian isolates formed a distinct genetic group among the Asian isolates. The Indian isolates were further classified into different genetic subgroups (< 5% divergence). Post-1995 isolates were divided into two subgroups while a few isolates which originated in the year 2005 from Andhra Pradesh formed a separate group. These isolates were closely related to the isolates of the 1970s. The FMDV isolates seem to undergo reverse mutation or convergent evolution wherein sequences identical to the ancestors are present in the isolates in circulation. The eight vaccine strains included in the study were not related to each other and belonged to different genetic groups. Recombination was detected in the Lpro region in one isolate (A IND 20/82) and in the VP1 coding 1D region in another isolate (A RAJ 21/96). Positive selection was identified at aa positions 23 in the Lpro ( < 0.05; 0.046*) and at aa 171 in the capsid protein VP1 ( < 0.01; 0.003**).

  7. Construction and Identification of Bait Vectors with VP1 Gene of Encephalomyocarditis Virus in Yeast Two-hybrid System%脑心肌炎病毒VP1蛋白酵母双杂交诱饵载体的构建及验证

    Institute of Scientific and Technical Information of China (English)

    谢晶莹; 冯若飞; 徐雷; 张海霞; 李向茸; 侯兰新; 马忠仁

    2015-01-01

    为筛选与脑心肌炎病毒VP1蛋白相互作用的靶细胞cDNA文库蛋白,构建VP1蛋白的诱饵载体pDHB1-VP1。扩增EMCV的VP1基因并克隆至pMD18-T载体中,经测序验证正确后定向克隆至酵母双杂交诱饵载体pDHB1。将重组pDHB1-VP1载体进行酶切验证和测序分析,并转化酵母报告菌株NMY51,检测其在酵母细胞中有无表达和自激活作用。结果表明,构建的pDHB1-VP1基因可以在酵母细胞中正确表达,产物大小约66 kD,而且可以与兔抗EMCV血清发生特异性结合,有较好免疫原性。成功构建了诱饵载体pDHB1-VP1,可以在酵母细胞中表达且其对报告基因无自激活作用,可以应用于酵母双杂交筛选试验中。%Bait vector pDHB1-VP1 was constructed for screening cellular proteins interacting with VP1 protein of encephalomyocarditis virus from yeast two-hybrid cDNA library of target cells in this study. VP1 gene was amplified and cloned into pMD18-T vector. After being verified by sequencing, it was directional cloning into bait vector pDHB1 of yeast two-hybrid system. Then the recombinant plasmid was identified by enzyme digestion and sequencing and transformed into yeast cells NMY51.The bait vectors’ expression and self-activation to reporter genes were tested.The results showed that the bait plasmid pDHB1-VP1 could express in yeast cells, and product size was 66 kD. It was specific binding with rabbit anti EMCV serum, which showed better immunogenicity. Bait plasmid pDHB1-VP1 was successfully constructed, could express in yeast cells and proved to be no self-activation to reporter genes.It could be used in the yeast two-hybrid system screening test.

  8. Development of a subunit vaccine containing recombinant chicken anemia virus VP1 and pigeon IFN-γ.

    Science.gov (United States)

    Shen, Sin Ying; Chang, Wei Chun; Yi, Hsiang Heng; Tsai, Shinn-Shong; Liu, Hung Jen; Liao, Pei-Chun; Chuang, Kuo Pin

    2015-10-15

    Chicken anemia virus (CAV) is a severe threat to the chicken industry and causes heavy economic losses worldwide. In this study, we evaluated the immune response and protective efficacy provided by a subunit vaccine containing recombinant VP1 (rVP1) and pigeon interferon-γ (rPiIFN-γ). Results indicated that rPiIFN-γ enhanced humoral immunity elicited by rVP1 as early as 10 day after primary immunization and reach the high titer after secondary immunization. When compared to chickens immunized with rVP1, inactivated vaccine, chickens immunized with rVP1+rPiIFN-γ showed faster and higher levels (pvaccine prevent the reducing of hematocrit values in comparison with the rVP1 or inactivated groups. The relative fold inductions of mRNA expression of Th1-type (IFN-γ), but not Th2-type (IL-4) cytokines in splenocytes isolated from chickens immunized with rVP1+rPiIFN-γ were significantly higher than those of the rVP1 or inactivated vaccine groups. In conclusion, our study found that rPiIFN-γ can enhance both humoral and cellular immunity elicited by an rVP1 vaccine. The rVP1+rPiIFN-γ vaccine may provide a new strategy vaccine against CAV in chicken.

  9. Phase transfer based synthesis and thermophysical properties of Au/Therminol VP-1 nanofluids

    Institute of Scientific and Technical Information of China (English)

    Caixia Wang; Jun Yang; Yulong Ding

    2013-01-01

    This paper reports a phase transfer based wet chemistry method for the preparation of Au/VP-1 nanofluids. The method involves the transfer of AuCl4- ions from water to the base liquid Therminol VP-1, followed by the reduction of AuCl4- ions using NaBH4. The prepared nanofluids are characterized for their thermophysical properties and stability. The results show that the mass concentration of Au particles has a significant effect on the stability of Au/VP-1 nanofluids. An increase in the Au concentration results in a higher extent of agglomeration among the particles, leading to a decrease in the nanofluid stability. The results also show that the introduction of 0.005-0.05%Au nanoparticles enhances the thermal conductivity of the fluids by up to 6.5%, whereas the viscosity increase is minimal.

  10. Large-scale functional purification of recombinant HIV-1 capsid.

    Directory of Open Access Journals (Sweden)

    Magdeleine Hung

    Full Text Available During human immunodeficiency virus type-1 (HIV-1 virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents.

  11. Vesicular stomatitis virus replicon expressing the VP2 outer capsid protein of bluetongue virus serotype 8 induces complete protection of sheep against challenge infection.

    Science.gov (United States)

    Kochinger, Stefanie; Renevey, Nathalie; Hofmann, Martin A; Zimmer, Gert

    2014-06-13

    Bluetongue virus (BTV) is an arthropod-borne pathogen that causes an often fatal, hemorrhagic disease in ruminants. Different BTV serotypes occur throughout many temperate and tropical regions of the world. In 2006, BTV serotype 8 (BTV-8) emerged in Central and Northern Europe for the first time. Although this outbreak was eventually controlled using inactivated virus vaccines, the epidemic caused significant economic losses not only from the disease in livestock but also from trade restrictions. To date, BTV vaccines that allow simple serological discrimination of infected and vaccinated animals (DIVA) have not been approved for use in livestock. In this study, we generated recombinant RNA replicon particles based on single-cycle vesicular stomatitis virus (VSV) vectors. Immunization of sheep with infectious VSV replicon particles expressing the outer capsid VP2 protein of BTV-8 resulted in induction of BTV-8 serotype-specific neutralizing antibodies. After challenge with a virulent BTV-8 strain, the vaccinated animals neither developed signs of disease nor showed viremia. In contrast, immunization of sheep with recombinant VP5 - the second outer capsid protein of BTV - did not confer protection. Discrimination of infected from vaccinated animals was readily achieved using an ELISA for detection of antibodies against the VP7 antigen. These data indicate that VSV replicon particles potentially represent a safe and efficacious vaccine platform with which to control future outbreaks by BTV-8 or other serotypes, especially in previously non-endemic regions where discrimination between vaccinated and infected animals is crucial.

  12. Vaccination of horses with a recombinant modified vaccinia Ankara virus (MVA) expressing African horse sickness (AHS) virus major capsid protein VP2 provides complete clinical protection against challenge.

    Science.gov (United States)

    Alberca, Berta; Bachanek-Bankowska, Katarzyna; Cabana, Marta; Calvo-Pinilla, Eva; Viaplana, Elisenda; Frost, Lorraine; Gubbins, Simon; Urniza, Alicia; Mertens, Peter; Castillo-Olivares, Javier

    2014-06-17

    African horse sickness virus (AHSV) is an arthropod-borne pathogen that infects all species of equidae and causes high mortality in horses. Previously, a recombinant modified vaccinia Ankara (MVA) virus expressing the protein VP2 of AHSV serotype 4 was shown to induce virus neutralising antibodies in horses and protected interferon alpha receptor gene knock-out mice (IFNAR -/-) against virulent AHSV challenge. This study builds on the previous work, examining the protective efficacy of MVA-VP2 vaccination in the natural host of AHSV infection. A study group of 4 horses was vaccinated twice with a recombinant MVA virus expressing the major capsid protein (VP2) of AHSV serotype 9. Vaccinated animals and a control group of unvaccinated horses were then challenged with a virulent strain of AHSV-9. The vaccinated animals were completely protected against clinical disease and also against viraemia as measured by standard end-point dilution assays. In contrast, all control horses presented viraemia after challenge and succumbed to the infection. These results demonstrate the potential of recombinant MVA viruses expressing the outer capsid VP2 of AHSV as a protective vaccine against AHSV infection in the field.

  13. [Functional analysis of promoters of Vibrio cholerea typing phage VP1 with reporter system].

    Science.gov (United States)

    Li, Yan-Ping; Liang, Wei-Li; Wang, Duo-Chun; Qi, Guo-Ming; Kan, Biao; Gao, Shou-Yi; Liu, Yan-Qing

    2005-12-01

    Phage VP1 infects and lyses Vibrio cholerae. The VP1 genome is a circular double-strand DNA and its size is 32176 base pairs. Analysis of the sequence of the VP1 genome revealed the presence of 15 putative promoter sequence. The activities of these putative promoters in V. cholerae were assayed by transformation of reporter gene plasmid and phage infection together. Promoter regions were ligated into pRS1274/BamH I/EcoR I. Then transformed into E. coli JM109 and all of clone display blue. The recombinant plasmids were transformed into V. cholerae 7743 deltaZ by electroporation, then bacteriophage VP1 infect transformant. The time-course expressing lacZ gene and detecting change of beta-galactosidase enzyme activity in V. cholerae transformants at latent period, indicated P17 probably is a early promoter; P2 and P3 and P9 etc are medium-term promoters; P18 is a late promoter.

  14. Antimicrobial properties of a lipid interactive -helical peptide VP1 against Staphylococcus aureus bacteria

    OpenAIRE

    Dennison, Sarah R.; Morton, Leslie H.G.; Harris, Frederick; Phoenix, David A.

    2007-01-01

    Antimicrobial properties of a lipid interactive -helical peptide VP1 against Staphylococcus aureus bacteria correspondance: Corresponding author. Tel: +44 1772 893481; fax: +44 1772 894981. (Phoenix, David A.) (Phoenix, David A.) Faculty of Science and Technology--> , University of Central Lancashire--> , Preston PR1 2HE--> - UNITED KINGDOM (Dennison, Sarah R) Department of Forensic and Inve...

  15. Characterization of the banana streak virus capsid protein and mapping of the immunodominant continuous B-cell epitopes to the surface-exposed N terminus.

    Science.gov (United States)

    Vo, Jenny N; Campbell, Paul R; Mahfuz, Nur N; Ramli, Ras; Pagendam, Daniel; Barnard, Ross; Geering, Andrew D W

    2016-12-01

    This study identified the structural proteins of two badnavirus species, Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV), and mapped the distribution of continuous B-cell epitopes. Two different capsid protein (CP) isoforms of about 44 and 40 kDa (CP1 and CP2) and the virion-associated protein (VAP) were consistently associated with purified virions. For both viral species, the N terminus of CP2 was successfully sequenced by Edman degradation but that of CP1 was chemically blocked. De novo peptide sequencing of tryptic digests suggested that CP1 and CP2 derive from the same region of the P3 polyprotein but differ in the length of either the N or the C terminus. A three-dimensional model of the BSMYV-CP was constructed, which showed that the CP is a multi-domain structure, containing homologues of the retroviral capsid and nucleocapsid proteins, as well as a third, intrinsically disordered protein region at the N terminus, henceforth called the NID domain. Using the Pepscan approach, the immunodominant continuous epitopes were mapped to the NID domain for five different species of banana streak virus. Anti-peptide antibodies raised against these epitopes in BSMYV were successfully used for detection of native virions and denatured CPs in serological assays. Immunoelectron microscopy analysis of the virion surface using the anti-peptide antibodies confirmed that the NID domain is exposed on the surface of virions, and that the difference in mass of the two CP isoforms is due to variation in length of the NID domain.

  16. Crystallization and X-ray analysis of the T = 4 particle of hepatitis B capsid protein with an N-terminal extension

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Wen Siang [Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); McNae, Iain W.; Ho, Kok Lian; Walkinshaw, Malcolm D., E-mail: m.walkinshaw@ed.ac.uk [Institute of Structural and Molecular Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, King’s Buildings, Mayfield Road, Edinburgh EH9 3JR,Scotland (United Kingdom); Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia)

    2007-08-01

    Hepatitis B virus capsids have significant potential as carriers for immunogenic peptides. The crystal structure of the T = 4 particle of hepatitis B core protein containing an N-terminal extension reveals that the fusion peptide is exposed on the exterior of the particle. Hepatitis B core (HBc) particles have been extensively exploited as carriers for foreign immunological epitopes in the development of multicomponent vaccines and diagnostic reagents. Crystals of the T = 4 HBc particle were grown in PEG 20 000, ammonium sulfate and various types of alcohols. A temperature jump from 277 or 283 to 290 K was found to enhance crystal growth. A crystal grown using MPD as a cryoprotectant diffracted X-rays to 7.7 Å resolution and data were collected to 99.6% completeness at 8.9 Å. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 352.3, b = 465.5, c = 645.0 Å. The electron-density map reveals a protrusion that is consistent with the N-terminus extending out from the surface of the capsid. The structure presented here supports the idea that N-terminal insertions can be exploited in the development of diagnostic reagents, multicomponent vaccines and delivery vehicles into mammalian cells.

  17. Analysis of SAT type foot-and-mouth disease virus capsid proteins and the identification of putative amino acid residues affecting virus stability.

    Science.gov (United States)

    Maree, Francois F; Blignaut, Belinda; de Beer, Tjaart A P; Rieder, Elizabeth

    2013-01-01

    Foot-and-mouth disease virus (FMDV) initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces.

  18. Analysis of SAT Type Foot-And-Mouth Disease Virus Capsid Proteins and the Identification of Putative Amino Acid Residues Affecting Virus Stability

    Science.gov (United States)

    Maree, Francois F.; Blignaut, Belinda; de Beer, Tjaart A. P.; Rieder, Elizabeth

    2013-01-01

    Foot-and-mouth disease virus (FMDV) initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces. PMID:23717387

  19. Analysis of SAT type foot-and-mouth disease virus capsid proteins and the identification of putative amino acid residues affecting virus stability.

    Directory of Open Access Journals (Sweden)

    Francois F Maree

    Full Text Available Foot-and-mouth disease virus (FMDV initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces.

  20. Capsid protein: evidences about the partial protective role of neutralizing antibody-independent immunity against dengue in monkeys.

    Science.gov (United States)

    Gil, Lázaro; Izquierdo, Alienys; Lazo, Laura; Valdés, Iris; Ambala, Peris; Ochola, Lucy; Marcos, Ernesto; Suzarte, Edith; Kariuki, Thomas; Guzmán, Guadalupe; Guillén, Gerardo; Hermida, Lisset

    2014-05-01

    The role of cellular immune response in dengue virus infection is not yet fully understood. Only few studies in murine models propose that CD8(+) T-cells are associated with protection from infection and disease. At the light of recent reports about the protective role of CD8(+) T-cells in humans and the no correlation between neutralizing antibodies and protection observed in several studies, a vaccine based on cell-mediated immunity constitute an attractive approach. Our group has developed a capsid-based vaccine as nucleocpasid-like particles from dengue-2 virus, which induced a protective CD4(+) and CD8(+) cell-mediated immunity in mice, without the contribution of neutralizing antibodies. Herein we evaluated the immunogenicity and protective efficacy of this molecule in monkeys. Neither IgG antibodies against the whole virus nor neutralizing antibodies were elicited after the antigen inoculation. However, animals developed a cell-mediated immunity, measured by gamma interferon secretion and cytotoxic capacity. Although only one out of three vaccinated animals was fully protected against viral challenge, a viral load reduction was observed in this group compared with the placebo one, suggesting that capsid could be the base on an attractive vaccine against dengue.

  1. Live cell imaging of interactions between replicase and capsid protein of Brome mosaic virus using Bimolecular Fluorescence Complementation: Implications for replication and genome packaging

    Energy Technology Data Exchange (ETDEWEB)

    Chaturvedi, Sonali; Rao, A.L.N., E-mail: arao@ucr.edu

    2014-09-15

    In Brome mosaic virus, it was hypothesized that a physical interaction between viral replicase and capsid protein (CP) is obligatory to confer genome packaging specificity. Here we tested this hypothesis by employing Bimolecular Fluorescent Complementation (BiFC) as a tool for evaluating protein–protein interactions in living cells. The efficacy of BiFC was validated by a known interaction between replicase protein 1a (p1a) and protein 2a (p2a) at the endoplasmic reticulum (ER) site of viral replication. Additionally, co-expression in planta of a bona fide pair of interacting protein partners of p1a and p2a had resulted in the assembly of a functional replicase. Subsequent BiFC assays in conjunction with mCherry labeled ER as a fluorescent cellular marker revealed that CP physically interacts with p2a, but not p1a, and this CP:p2a interaction occurs at the cytoplasmic phase of the ER. The significance of the CP:p2a interaction in BMV replication and genome packaging is discussed. - Highlights: • YFP fusion proteins of BMV p1a and p2a are biologically active. • Self-interaction was observed for p1a, p2a and CP. • CP interacts with p2a but not p1a. • Majority of reconstituted YFP resulting from bona fide fusion protein partners localized on ER.

  2. Dual inhibitor of PDE7 and GSK-3-VP1.15 acts as antipsychotic and cognitive enhancer in C57BL/6J mice.

    Science.gov (United States)

    Lipina, Tatiana V; Palomo, Valle; Gil, Carmen; Martinez, Ana; Roder, John C

    2013-01-01

    Cognitive deficit is a core of schizophrenia and it is not effectively treated by the available antipsychotic drugs, hence new and more effective therapy is needed. Schizophrenia is considered as a pathway disorder where Disrupted-In-Schizophrenia-1 (DISC1) is important molecular player that regulates multiple cellular cascades. We recently reported synergistic action between phosphodiesterase-4 (PDE4) and glycogen synthase kinase-3 (GSK-3) as DISC1 interacting proteins. In the current study we characterized behavioural effects of a newly developed compound, VP1.15 that inhibits both PDE7 and GSK-3 with main focus on its antipsychotic and cognitive capacities. VP1.15 reduced ambulation in C57BL/6J mice in a dose-dependent manner (7.5 mg/kg and 3 mg/kg, respectively) and, hence, lower dose was chosen for the further analysis. VP1.1.5 facilitated pre-pulse inhibition (PPI), reversed amphetamine- but not MK-801-induced PPI deficit. The drug was able to ameliorate the disrupted latent inhibition (LI) induced by the increased number of conditioning trials and reversed amphetamine-induced LI deficit, supporting further its antipsychotic effects. The drug also significantly improved episodic memory in the spatial object recognition test, facilitated working memory in Y-maze and enhanced cued fear memory, but had no effect on executive function in the Puzzle box and contextual fear conditioning. Taken together, VP1.15 elicited antipsychotic effects and also facilitated cognitive domains in mice, suggesting that multitarget drugs, affecting molecular substrates from the same pathway, perhaps could be antipsychotics of new-generation that open a new possibilities in drug discoveries. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

  3. T-cell responses to oncogenic merkel cell polyomavirus proteins distinguish patients with merkel cell carcinoma from healthy donors

    DEFF Research Database (Denmark)

    Skou, Rikke Birgitte Lyngaa; Pedersen, Natasja Wulff; Schrama, David

    2014-01-01

    PURPOSE: Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with strong evidence of viral carcinogenesis. The association of MCC with the Merkel cell polyomavirus (MCPyV) may explain the explicit immunogenicity of MCC. Indeed, MCPyV-encoded proteins are likely targets for cytotoxic...... the MCPyV oncoprotein large T and small T antigens and the virus capsid protein VP1 for potential T-cell epitopes, and tested for MHC class I affinity. We confirmed the relevance of these epitopes using a high-throughput platform for T-cell enrichment and combinatorial encoding of MHC class I multimers...

  4. Role of electrostatic interactions in the assembly of empty spherical viral capsids

    CERN Document Server

    Siber, Antonio

    2007-01-01

    We examine the role of electrostatic interactions in the assembly of empty spherical viral capsids. The charges on the protein subunits that make the viral capsid mutually interact and are expected to yield electrostatic repulsion acting against the assembly of capsids. Thus, attractive protein-protein interactions of non-electrostatic origin must act to enable the capsid formation. We investigate whether the interplay of repulsive electrostatic and attractive interactions between the protein subunits can result in the formation of spherical viral capsids of a preferred radius. For this to be the case, we find that the attractive interactions must depend on the angle between the neighboring protein subunits (i.e. on the mean curvature of the viral capsid) so that a particular angle(s) is (are) preferred energywise. Our results for the electrostatic contributions to energetics of viral capsids nicely correlate with recent experimental determinations of the energetics of protein-protein contacts in Hepatitis B ...

  5. Imunogenicidade de proteínas do capsídeo do Cowpea severe mosaic virus (CPSMV Capsid protein immunogenicity of Cowpea severe mosaic virus (CPSMV

    Directory of Open Access Journals (Sweden)

    José Evando Aguiar Beserra Júnior

    2009-02-01

    Full Text Available A análise SDS-PAGE do Cowpea severe mosaic virus (CPSMV purificado revelou a migração de três frações protéicas estimadas em 43, 23 e 21 kDa, correspondentes às proteínas do capsídeo: denominadas proteína maior (43 kDa e menor (23 kDa; intacta e 21 kDa; clivada. As proteínas do capsídeo, na sua forma nativa, foram utilizadas na imunização de camundongos pelas vias oral e nasal, durante 10 dias consecutivos. As frações protéicas de 43 e 23 kDa, em sua forma desnaturada, foram utilizadas para imunização subcutânea. A resposta imunológica da mucosa foi avaliada pela proliferação celular das placas de Peyer de camundongos imunizados pela via oral com o CPSMV purificado. Ficou demonstrado que o CPSMV induz resposta imunológica, evidenciada pela síntese de anticorpos séricos, quando administrado na sua forma nativa pelas vias oral e nasal ou através de suas proteínas do capsídeo desnaturadas, pela via subcutânea. Não foi necessário o uso de adjuvantes, quer por via oral quer por via nasal. As frações protéicas de 43 e 23 kDa mostraram-se responsáveis pela imunogenicidade do vírus, como foi evidenciado pela síntese de anticorpos específicos detectados por ELISA. A análise da proliferação celular da placas de Peyer revelou um aumento (r=0,88 do número de leucócitos ao longo de 42 dias após a imunização. Esses resultados reforçam a possibilidade do uso do CPSMV como vetor seguro de antígenos de doenças humanas/animais pouco imunogênicos para produção de vacinas.SDS-PAGE analysis of purified Cowpea severe mosaic virus (CPSMV revealed the migration of three protein fractions of 43, 23 and 21 kDa, corresponding to the capsid protein called large protein (43 kDa and small protein (23 kDa; intact and 21 kDa; cleaved. The capsid proteins, in their native form, were used to immunize mice through oral and nasal routes for ten consecutive days. The denatured form of the 43 and 23 kDa protein fractions were

  6. Impact of Capsid Conformation and Rep-Capsid Interactions on Adeno-Associated Virus Type 2 Genome Packaging

    OpenAIRE

    Bleker, Svenja; Pawlita, Michael; Kleinschmidt, Jürgen A.

    2006-01-01

    Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefol...

  7. Structure of the HIV-1 Full-Length Capsid Protein in a Conformationally Trapped Unassembled State Induced by Small-Molecule Binding

    Energy Technology Data Exchange (ETDEWEB)

    Du, Shoucheng; Betts, Laurie; Yang, Ruifeng; Shi, Haibin; Concel, Jason; Ahn, Jinwoo; Aiken, Christopher; Zhang, Peijun; Yeh, Joanne I. (Pitt); (Vanderbilt); (UNC)

    2012-11-26

    The capsid (CA) protein plays crucial roles in HIV infection and replication, essential to viral maturation. The absence of high-resolution structural data on unassembled CA hinders the development of antivirals effective in inhibiting assembly. Unlike enzymes that have targetable, functional substrate-binding sites, the CA does not have a known site that affects catalytic or other innate activity, which can be more readily targeted in drug development efforts. We report the crystal structure of the HIV-1 CA, revealing the domain organization in the context of the wild-type full-length (FL) unassembled CA. The FL CA adopts an antiparallel dimer configuration, exhibiting a domain organization sterically incompatible with capsid assembly. A small compound, generated in situ during crystallization, is bound tightly at a hinge site ('H site'), indicating that binding at this interdomain region stabilizes the ADP conformation. Electron microscopy studies on nascent crystals reveal both dimeric and hexameric lattices coexisting within a single condition, in agreement with the interconvertibility of oligomeric forms and supporting the feasibility of promoting assembly-incompetent dimeric states. Solution characterization in the presence of the H-site ligand shows predominantly unassembled dimeric CA, even under conditions that promote assembly. Our structure elucidation of the HIV-1 FL CA and characterization of a potential allosteric binding site provides three-dimensional views of an assembly-defective conformation, a state targeted in, and thus directly relevant to, inhibitor development. Based on our findings, we propose an unprecedented means of preventing CA assembly, by 'conformationally trapping' CA in assembly-incompetent conformational states induced by H-site binding.

  8. Sizing up large protein complexes by electrospray ionisation-based electrophoretic mobility and native mass spectrometry : morphology selective binding of Fabs to hepatitis B virus capsids

    NARCIS (Netherlands)

    Bereszczak, Jessica Z; Havlik, Marlene; Weiss, Victor U; Marchetti-Deschmann, Martina; van Duijn, Esther; Watts, Norman R; Wingfield, Paul T; Allmaier, Guenter; Steven, Alasdair C; Heck, Albert J R

    2014-01-01

    The capsid of hepatitis B virus (HBV) is a major viral antigen and important diagnostic indicator. HBV capsids have prominent protrusions ('spikes') on their surface and are unique in having either T = 3 or T = 4 icosahedral symmetry. Mouse monoclonal and also human polyclonal antibodies bind either

  9. Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core*

    Science.gov (United States)

    Camus, Gregory; Herker, Eva; Modi, Ankit A.; Haas, Joel T.; Ramage, Holly R.; Farese, Robert V.; Ott, Melanie

    2013-01-01

    The triglyceride-synthesizing enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) plays a critical role in hepatitis C virus (HCV) infection by recruiting the HCV capsid protein core onto the surface of cellular lipid droplets (LDs). Here we find a new interaction between the non-structural protein NS5A and DGAT1 and show that the trafficking of NS5A to LDs depends on DGAT1 activity. DGAT1 forms a complex with NS5A and core and facilitates the interaction between both viral proteins. A catalytically inactive mutant of DGAT1 (H426A) blocks the localization of NS5A, but not core, to LDs in a dominant-negative manner and impairs the release of infectious viral particles, underscoring the importance of DGAT1-mediated translocation of NS5A to LDs in viral particle production. We propose a model whereby DGAT1 serves as a cellular hub for HCV core and NS5A proteins, guiding both onto the surface of the same subset of LDs, those generated by DGAT1. These results highlight the critical role of DGAT1 as a host factor for HCV infection and as a potential drug target for antiviral therapy. PMID:23420847

  10. Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using the Pichia pastoris PGK1 Promoter

    Directory of Open Access Journals (Sweden)

    F. C. Mariz

    2015-01-01

    Full Text Available The human papillomavirus (HPV L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive PGK1 promoter (PPGK1 from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of PPGK1 into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris.

  11. Crosslinking in viral capsids via tiling theory.

    Science.gov (United States)

    Twarock, R; Hendrix, R W

    2006-06-07

    A vital part of a virus is its protein shell, called the viral capsid, that encapsulates and hence protects the viral genome. It has been shown in Twarock [2004. A tiling approach to vius capsids assembly explaining a structural puzzle in virology. J. Theor. Biol. 226, 477-482] that the surface structures of viruses with icosahedrally symmetric capsids can be modelled in terms of tilings that encode the locations of the protein subunits. This theory is extended here to multi-level tilings in order to model crosslinking structures. The new framework is demonstrated for the case of bacteriophage HK97, and it is shown, how the theory can be used in general to decide if crosslinking, and what type of crosslinking, is compatible from a mathematical point of view with the geometrical surface structure of a virus.

  12. Capsid proteins from human immunodeficiency virus type 1 and simian immunodeficiency virus SIVmac can coassemble into mature cores of infectious viruses.

    Science.gov (United States)

    Chen, Jianbo; Pathak, Vinay K; Peng, Weiqun; Hu, Wei-Shau

    2008-09-01

    We have recently shown that the Gag polyproteins from human immunodeficiency virus type 1 (HIV-1) and HIV-2 can coassemble and functionally complement each other. During virion maturation, the Gag polyproteins undergo proteolytic cleavage to release mature proteins including capsid (CA), which refolds and forms the outer shell of a cone-shaped mature core. Less than one-half of the CA proteins present within the HIV-1 virion are required to form the mature core. Therefore, it is unclear whether the mature core in virions containing both HIV-1 and HIV-2 Gag consists of CA proteins from a single virus or from both viruses. To determine whether CA proteins from two different viruses can coassemble into mature cores of infectious viruses, we exploited the specificity of the tripartite motif 5alpha protein from the rhesus monkey (rhTRIM5alpha) for cores containing HIV-1 CA (hCA) but not the simian immunodeficiency virus SIV(mac) CA protein (sCA). If hCA and sCA cannot coassemble into the same core when equal amounts of sCA and hCA are coexpressed, the infectivities of such virus preparations in cells should be inhibited less than twofold by rhTRIM5alpha. However, if hCA and sCA can coassemble into the same core structure to form a mixed core, rhTRIM5alpha would be able to recognize such cores and significantly restrict virus infectivity. We examined the restriction phenotypes of viruses containing both hCA and sCA. Our results indicate that hCA and sCA can coassemble into the same mature core to produce infectious virus. To our knowledge, this is the first demonstration of functional coassembly of heterologous CA protein into the retroviral core.

  13. Nucleic localization of human papillomavirus minor capsid protein L2%人乳头瘤病毒次要衣壳蛋白L2核定位

    Institute of Scientific and Technical Information of China (English)

    陈卫东; 井申荣

    2012-01-01

    在人乳头瘤病毒(human papillomavirus,HPV)次要衣壳蛋白L2的N端和C端,有大量带正电荷的氨基酸残基组成核定位信号(nuclear localization signal,NLS).细胞的核结构域10 (nuclear domain 10,ND10)是细胞周期和病毒生活周期的重要调节者.L2定位到ND10的过程不仅会受到早幼粒细胞白血病蛋白(promyleocytic leukaemia protein,PML)、死亡结构域相关蛋白(death domain-associated protein,Daxx)、Sp100核抗原(Sp100 nuclear antigen)等细胞蛋白的影响,也会与L1在ND10发生相互作用.在HPV感染和组装过程中,L2的核定位信号有着重要作用.%The nuclear localization signal, NLS, which is composed of many amino acids with positive charge residue, is located in both ends of N terminus and C terminus of the human papillomavirus minor capsid protein L2. The nuclear domain 10, ND-10, which is in the cell nucleus, is an important modulator of both cell cycle and virus cyclogeny. The process of the locating of L2 to ND-10, is not only impacted by the cell proteins, such as the promyelocytic leukemia protein, death domain-associated protein Daxx, nuclear antigen Sp100, but also interacts with L1 in ND-10. The NLS of L2 plays a significant role during the HPV virus assembly course.

  14. Self-assembly of virus-like particles of rabbit hemorrhagic disease virus capsid protein expressed in Escherichia coli and their immunogenicity in rabbits.

    Science.gov (United States)

    Guo, Huimin; Zhu, Jie; Tan, Yonggui; Li, Chuanfeng; Chen, Zongyan; Sun, Shiqi; Liu, Guangqing

    2016-07-01

    In this study, virus-like particles (VLPs) derived from rabbit hemorrhagic disease virus (RHDV) were evaluated for the development of a vaccine against RHDV infection. The VP60 gene was cloned and inserted into a pSMK expression vector containing a small ubiquitin-like modifier (SUMO) tag that can promote the soluble expression of heterologous proteins in Escherichia coli cells. After expression and purification of His-SUMO-VP60 and cleavage of the SUMO tag, we found that the RHDV VP60 protein had self-assembled into VLPs with a similar shape and smaller size compared with authentic RHDV capsid. Next, the antigenicity and immunogenicity of the VLPs were examined. The results showed that RHDV-specific responses were clearly induced in rabbits and that all rabbits in the VLP group survived while those in the negative control group died within 72 h post-infection. These results suggest that VLP-based RHDV could be a promising RHDV vaccine candidate.

  15. Cleavage of the HPV16 Minor Capsid Protein L2 during Virion Morphogenesis Ablates the Requirement for Cellular Furin during De Novo Infection

    Directory of Open Access Journals (Sweden)

    Linda Cruz

    2015-11-01

    Full Text Available Infections by high-risk human papillomaviruses (HPV are the causative agents for the development of cervical cancer. As with other non-enveloped viruses, HPVs are taken up by the cell through endocytosis following primary attachment to the host cell. Through studies using recombinant pseudovirus particles (PsV, many host cellular proteins have been implicated in the process. The proprotein convertase furin has been demonstrated to cleave the minor capsid protein, L2, post-attachment to host cells and is required for infectious entry by HPV16 PsV. In contrast, using biochemical inhibition by a furin inhibitor and furin-negative cells, we show that tissue-derived HPV16 native virus (NV initiates infection independent of cellular furin. We show that HPV16 L2 is cleaved during virion morphogenesis in differentiated tissue. In addition, HPV45 is also not dependent on cellular furin, but two other alpha papillomaviruses, HPV18 and HPV31, are dependent on the activity of cellular furin for infection.

  16. Detection of Foot-and-mouth Disease Virus RNA and Capsid Protein in Lymphoid Tissues of Convalescent Pigs Does Not Indicate Existence of a Carrier State.

    Science.gov (United States)

    Stenfeldt, C; Pacheco, J M; Smoliga, G R; Bishop, E; Pauszek, S J; Hartwig, E J; Rodriguez, L L; Arzt, J

    2016-04-01

    A systematic study was performed to investigate the potential of pigs to establish and maintain persistent foot-and-mouth disease virus (FMDV) infection. Infectious virus could not be recovered from sera, oral, nasal or oropharyngeal fluids obtained after resolution of clinical infection with any of five FMDV strains within serotypes A, O and Asia-1. Furthermore, there was no isolation of live virus from tissue samples harvested at 28-100 days post-infection from convalescent pigs recovered from clinical or subclinical FMD. Despite lack of detection of infectious FMDV, there was a high prevalence of FMDV RNA detection in lymph nodes draining lesion sites harvested at 35 days post-infection, with the most frequent detection recorded in popliteal lymph nodes (positive detection in 88% of samples obtained from non-vaccinated pigs). Likewise, at 35 dpi, FMDV capsid antigen was localized within follicles of draining lymph nodes, but without concurrent detection of FMDV non-structural protein. There was a marked decline in the detection of FMDV RNA and antigen in tissue samples by 60 dpi, and no antigen or viral RNA could be detected in samples obtained at 100 dpi. The data presented herein provide the most extensive investigation of FMDV persistence in pigs. The overall conclusion is that domestic pigs are unlikely to be competent long-term carriers of infectious FMDV; however, transient persistence of FMDV protein and RNA in lymphoid tissues is common following clinical or subclinical infection.

  17. Self-assembly of virus-like particles of canine parvovirus capsid protein expressed from Escherichia coli and application as virus-like particle vaccine.

    Science.gov (United States)

    Xu, Jin; Guo, Hui-Chen; Wei, Yan-Quan; Dong, Hu; Han, Shi-Chong; Ao, Da; Sun, De-Hui; Wang, Hai-Ming; Cao, Sui-Zhong; Sun, Shi-Qi

    2014-04-01

    Canine parvovirus disease is an acute infectious disease caused by canine parvovirus (CPV). Current commercial vaccines are mainly attenuated and inactivated; as such, problems concerning safety may occur. To resolve this problem, researchers developed virus-like particles (VLPs) as biological nanoparticles resembling natural virions and showing high bio-safety. This property allows the use of VLPs for vaccine development and mechanism studies of viral infections. Tissue-specific drug delivery also employs VLPs as biological nanomaterials. Therefore, VLPs derived from CPV have a great potential in medicine and diagnostics. In this study, small ubiquitin-like modifier (SUMO) fusion motif was utilized to express a whole, naturalVP2 protein of CPV in Escherichia coli. After the cleavage of the fusion motif, the CPV VP2 protein has self-assembled into VLPs. The VLPs had a size and shape that resembled the authentic virus capsid. However, the self-assembly efficiency of VLPs can be affected by different pH levels and ionic strengths. The mice vaccinated subcutaneously with CPV VLPs and CPV-specific immune responses were compared with those immunized with the natural virus. This result showed that VLPs can effectively induce anti-CPV specific antibody and lymphocyte proliferation as a whole virus. This result further suggested that the antigen epitope of CPV was correctly present on VLPs, thereby showing the potential application of a VLP-based CPV vaccine.

  18. A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2.

    Directory of Open Access Journals (Sweden)

    Ebenezer Tumban

    Full Text Available BACKGROUND: Current human papillomavirus (HPV vaccines that are based on virus-like particles (VLPs of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin. METHODOLOGY/PRINCIPAL FINDINGS: L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV. CONCLUSION/SIGNIFICANCE: VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts.

  19. Coarse-grained simulation reveals key features of HIV-1 capsid self-assembly

    Science.gov (United States)

    Grime, John M. A.; Dama, James F.; Ganser-Pornillos, Barbie K.; Woodward, Cora L.; Jensen, Grant J.; Yeager, Mark; Voth, Gregory A.

    2016-05-01

    The maturation of HIV-1 viral particles is essential for viral infectivity. During maturation, many copies of the capsid protein (CA) self-assemble into a capsid shell to enclose the viral RNA. The mechanistic details of the initiation and early stages of capsid assembly remain to be delineated. We present coarse-grained simulations of capsid assembly under various conditions, considering not only capsid lattice self-assembly but also the potential disassembly of capsid upon delivery to the cytoplasm of a target cell. The effects of CA concentration, molecular crowding, and the conformational variability of CA are described, with results indicating that capsid nucleation and growth is a multi-stage process requiring well-defined metastable intermediates. Generation of the mature capsid lattice is sensitive to local conditions, with relatively subtle changes in CA concentration and molecular crowding influencing self-assembly and the ensemble of structural morphologies.

  20. Whole-Chain Tick Saliva Proteins Presented on Hepatitis B Virus Capsid-Like Particles Induce High-Titered Antibodies with Neutralizing Potential.

    Directory of Open Access Journals (Sweden)

    Philipp Kolb

    Full Text Available Ticks are vectors for various, including pathogenic, microbes. Tick saliva contains multiple anti-host defense factors that enable ticks their bloodmeals yet also facilitate microbe transmission. Lyme disease-causing borreliae profit specifically from the broadly conserved tick histamine release factor (tHRF, and from cysteine-rich glycoproteins represented by Salp15 from Ixodes scapularis and Iric-1 from Ixodes ricinus ticks which they recruit to their outer surface protein C (OspC. Hence these tick proteins are attractive targets for anti-tick vaccines that simultaneously impair borrelia transmission. Main obstacles are the tick proteins´ immunosuppressive activities, and for Salp15 orthologs, the lack of efficient recombinant expression systems. Here, we exploited the immune-enhancing properties of hepatitis B virus core protein (HBc derived capsid-like particles (CLPs to generate, in E. coli, nanoparticulate vaccines presenting tHRF and, as surrogates for the barely soluble wild-type proteins, cysteine-free Salp15 and Iric-1 variants. The latter CLPs were exclusively accessible in the less sterically constrained SplitCore system. Mice immunized with tHRF CLPs mounted a strong anti-tHRF antibody response. CLPs presenting cysteine-free Salp15 and Iric-1 induced antibodies to wild-type, including glycosylated, Salp15 and Iric-1. The broadly distributed epitopes included the OspC interaction sites. In vitro, the anti-Salp15 antibodies interfered with OspC binding and enhanced human complement-mediated killing of Salp15 decorated borreliae. A mixture of all three CLPs induced high titered antibodies against all three targets, suggesting the feasibility of combination vaccines. These data warrant in vivo validation of the new candidate vaccines´ protective potential against tick infestation and Borrelia transmission.

  1. Spatial and temporal changes of cyanophage communities in paddy field soils as revealed by the capsid assembly protein gene g20.

    Science.gov (United States)

    Wang, Guanghua; Asakawa, Susumu; Kimura, Makoto

    2011-05-01

    Bacteriophages are ubiquitous in various environments. Our previous study revealed the diversity of the cyanophage community in paddy floodwater. In this study, the phylogeny and genetic diversity of cyanophage communities in paddy field soils were reported. The viral capsid assembly protein gene (g20) of cyanophage was amplified with the primers CPS1 and CPS8 from soil DNA extracted during two different sampling times at three sampling sites in Japan. The sequencing results indicated that about 93% of the clones were g20 genes. In total, 70 clones of g20 genes were obtained in this study, of which 69 clones were of cyanophage origin. As evaluated by g20 sequence assemblages in paddy field soils, the unifrac analyses results indicated that cyanophage communities changed among the sampling sites and times and differed from those communities detected in paddy floodwater. The phylogenetic analysis showed that the g20 sequences in paddy field soils were very diverse and distributed into Clusters α, β and ɛ, as well as four newly formed clusters. Within Clusters β and ɛ, four unique subclusters were formed from the g20 clones that were only observed in this study. These findings suggested that the cyanophage communities in paddy field soils are different from those found in freshwater, marine water and paddy floodwater.

  2. Phylogenetic diversity of marine cyanophage isolates and natural virus communities as revealed by sequences of viral capsid assembly protein gene g20.

    Science.gov (United States)

    Zhong, Yan; Chen, Feng; Wilhelm, Steven W; Poorvin, Leo; Hodson, Robert E

    2002-04-01

    In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to specifically amplify ca. 592-bp fragments of the gene for viral capsid assembly protein g20. Phylogenetic analysis of isolated cyanophages revealed that the marine cyanophages were highly diverse yet more closely related to each other than to enteric coliphage T4. Genetically related marine cyanophage isolates were widely distributed without significant geographic segregation (i.e., no correlation between genetic variation and geographic distance). Cloning and sequencing analysis of six natural virus concentrates from estuarine and oligotrophic offshore environments revealed nine phylogenetic groups in a total of 114 different g20 homologs, with up to six clusters and 29 genotypes encountered in a single sample. The composition and structure of natural cyanophage communities in the estuary and open-ocean samples were different from each other, with unique phylogenetic clusters found for each environment. Changes in clonal diversity were also observed from the surface waters to the deep chlorophyll maximum layer in the open ocean. Only three clusters contained known cyanophage isolates, while the identities of the other six clusters remain unknown. Whether or not these unidentified groups are composed of bacteriophages that infect different Synechococcus groups or other closely related cyanobacteria remains to be determined. The high genetic diversity of marine cyanophage assemblages revealed by the g20 sequences suggests that marine viruses can potentially play important roles in regulating microbial genetic diversity.

  3. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.

    Science.gov (United States)

    Purcell, Maureen K; Pearman-Gillman, Schuyler; Thompson, Rachel L; Gregg, Jacob L; Hart, Lucas M; Winton, James R; Emmenegger, Eveline J; Hershberger, Paul K

    2016-07-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  4. Development of an in process control filtration-assisted chemiluminometric immunoassay to quantify foot and mouth disease virus (FMDV) non-capsid proteins in vaccine-antigen batches.

    Science.gov (United States)

    Capozzo, Alejandra Victoria; Martínez, Manuel Rosendo; Schielen, Wilhelmus Joseph Gerardus

    2010-09-14

    In many countries, foot and mouth disease (FMD) is controlled by vaccination and surveillance against non-capsid proteins (NCP); therefore vaccines are required not to induce antibodies against NCP. Vaccine purity is evaluated by repeated inoculation of naïve cattle, an expensive and time consuming protocol that raises several animal welfare concerns. We have developed an in process control filtration-assisted chemiluminometric immunoassay (FAL-ELISA), to detect and quantify NCP in vaccine-antigen batches regardless of its volume and composition. Samples are filtered through PVDF-filter microplates pre-coated with a monoclonal antibody against NCP. Filtration removes all unbound components in the sample and captured NCP are detected by anti-NCP conjugate followed by incubation with the substrate, luminol/peroxide. Analytical detection limit was 2 ng for purified NCP and 4 ng for vaccine-antigen batches spiked with NCP, which makes this assay sensitive enough to be applied to purity control of FMD vaccines. Vaccine components did not interfere with the antibody and substrate reactions in the assay. FAL-ELISA is an alternative for the in vivo tests, observing the objective to Replace, Reduce and Refine the use of animals for quality control of immunobiologicals.

  5. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

    Science.gov (United States)

    Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

    2016-01-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  6. Computational studies on the interaction of ABO-active saccharides with the norovirus VA387 capsid protein can explain experimental binding data

    Science.gov (United States)

    Koppisetty, Chaitanya A. K.; Nasir, Waqas; Strino, Francesco; Rydell, Gustaf E.; Larson, Göran; Nyholm, Per-Georg

    2010-05-01

    Norovirus strains are known to cause recurring epidemics of winter vomiting disease. The crystal structure of the capsid protein of VA387, a representative of the clinically important GII.4 genocluster, was recently solved in complex with histo-blood group A- and B-trisaccharides. However, the VA387 strain is known to bind also to other natural carbohydrates for which detailed structural information of the complexes is not available. In this study we have computationally explored the fit of the VA387 with a set of naturally occurring carbohydrate ligands containing a terminal α1,2-linked fucose. MD simulations both with explicit and implicit solvent models indicate that type 1 and 3 extensions of the ABO-determinant including ALeb and BLeb pentasaccharides can be well accommodated in the site. Scoring with Glide XP indicates that the downstream extensions of the ABO-determinants give an increase in binding strength, although the α1,2-linked fucose is the single strongest interacting residue. An error was discovered in the geometry of the GalNAc-Gal moiety of the published crystal structure of the A-trisaccharide/VA387 complex. The present modeling of the complexes with histo-blood group A-active structures shows some contacts which provide insight into mutational data, explaining the involvement of I389 and Q331. Our results can be applicable in structure-based design of adhesion inhibitors of noroviruses.

  7. Infectious RNA transcripts derived from cloned cDNA of papaya mosaic virus: effect of mutations to the capsid and polymerase proteins.

    Science.gov (United States)

    Sit, T L; AbouHaidar, M G

    1993-06-01

    Genomic length cDNAs of papaya mosaic virus (PMV) RNA were generated utilizing reverse transcriptase (RNase H-) for first strand synthesis, Sequenase for second strand synthesis and primers specific for the 5' and 3' termini of the viral genome. These cDNAs were cloned into plasmid pUC18 and infectious RNA transcripts were synthesized in vitro from a bacteriophage T7 RNA polymerase promoter incorporated into the 5' specific primer. The infectivity of transcripts was 16% that of native PMV RNA. Increasing the poly(A) tail length from A24 to A71 produced a 43% increase in infectivity. Transcripts synthesized with or without an m7GpppG cap structure were biologically active although uncapped transcripts were much less infectious. The addition of up to 2434 non-viral nucleotides at the 3' end of transcripts decreased but did not abolish infectivity. Insertions of two amino acid residues within the polymerase coding region inactivated viral transcripts. A single amino acid deletion within the capsid protein (CP) produced local lesions of a reduced size as compared to native PMV RNA. Viral particles could not be observed in crude extracts from lesions produced by this deletion mutant suggesting that it exists as a naked RNA species within the host. Mutations to the CP suggest that it is required not only for viral assembly but also for some other unidentified function(s) during the replication cycle.

  8. Protective immunization of horses with a recombinant canarypox virus vectored vaccine co-expressing genes encoding the outer capsid proteins of African horse sickness virus.

    Science.gov (United States)

    Guthrie, Alan J; Quan, Melvyn; Lourens, Carina W; Audonnet, Jean-Christophe; Minke, Jules M; Yao, Jiansheng; He, Ling; Nordgren, Robert; Gardner, Ian A; Maclachlan, N James

    2009-07-16

    We describe the development and preliminary characterization of a recombinant canarypox virus vectored (ALVAC) vaccine for protective immunization of equids against African horse sickness virus (AHSV) infection. Horses (n=8) immunized with either of two concentrations of recombinant canarypox virus vector (ALVAC-AHSV) co-expressing synthetic genes encoding the outer capsid proteins (VP2 and VP5) of AHSV serotype 4 (AHSV-4) developed variable titres (horse immunized with a commercial recombinant canarypox virus vectored vaccine expressing the haemagglutinin genes of two equine influenza H3N8 viruses was seronegative to AHSV and following infection with virulent AHSV-4 developed pyrexia, thrombocytopenia and marked oedema of the supraorbital fossae typical of the "dikkop" or cardiac form of African horse sickness. AHSV was detected by virus isolation and quantitative reverse transcriptase polymerase chain reaction in the blood of the control horse from 8 days onwards after challenge infection whereas AHSV was not detected at any time in the blood of the ALVAC-AHSV vaccinated horses. The control horse seroconverted to AHSV by 2 weeks after challenge infection as determined by both virus neutralization and ELISA assays, whereas six of eight of the ALVAC-AHSV vaccinated horses did not seroconvert by either assay following challenge infection with virulent AHSV-4. These data confirm that the ALVAC-AHSV vaccine will be useful for the protective immunization of equids against African horse sickness, and avoids many of the problems inherent to live-attenuated AHSV vaccines.

  9. Multivalent viral capsids with internal cargo for fibrin imaging.

    Directory of Open Access Journals (Sweden)

    Allie C Obermeyer

    Full Text Available Thrombosis is the cause of many cardiovascular syndromes and is a significant contributor to life-threatening diseases, such as myocardial infarction and stroke. Thrombus targeted imaging agents have the capability to provide molecular information about pathological clots, potentially improving detection, risk stratification, and therapy of thrombosis-related diseases. Nanocarriers are a promising platform for the development of molecular imaging agents as they can be modified to have external targeting ligands and internal functional cargo. In this work, we report the synthesis and use of chemically functionalized bacteriophage MS2 capsids as biomolecule-based nanoparticles for fibrin imaging. The capsids were modified using an oxidative coupling reaction, conjugating ∼90 copies of a fibrin targeting peptide to the exterior of each protein shell. The ability of the multivalent, targeted capsids to bind fibrin was first demonstrated by determining the impact on thrombin-mediated clot formation. The modified capsids out-performed the free peptides and were shown to inhibit clot formation at effective concentrations over ten-fold lower than the monomeric peptide alone. The installation of near-infrared fluorophores on the interior surface of the capsids enabled optical detection of binding to fibrin clots. The targeted capsids bound to fibrin, exhibiting higher signal-to-background than control, non-targeted MS2-based nanoagents. The in vitro assessment of the capsids suggests that fibrin-targeted MS2 capsids could be used as delivery agents to thrombi for diagnostic or therapeutic applications.

  10. Comparison of Immune Responses against FMD by a DNA Vaccine Encoding the FMDV/O/IRN/2007 VP1 Gene and the Conventional Inactivated Vaccine in an Animal Model

    Institute of Scientific and Technical Information of China (English)

    Farahnaz Motamedi Sedeh; Hoorieh Soleimanjahi; AmirReza Jalilian; Homayoon Mahravani

    2012-01-01

    Foot-and-mouth disease virus (FMDV) is highly contagious and responsible for huge outbreaks among cloven hoofed animals.The aim of the present study is to evaluate a plasmid DNA immunization system that expresses the FMDV/O/IRN/2007 VP1 gene and compare it with the conventional inactivated vaccine in an animal model.The VP1 gene was sub-cloned into the unique Kpn I and BamH I cloning sites of the pcDNA3.1+ and pEGFP-N1 vectors to construct the VP1 gene cassettes.The transfected BHKT7 cells with sub-cloned pEGFP-N1-VP1 vector expressed GFP-VP1 fusion protein and displayed more green fluorescence spots than the transfected BHKT7 cells with pEGFP-N1 vector,which solely expressed the GFP protein.Six mice groups were respectively immunized by the sub-cloned pcDNA3.1+-VP1 gene cassette as the DNA vaccine,DNA vaccine and PCMV-SPORT-GMCSF vector (as molecular adjuvant) together,conventional vaccine,PBS (as negative control),pcDNA3.1+ vector (as control group) and PCMV-SPORT vector that contained the GMCSF gene (as control group).Significant neutralizing antibody responses were induced in the mice which were immunized using plasmid vectors expressing the VP1 and GMCSF genes together,the DNA vaccine alone and the conventional inactivated vaccine (P<0.05).Co-administration of DNA vaccine and GMCSF gene improved neutralizing antibody response in comparison with administration of the DNA vaccine alone,but this response was the most for the conventional vaccine group.However,induction of humeral immunity response in the conventional vaccine group was more protective than for the DNA vaccine,but T-cell proliferation and IFN-γ concentration were the most in DNA vaccine with the GMCSF gene.Therefore the group that was vaccinated by DNA vaccine with the GMCSF gene,showed protective neutralizing antibody response and the most Th1 cellular immunity.

  11. 蓝狐细小病毒的分离鉴定及其VP1基因序列分析%Isolation and identification of blue fox parvovirus and sequence analysis of its VP1 gene

    Institute of Scientific and Technical Information of China (English)

    刘海防; 胡传伟; 谢之景; 倪长鹏; 贾赟; 姜世金; 张兴晓; 杨笃宝

    2009-01-01

    从泰安地区送检的疑是细小病毒感染的蓝狐粪便中分离到一株病毒.经理化特性鉴定、血凝谱鉴定、人工感染蓝狐等鉴定,表明所分离病毒为细小病毒.并且根据GenBank上发表的犬细小病毒(Canine parvovirus,CPV)、猫细小病毒(Feline parvovirus,FPV)核酸序列,设计扩增VP1基因的引物,采用PCR技术扩增所分离细小病毒的VP1全基因,将PCR产物克隆入pMD18-T载体,进行测序分析.结果, 所分离细小病毒的VP1基因全长2 256 bp,编码727个氨基酸,与CPV和FPV参照株的VP1基因同源性在98.7%~99.5%.VP1基因的系统发生分析表明所分离病毒与FPV的亲源关系最为密切.所分离病毒VP1蛋白375位氨基酸残基与CPV的VP1蛋白氨基酸残基一致,但其223位、236位、246位、466位、707位、711位氨基酸残基与FPV VP1蛋白的氨基酸残基一致,该病毒VP1蛋白序列表现出了过渡型序列特征,介于FPV与CPV间的过渡类型,这说明所分离病毒为蓝狐细小病毒(Blue fox parvovirus,BFPV),命名为BFPV-TA,蓝狐可能在CPV的起源过程起到重要的作用.

  12. Sequence Analysis of Segment 8 of Five Chinese Isolates of Rice Gall Dwarf Virus and Expression of a Main Outer Capsid Protein in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The rice gall dwarf disease, caused by the Rice gall dwarf virus (RGDV) is a serious disease occurring in rice in many regions of Guangdong province. As a basis to control the disease we have studied the genomic diversity of a variety of isolates from different locations. Genome segment 8(S8), encoding a main outer capsid protein (Pns8) of RGDV five isolates (BL, CH, DQ, GZ, XY) from Guangdong province was cloned and sequenced. The results revealed that all the S8 segments of the five isolates consisted of 1 578 nucleotides and had a single open reading frame (ORF) extending for 1 301 nucleotides from nucleotide 21 which encoded a polypeptide of 426 amino acids with an estimated molecular weight of 47.4 kDa. The S8 full-length sequence and the ORF sequence shared 97.3%-98.8% and 97.3%-99.1% nucleotide sequence identities within the five Chinese isolates, and shared 94.8%-95.6% and 95.0%-96.0% identities with those of the Thailand isolate respectively. The deduced amino acid sequence of Pns8 in GZ isolate was identical to that in the Thailand isolate, while the amino acid sequence variability of Pns8 within five Chinese isolates ranged from 0.5% to 2.1%. These results indicate that the S8 segment of RGDV is highly conserved in different isolates from different locations. The S8 cDNA from the XY isolate was cloned into the plasmid vector pET-28b(+) and a fused expression protein with an apparent molecular mass of 51kDa was specifically detected in an analysis of Escherichia coli Rossetta(DE3)Ⅱcells. To our knowledge, this is the first report on analysis of the RGDV segment 8 sequence and genetic comparison of different RGDV isolates and their protein expression.

  13. Assembly and Immunogenicity of Human Papillomavirus Type 16 Major Capsid Protein ( HPV16 L1 ) in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In this study, a recombinant Pichia pastoris expression system was developed to express HPV16 L1 protein that was driven by a strong AOX1 promoter. HPV16L1 gene was cloned into vector pPICZαB. HPV16 L1 protein expression induced by methanol was screened by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( SDSPAGE) and Western blotting. The results indicate that the HPV16 L1 protein is secreted by the recombinant P. pastotis, and the purified HPV16 L1 protein can self-assemble into virus-like particles(VLPs), which show a good immunogenicity and induces high-titer antibody in mice.

  14. Virus-binding proteins recovered from bacterial culture derived from activated sludge by affinity chromatography assay using a viral capsid peptide.

    Science.gov (United States)

    Sano, Daisuke; Matsuo, Takahiro; Omura, Tatsuo

    2004-06-01

    The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral infectious diseases in our society. Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed. In this study, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered. The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from the poliovirus type 1 (PV1) Mahoney strain (H(2)N-DNPASTTNKDKL-COOH) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin 1 as determined by enzyme-linked immunosorbent assay. The evaluation of surface charges of VBPs with ion-exchange chromatography found that a majority of VBP molecules had a net negative charge under the conditions of affinity chromatography. On the other hand, a calculated isoelectric point implied that the viral peptide in the affinity column was also charged negatively. As a result, the adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force that was able to overcome the electrostatic repulsive force. Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular masses were widely distributed but smaller than 100 kDa. Amino acid sequences of N termini of five VBPs were determined. Homology searches for the N termini against all protein sequences in the National Center for Biotechnology Information (NCBI) database showed that the isolated VBPs in this study were newly discovered proteins. These VBPs that originated with bacteria in activated sludge might be stable, because they are existing in the environment of wastewater treatments. Therefore, a virus removal technology

  15. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    Science.gov (United States)

    Andoh, Y.; Yoshii, N.; Yamada, A.; Fujimoto, K.; Kojima, H.; Mizutani, K.; Nakagawa, A.; Nomoto, A.; Okazaki, S.

    2014-10-01

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 106 all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  16. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    Energy Technology Data Exchange (ETDEWEB)

    Andoh, Y.; Yoshii, N.; Yamada, A.; Kojima, H.; Mizutani, K.; Okazaki, S., E-mail: okazaki@apchem.nagoya-u.ac.jp [Department of Applied Chemistry, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan); Fujimoto, K. [Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Nakagawa, A. [Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871 (Japan); Nomoto, A. [Institute of Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo 141-0021 (Japan)

    2014-10-28

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 10{sup 6} all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  17. Lentiviral Gag assembly analyzed through the functional characterization of chimeric simian immunodeficiency viruses expressing different domains of the feline immunodeficiency virus capsid protein.

    Directory of Open Access Journals (Sweden)

    María J Esteva

    Full Text Available To gain insight into the functional relationship between the capsid (CA domains of the Gag polyproteins of simian and feline immunodeficiency viruses (SIV and FIV, respectively, we constructed chimeric SIVs in which the CA-coding region was partially or totally replaced by the equivalent region of the FIV CA. The phenotypic characterization of the chimeras allowed us to group them into three categories: the chimeric viruses that, while being assembly-competent, exhibit a virion-associated unstable FIV CA; a second group represented only by the chimeric SIV carrying the N-terminal domain (NTD of the FIV CA which proved to be assembly-defective; and a third group constituted by the chimeric viruses that produce virions exhibiting a mature and stable FIV CA protein, and which incorporate the envelope glycoprotein and contain wild-type levels of viral genome RNA and reverse transcriptase. Further analysis of the latter group of chimeric SIVs demonstrated that they are non-infectious due to a post-entry impairment, such as uncoating of the viral core, reverse transcription or nuclear import of the preintegration complex. Furthermore, we show here that the carboxyl-terminus domain (CTD of the FIV CA has an intrinsic ability to dimerize in vitro and form high-molecular-weight oligomers, which, together with our finding that the FIV CA-CTD is sufficient to confer assembly competence to the resulting chimeric SIV Gag polyprotein, provides evidence that the CA-CTD exhibits more functional plasticity than the CA-NTD. Taken together, our results provide relevant information on the biological relationship between the CA proteins of primate and nonprimate lentiviruses.

  18. Insights into minor group rhinovirus uncoating: the X-ray structure of the HRV2 empty capsid.

    Directory of Open Access Journals (Sweden)

    Damià Garriga

    2012-01-01

    Full Text Available Upon attachment to their respective receptor, human rhinoviruses (HRVs are internalized into the host cell via different pathways but undergo similar structural changes. This ultimately results in the delivery of the viral RNA into the cytoplasm for replication. To improve our understanding of the conformational modifications associated with the release of the viral genome, we have determined the X-ray structure at 3.0 Å resolution of the end-stage of HRV2 uncoating, the empty capsid. The structure shows important conformational changes in the capsid protomer. In particular, a hinge movement around the hydrophobic pocket of VP1 allows a coordinated shift of VP2 and VP3. This overall displacement forces a reorganization of the inter-protomer interfaces, resulting in a particle expansion and in the opening of new channels in the capsid core. These new breaches in the capsid, opening one at the base of the canyon and the second at the particle two-fold axes, might act as gates for the externalization of the VP1 N-terminus and the extrusion of the viral RNA, respectively. The structural comparison between native and empty HRV2 particles unveils a number of pH-sensitive amino acid residues, conserved in rhinoviruses, which participate in the structural rearrangements involved in the uncoating process.

  19. Insights into minor group rhinovirus uncoating: the X-ray structure of the HRV2 empty capsid.

    Science.gov (United States)

    Garriga, Damià; Pickl-Herk, Angela; Luque, Daniel; Wruss, Jürgen; Castón, José R; Blaas, Dieter; Verdaguer, Núria

    2012-01-01

    Upon attachment to their respective receptor, human rhinoviruses (HRVs) are internalized into the host cell via different pathways but undergo similar structural changes. This ultimately results in the delivery of the viral RNA into the cytoplasm for replication. To improve our understanding of the conformational modifications associated with the release of the viral genome, we have determined the X-ray structure at 3.0 Å resolution of the end-stage of HRV2 uncoating, the empty capsid. The structure shows important conformational changes in the capsid protomer. In particular, a hinge movement around the hydrophobic pocket of VP1 allows a coordinated shift of VP2 and VP3. This overall displacement forces a reorganization of the inter-protomer interfaces, resulting in a particle expansion and in the opening of new channels in the capsid core. These new breaches in the capsid, opening one at the base of the canyon and the second at the particle two-fold axes, might act as gates for the externalization of the VP1 N-terminus and the extrusion of the viral RNA, respectively. The structural comparison between native and empty HRV2 particles unveils a number of pH-sensitive amino acid residues, conserved in rhinoviruses, which participate in the structural rearrangements involved in the uncoating process.

  20. Immunodominant epitopes mapped by synthetic peptides on the capsid protein of avian hepatitis E virus are non-protective.

    Science.gov (United States)

    Guo, Hailong; Zhou, E M; Sun, Z F; Meng, X J

    2008-03-01

    Avian hepatitis E virus (avian HEV) was recently discovered in chickens with hepatitis-splenomegaly syndrome in the United States. The open reading frame 2 (ORF2) protein of avian HEV has been shown to cross-react with human and swine HEV ORF2 proteins, and immunodominant antigenic epitopes on avian HEV ORF2 protein were identified in the predicted antigenic domains by synthetic peptides. However, whether these epitopes are protective against avian HEV infection has not been investigated. In this study, groups of chickens were immunized with keyhole limpet hemocyanin (KLH)-conjugated peptides and recombinant avian HEV ORF2 antigen followed by challenge with avian HEV virus to assess the protective capacity of these peptides containing the epitopes. While avian HEV ORF2 protein showed complete protection against infection, viremia and fecal virus shedding were found in all peptide-immunized chickens. Using purified IgY from normal, anti-peptide, and anti-avian HEV ORF2 chicken sera, an in-vitro neutralization and in-vivo monitoring assay was performed to further evaluate the neutralizing ability of anti-peptide IgY. Results showed that none of the anti-peptide IgY can neutralize avian HEV in vitro, as viremia, fecal virus shedding, and seroconversion appeared similarly in chickens inoculated with avian HEV mixed with anti-peptide IgY and chickens inoculated with avian HEV mixed with normal IgY. As expected, chickens inoculated with the avian HEV and anti-avian HEV ORF2 IgY mixture did not show detectable avian HEV infection. Taken together, the results of this study demonstrated that immunodominant epitopes on avian HEV ORF2 protein identified by synthetic peptides are non-protective, suggesting protective neutralizing epitope on avian HEV ORF2 may not be linear as is human HEV.

  1. Co-expression of Ubiquitin gene and capsid protein gene enhances the potency of DNA immunization of PCV2 in mice

    Directory of Open Access Journals (Sweden)

    Zhou Yanjun

    2011-05-01

    Full Text Available Abstract A recombinant plasmid that co-expressed ubiquitin and porcine circovirus type 2 (PCV2 virus capsid protein (Cap, denoted as pc-Ub-Cap, and a plasmid encoding PCV2 virus Cap alone, denoted as pc-Cap, were transfected into 293T cells. Indirect immunofluorescence (IIF and confocal microscopy were performed to measure the cellular expression of Cap. Three groups of mice were then vaccinated once every three weeks for a total of three doses with pc-Ub-Cap, pc-Cap or the empty vector pCAGGS, followed by challenging all mice intraperitoneally with 0.5 mL 106.5 TCID50/mL PCV2. To characterize the protective immune response against PCV2 infection in mice, assays of antibody titer (including different IgG isotypes, flow cytometric analysis (FCM, lymphocyte proliferation, cytokine production and viremia were evaluated. The results showed that pc-Ub-Cap and pc-Cap were efficiently expressed in 293T cells. However, pc-Ub-Cap-vaccinated animals had a significantly higher level of Cap-specific antibody and induced a stronger Th1 type cellular immune response than did pc-Cap-vaccinated animals, suggesting that ubiquitin conjugation improved both the cellular and humoral immune responses. Additionally, viral replication in blood was lower in the pc-Ub-Cap-vaccinated group than in the pc-Cap and empty vector groups, suggesting that the protective immunity induced by pc-Ub-Cap is superior to that induced by pc-Cap.

  2. Construction of Prophylactic Human Papillomavirus Type 16 L1 Capsid Protein Vaccine Delivered by Live Attenuated Shigella flexneri Strain sh42

    Institute of Scientific and Technical Information of China (English)

    Xiao-Feng YANG; Xin-Zhong QU; Kai WANG; Jin ZHENG; Lü-Sheng SI; Xiao-Ping DONG; Yi-Li WANG

    2005-01-01

    To express human papillomavirus (HPV) L1 capsid protein in the recombinant strain of Shigella and study the potential of a live attenuated Shigella-based HPV prophylactic vaccine in preventing HPV infection, the icsA/virG fragment of Shigella-based prokaryotic expression plasmid pHS3199 was constructed.HPV type 16 L 1 (HPV 16L 1) gene was inserted into plasmid pHS 3199 to form the pHS3199-HPV 16L1construct, and pHS3199-HPV16L1 was electroporated into a live attenuated Shigella strain sh42. Western blotting analysis showed that HPV 16L1 could be expressed stably in the recombinant strain sh42-HPV 16L1.Sereny test results were negative, which showed that the sh42-HPV16L1 lost virulence. However, the attenuated recombinant strain partially maintained the invasive property as indicated by the HeLa cell infection assay. Specific IgG, IgA antibody against HPV16L1 virus-like particles (VLPs) were detected in the sera,intestinal lavage and vaginal lavage from animals immunized by sh42-HPV 16L 1. The number of antibodysecreting cells in the spleen and draining lymph nodes were increased significantly compared with the control group. Sera from immunized animals inhibited murine hemagglutination induced by HPV16L1 VLPs, which indicated that the candidate vaccine could stimulate an efficient immune response in guinea pig's mucosal sites. This may be an effective strategy for the development of an HPV prophylactic oral vaccine.

  3. Enhancing mucosal immunity in mice by recombinant adenovirus expressing major epitopes of porcine circovirus-2 capsid protein delivered with cytosine-phosphate-guanosine oligodeoxynucleotides.

    Science.gov (United States)

    Chang, Hong-Tao; He, Xiu-Yuan; Liu, Yu-Feng; Chen, Lu; Guo, Quan-Hai; Yu, Qiu-Ying; Zhao, Jun; Wang, Xin-Wei; Yang, Xia; Wang, Chuan-Qing

    2014-01-01

    A recombinant replication-defective adenovirus expressing the major epitopes of porcine circovirus-2 (PCV-2) capsid protein (rAd/Cap/518) was previously constructed and shown to induce mucosal immunity in mice following intranasal delivery. In the present study, immune responses induced by intranasal immunization with a combination of rAd/Cap/518 and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) were evaluated in mice. The levels of PCV-2-specific IgG in serum and IgA in saliva, lung, and intestinal fluids were significantly higher in the group immunized with rAd/Cap/518 and CpG ODN than animals immunized with rAd/Cap/518 alone. The frequencies of IL-2-secreting CD4⁺ T cells and IFN-γ-producing CD8⁺ T cells were significantly higher in the combined immunization group than mice immunized with rAd/Cap/518 alone. The frequencies of CD3⁺, CD3⁺CD4⁺CD8⁻, and CD3⁺CD4⁻CD8⁺ T cells in the combined immunization group were similar to that treated with CpG ODN alone, but significantly higher than mice that did not receive CpG ODN. PCV-2 load after challenge in the combined immunization group was significantly lower than that in the phosphate-buffered saline placebo group and approximately 7-fold lower in the group treated with CpG ODN alone. These results indicate that rAd/Cap/518 combined with CpG ODN can enhance systemic and local mucosal immunity in mice, and represent a promising synergetic mucosal vaccine against PCV-2.

  4. Kinetics versus Thermodynamics in Virus Capsid Polymorphism.

    Science.gov (United States)

    Moerman, Pepijn; van der Schoot, Paul; Kegel, Willem

    2016-07-07

    Virus coat proteins spontaneously self-assemble into empty shells in aqueous solution under the appropriate physicochemical conditions, driven by an interaction free energy per bond on the order of 2-5 times the thermal energy kBT. For this seemingly modest interaction strength, each protein building block nonetheless gains a very large binding free energy, between 10 and 20 kBT. Because of this, there is debate about whether the assembly process is reversible or irreversible. Here we discuss capsid polymorphism observed in in vitro experiments from the perspective of nucleation theory and of the thermodynamics of mass action. We specifically consider the potential contribution of a curvature free energy term to the effective interaction potential between the proteins. From these models, we propose experiments that may conclusively reveal whether virus capsid assembly into a mixture of polymorphs is a reversible or an irreversible process.

  5. Use of recombinant capsid proteins in the development of a vaccine against foot-and-mouth disease virus (FMDV)

    DEFF Research Database (Denmark)

    Belsham, Graham; Bøtner, Anette

    2015-01-01

    -scale culling of infected, and potentially infected, animals there has been significant effort to develop new vaccines against this disease which avoid some, or all, of the deficiencies of current vaccines. A major focus has been on the use of systems that express the structural proteins of the virus that self....... The development and use of such improved vaccines should assist in the global efforts to control this important disease...

  6. Molecular studies on bromovirus capsid protein. VII. Selective packaging on BMV RNA4 by specific N-terminal arginine residuals.

    Science.gov (United States)

    Choi, Y G; Rao, A L

    2000-09-15

    An arginine-rich RNA-binding motif (ARM) found at the N-proximal region of Brome mosaic virus (BMV) coat protein (CP) adopts alpha-helical conformation and shares homology with CPs of plant and insect RNA viruses, HIV-Rev and Tat proteins, bacterial antiterminators, and ribosomal splicing factors. The ARM of BMV CP, consisting of amino acids 9 through 21 with six arginine residues, is essential for RNA binding and subsequent packaging. In this study analysis of the alpha-helical contents of wild-type and mutant peptides by circular dichroism spectra identified protein determinants required for such conformation. Electrophoretic mobility-shift assays between viral RNA and BMV CP peptides with either proline or alanine substitutions revealed that the interaction is nonspecific. Expression in vivo of mature full-length BMV CP subunits, having the same substitutions for each arginine within the ARM, derived from biologically active clones was found to be competent to assemble into infectious virions and cause visible symptom phenotypes in whole plants. However, analysis of virion progeny RNA profiles of CP variants and subsequent in vitro reassembly assays between mutant CP and four BMV RNAs unveiled the ability of arginine residues at positions 10, 13, or 14 of the ARM to confer selective packaging of BMV RNA4. Thus, BMV CP contains determinants that specifically interact with RNA4 to ensure selective packaging.

  7. Capsid, membrane and NS3 are the major viral proteins involved in autophagy induced by Japanese encephalitis virus.

    Science.gov (United States)

    Wang, Xiujin; Hou, Lei; Du, Jige; Zhou, Lei; Ge, Xinna; Guo, Xin; Yang, Hanchun

    2015-08-05

    Japanese encephalitis virus (JEV) is an important zoonotic pathogen causing viral encephalitis in human and reproductive failure in pigs. In the present study, we first examined the autophagy induced by JEV infection in host cells, and then analyzed the JEV proteins involving in autophagy induction, and further investigated the relationship between viral protein and immunity-related GTPases M (IRGM). Our results showed that JEV infection could induce autophagy in host cells and autophagy promoted the replication of JEV in vitro; the cells transfected with individual plasmid that was expressing C, M and NS3 had a significantly higher conversion of LC3-I/II, and enhanced LC3 signals with the fluorescence punctuates accumulation which was completely co-localized with LC3 and increased number of autophagosomes-like vesicles, suggesting that C, M and NS3 are the major viral proteins involving in autophagy induction upon JEV infection; the virus titer in the cells treated by the siRNA specific for IRGM had a significant decrease, and the NS3 signals in the cells transfected with the plasmid that was expressing NS3 were completely co-localized with the IRGM signals, suggesting that the NS3 of JEV could target IRGM which may play a role in the replication of JEV. Our findings help to understand the role of autophagy in JEV and other flaviviruses infections.

  8. Liposomal leakage induced by virus-derived peptides, viral proteins, and entire virions: rapid analysis by chip electrophoresis.

    Science.gov (United States)

    Weiss, Victor U; Bilek, Gerhard; Pickl-Herk, Angela; Subirats, Xavier; Niespodziana, Katarzyna; Valenta, Rudolf; Blaas, Dieter; Kenndler, Ernst

    2010-10-01

    Permeabilization of model lipid membranes by virus-derived peptides, viral proteins, and entire virions of human rhinovirus was assessed by quantifying the release of a fluorescent dye from liposomes via a novel chip electrophoretic assay. Liposomal leakage readily occurred upon incubation with the pH-sensitive synthetic fusogenic peptide GALA and, less efficiently, with a 24mer peptide (P1-N) derived from the N-terminus of the capsid protein VP1 of human rhinovirus 2 (HRV2) at acidic pH. Negative stain transmission electron microscopy showed that liposomes incubated with the rhinovirus-derived peptide remained largely intact. At similar concentrations, the GALA peptide caused gross morphological changes of the liposomes. On a molar basis, the leakage-inducing efficiency of the P1 peptide was by about 2 orders of magnitude inferior to that of recombinant VP1 (from HRV89) and entire HRV2. This underscores the role in membrane destabilization of VP1 domains remote from the N-terminus and the arrangement of the peptide in the context of the icosahedral virion. Our method is rapid, requires tiny amounts of sample, and allows for the parallel determination of released and retained liposomal cargo.

  9. Expression and Assembly Mechanism of the Capsid Proteins of a Satellite Virus (XSV) Associated with Macrobrachium rosenbergii Nodavirus

    Institute of Scientific and Technical Information of China (English)

    Jian-min WANG; Hua-jun ZHANG; Zheng-li SHI

    2008-01-01

    The extra small virus (XSV) is a satellite virus associated with Macrobrachium rosenbergii nodavirus (MrNV) and its genome consists of two overlapping ORFs, CP17 and CP16. Here we demonstrate that CP16 is expressed from the second AUG of the CP17 gene and is not a proteinase cleavage result of CP17. We further expressed CP17 and several truncated CP17s (in which the N- or C-terminus or both was deleted), respectively, in Escherichia coli. Except for the recombinant plasmid CP17ΔC10, all recombinant plasmids expressed soluble protein which assembled into virus-like particles (VLPs), suggesting that the C-terminus is important for VLP formation.

  10. Signaling from the embryo conditions Vp1-mediated repression of alpha-amylase genes in the aleurone of developing maize seeds.

    Science.gov (United States)

    Hoecker, U; Vasil, I K; McCarty, D R

    1999-08-01

    The VP1 transcription factor functions as both a repressor and an activator of gene expression in the developing aleurone. Vp1 activation of the anthocyanin pathway exhibits strict cell autonomy in aleurone. In contrast, Vp1-mediated repression of hydrolase genes in aleurone cells during seed development is determined by a combination of cell autonomous and cell non-autonomous signals. To analyze signaling between the embryo and aleurone during seed development, a T-B3La chromosome translocation was used to create seed that has non-concordant embryo and endosperm genotypes. We show that de-repression of an Amy-GUS reporter gene in developing vp1 mutant aleurone cells strongly depends on the presence of a viviparous embryo. Genetic ablation of the developing embryo in vp1 mutant and Vp1 seeds through the introduction of an early embryo mutation caused a similar enhancement of Amy-GUS expression in the aleurone, suggesting that the quiescent embryo present in normal seed is a critical source of inhibitory signals. Analysis of an ABA deficient vp1 vp5 double mutant indicates that ABA synthesized in the embyro interacts additively with Vp1 to prevent precocious induction of alpha-amylase genes in the aleurone of the developing seed. A lack of ABA synthesis, however, does not account for the strongly synergistic interaction between a viviparous vp1 embryo and mutant aleurone suggesting that a quiescent embyro is a source of other inhibitory signals.

  11. Use of recombinant capsid proteins in the development of a vaccine against the foot-and-mouth disease virus

    Directory of Open Access Journals (Sweden)

    Belsham GJ

    2015-02-01

    Full Text Available Graham J Belsham, Anette Bøtner National Veterinary Institute, Technical University of Denmark, Kalvehave, Denmark Abstract: Foot-and-mouth disease remains one of the world's most economically important diseases of livestock. It is caused by foot-and-mouth disease virus, a member of the picornavirus family. The virus replicates very rapidly and can be efficiently transmitted between hosts by a variety of routes. The disease has been effectively controlled in some parts of the world but remains endemic in many others, thus there is a constant risk of introduction of the disease into areas that are normally free of foot-and-mouth disease with potentially huge economic consequences. To reduce the need for large-scale culling of infected, and potentially infected, animals there has been significant effort to develop new vaccines against this disease which avoid some, or all, of the deficiencies of current vaccines. A major focus has been on the use of systems that express the structural proteins of the virus that self-assemble to generate “empty capsid” particles which share many features with the intact virus but lack the ribonucleic acid genome and are therefore non-infectious. Such particles can be “designed” to improve their stability or modify their antigenicity and can be produced without “high containment” facilities. The development and use of such improved vaccines should assist in the global efforts to control this important disease. Keywords: picornavirus, diagnostic assays, virus structure, infection, immune responses

  12. Rhinovirus-induced VP1-specific Antibodies are Group-specific and Associated With Severity of Respiratory Symptoms

    Directory of Open Access Journals (Sweden)

    Katarzyna Niespodziana

    2015-01-01

    Interpretation: Our results demonstrate that increases of antibodies towards the VP1 N-terminus are group-specific and associated with severity of respiratory symptoms and suggest that it may be possible to develop serological tests for identifying causative RV groups.

  13. The Suramin Derivative NF449 Interacts with the 5-fold Vertex of the Enterovirus A71 Capsid to Prevent Virus Attachment to PSGL-1 and Heparan Sulfate.

    Directory of Open Access Journals (Sweden)

    Yorihiro Nishimura

    2015-10-01

    Full Text Available NF449, a sulfated compound derived from the antiparasitic drug suramin, was previously reported to inhibit infection by enterovirus A71 (EV-A71. In the current work, we found that NF449 inhibits virus attachment to target cells, and specifically blocks virus interaction with two identified receptors--the P-selectin ligand, PSGL-1, and heparan sulfate glycosaminoglycan--with no effect on virus binding to a third receptor, the scavenger receptor SCARB2. We also examined a number of commercially available suramin analogues, and newly synthesized derivatives of NF449; among these, NF110 and NM16, like NF449, inhibited virus attachment at submicromolar concentrations. PSGL-1 and heparan sulfate, but not SCARB2, are both sulfated molecules, and their interaction with EV-A71 is thought to involve positively charged capsid residues, including a conserved lysine at VP1-244, near the icosahedral 5-fold vertex. We found that mutation of VP1-244 resulted in resistance to NF449, suggesting that this residue is involved in NF449 interaction with the virus capsid. Consistent with this idea, NF449 and NF110 prevented virus interaction with monoclonal antibody MA28-7, which specifically recognizes an epitope overlapping VP1-244 at the 5-fold vertex. Based on these observations we propose that NF449 and related compounds compete with sulfated receptor molecules for a binding site at the 5-fold vertex of the EV-A71 capsid.

  14. Phylogenetic analysis of VP1 gene sequences of waterfowl parvoviruses from the Mainland of China revealed genetic diversity and recombination.

    Science.gov (United States)

    Wang, Shao; Cheng, Xiao-Xia; Chen, Shao-Ying; Lin, Feng-Qiang; Chen, Shi-Long; Zhu, Xiao-Li; Wang, Jin-Xiang; Huang, Mei-Qing; Zheng, Min

    2016-03-01

    To determine the origin and evolution of goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) in the Mainland of China, phylogenetic and recombination analyses in the present study were performed on 32 complete VP1 gene sequences from China and other countries. Based on the phylogenetic analysis of the VP1 gene, GPV strains studied here from Mainland China (PRC) could be divided into three genotypes, namely PRC-I, PRC-II and PRC-III. Genotype PRC-I is indigenous to Mainland China. Only one GPV strain from Northeast China was of Genotype PRC-II and was thought to be imported from Europe. Genotype PRC-III, which was the most isolated genotype during 1999-2012, is related to GPVs in Taiwan and has been the predominant pathogen responsible for recent Derzy's disease outbreaks in Mainland China. Current vaccine strains used in Mainland China belong to Genotype PRC-I that is evolutionary distant from Genotypes PRC-II and PRC-III. In comparison, MDPV strains herein from Mainland China are clustered in a single group which is closely related to Taiwanese MDPV strains, and the full-length sequences of the VP1 gene of China MDPVs are phylogenetic closely related to the VP1 sequence of a Hungarian MDPV strain. Moreover, We also found that homologous recombination within VP1 gene plays a role in generating genetic diversity in GPV evolution. The GPV GDFSh from Guangdong Province appears to be the evolutionary product of a recombination event between parental GPV strains GD and B, while the major parent B proved to be a reference strain for virulent European GPVs. Our findings provide valuable information on waterfowl parvoviral evolution in Mainland China.

  15. Analysis on genomic characteristics of enterovirus 71 types of VP1 gene in Henan of 2010%河南省2010年肠道病毒71型VP1基因特征分析

    Institute of Scientific and Technical Information of China (English)

    卫海燕; 黄学勇; 许玉玲; 马宏; 陈豪敏; 许汴利

    2012-01-01

    OBJECTIVE To reveal the genomic characteristics of enterovirus 71 strains of VP1 gene from Hand-Foot-Mouth Disease, and analyze their molecular epidemiology. METHODS A total of 840 clinical specimens were collected and isolated, and 34 enterovirus among 71 types of VP1 gene were analyzed by bioinformatics software. RESULTS The VP1 of 34 isolated specimens was gained by sequencing. The nucleotide similarities of VP1 gene were at the range of 96.3% to 100%. Phylogenetic analysis based on VP1 regions revealed that the Henan enterovirus 71 in 2010 belonged to C4a in subgenotype C4, and no difference was found between the severe and mild illness. Cluster C4a enterovirus 71 was also found in henan and other seven provinces during 2003-2009. CONCLUSION 71 isolated strains of enterovirus in Henan of 2008 belong to C4a in the C4 subgenotype, which is in line with the predominant virus circulating in mainland China since 2004.%目的 对河南省2010年手足口病监测标本进行病原分离及VP1基因测序,了解分离病毒的基因特征及分子流行病学特点.方法 将2010年收集的手足口病患者粪便标本和肛拭子标本840份进行病毒分离鉴定并对34株病毒分离株测定肠道病毒71型VP1全序,利用生物信息学软件对序列分析,构建序列系统进化树.结果 测序获得34株来自河南省11个地市的VP1全长序列,分离株间的VP1区核苷酸相似性为96.3%~100%,系统进化分析显示属于C4基因型的C4a亚群,所有分离株均处于同一进化分支,轻重症间无明显差别.在2003~2009年的河南省和其他7省亦发现有C4a亚群存在.结论 2010年河南EV71分离株为C4基因型的C4a亚群,河南省2008年以来的分离株与2004年以来的中国大陆优势株流行趋势完全一致.

  16. Isolation and characterization of TgVP1, a type I vacuolar H+-translocating pyrophosphatase from Toxoplasma gondii. The dynamics of its subcellular localization and the cellular effects of a diphosphonate inhibitor.

    Science.gov (United States)

    Drozdowicz, Yolanda M; Shaw, Michael; Nishi, Manami; Striepen, Boris; Liwinski, Helene A; Roos, David S; Rea, Philip A

    2003-01-10

    Here we report the isolation and characterization of a type I vacuolar-type H(+)-pyrophosphatase (V-PPase), TgVP1, from an apicomplexan, Toxoplasma gondii, a parasitic protist that is particularly amenable to molecular and genetic manipulation. The 816-amino acid TgVP1 polypeptide is 50% sequence-identical (65% similar) to the prototypical type I V-PPase from Arabidopsis thaliana, AVP1, and contains all the sequence motifs characteristic of this pump category. Unlike AVP1 and other known type I enzymes, however, TgVP1 contains a 74-residue N-terminal extension encompassing a 42-residue N-terminal signal peptide sequence, sufficient for targeting proteins to the secretory pathway of T. gondii. Providing that the coding sequence for the entire N-terminal extension is omitted from the plasmid, transformation of Saccharomyces cerevisiae with plasmid-borne TgVP1 yields a stable and functional translation product that is competent in aminomethylenediphosphonate (AMDP)-inhibitable K(+)-activated pyrophosphate (PP(i)) hydrolysis and PP(i)-energized H(+) translocation. Immunofluorescence microscopy of both free and intracellular T. gondii tachyzoites using purified universal V-PPase polyclonal antibodies reveals a punctate apical distribution for the enzyme. Equivalent studies of the tachyzoites during host cell invasion, by contrast, disclose a transverse radial distribution in which the V-PPase is associated with a collar-like structure that migrates along the length of the parasite in synchrony with and in close apposition to the penetration furrow. Although treatment of T. gondii with AMDP concentrations as high as 100 microm had no discernible effect on the efficiency of host cell invasion and integration, concentrations commensurate with the I(50) for the inhibition of TgVP1 activity in vitro (0.9 microm) do inhibit cell division and elicit nuclear enlargement concomitant with the inflation and eventual disintegration of acidocalcisome-like vesicular structures. A

  17. Nonlinear Finite Element Analysis of Nanoindentation of Viral Capsids

    CERN Document Server

    Gibbons, M M; Gibbons, Melissa M.; Klug, William S.

    2006-01-01

    Recent Atomic Force Microscope (AFM) nanoindentation experiments measuring mechanical response of the protein shells of viruses have provided a quantitative description of their strength and elasticity. To better understand and interpret these measurements, and to elucidate the underlying mechanisms, this paper adopts a course-grained modeling approach within the framework of three-dimensional nonlinear continuum elasticity. Homogeneous, isotropic, elastic, thick shell models are proposed for two capsids: the spherical Cowpea Chlorotic Mottle Virus (CCMV), and the ellipsocylindrical bacteriophage $\\phi 29$. As analyzed by the finite element method, these models enable parametric characterization of the effects of AFM tip geometry, capsid dimensions, and capsid constitutive descriptions. The generally nonlinear force response of capsids to indentation is shown to be insensitive to constitutive details, and greatly influenced by geometry. Nonlinear stiffening and softening of the force response is dependent on ...

  18. RECOVIR: An application package to automatically identify some single stranded RNA viruses using capsid protein residues that uniquely distinguish among these viruses

    Directory of Open Access Journals (Sweden)

    Fox George E

    2007-10-01

    Full Text Available Abstract Background Most single stranded RNA (ssRNA viruses mutate rapidly to generate large number of strains having highly divergent capsid sequences. Accurate strain recognition in uncharacterized target capsid sequences is essential for epidemiology, diagnostics, and vaccine development. Strain recognition based on similarity scores between target sequences and sequences of homology matched reference strains is often time consuming and ambiguous. This is especially true if only partial target sequences are available or if different ssRNA virus families are jointly analyzed. In such cases, knowledge of residues that uniquely distinguish among known reference strains is critical for rapid and unambiguous strain identification. Conventional sequence comparisons are unable to identify such capsid residues due to high sequence divergence among the ssRNA virus reference strains. Consequently, automated general methods to reliably identify strains using strain distinguishing residues are not currently available. Results We present here RECOVIR ("recognize viruses", a software tool to automatically detect strains of caliciviruses and picornaviruses by comparing their capsid residues with built-in databases of residues that uniquely distinguish among known reference strains of these viruses. The databases were created by constructing partitioned phylogenetic trees of complete capsid sequences of these viruses. Strains were correctly identified for more than 300 complete and partial target sequences by comparing the database residues with the aligned residues of these sequences. It required about 5 seconds of real time to process each sequence. A Java-based user interface coupled with Perl-coded computational modules ensures high portability of the software. RECOVIR currently runs on Windows XP and Linux platforms. The software generalizes a manual method briefly outlined earlier for human caliciviruses. Conclusion This study shows implementation of

  19. Natural Type 3/Type 2 Intertypic Vaccine-Related Poliovirus Recombinants with the First Crossover Sites within the VP1 Capsid Coding Region

    DEFF Research Database (Denmark)

    Zhang, Yong; Zhu, Shuangli; Yan, Dongmei;

    2010-01-01

    Ten uncommon natural type 3/type 2 intertypic poliovirus recombinants were isolated from stool specimens from nine acute flaccid paralysis case patients and one healthy vaccinee in China from 2001 to 2008.......Ten uncommon natural type 3/type 2 intertypic poliovirus recombinants were isolated from stool specimens from nine acute flaccid paralysis case patients and one healthy vaccinee in China from 2001 to 2008....

  20. A single amino acid of human immunodeficiency virus type 2 capsid protein affects conformation of two external loops and viral sensitivity to TRIM5α.

    Directory of Open Access Journals (Sweden)

    Tadashi Miyamoto

    Full Text Available We previously reported that human immunodeficiency virus type 2 (HIV-2 carrying alanine or glutamine but not proline at position 120 of the capsid protein (CA could grow in the presence of anti-viral factor TRIM5α of cynomolgus monkey (CM. To elucidate details of the interaction between the CA and TRIM5α, we generated mutant HIV-2 viruses, each carrying one of the remaining 17 possible amino acid residues, and examined their sensitivity to CM TRIM5α-mediated restriction. Results showed that hydrophobic residues or those with ring structures were associated with sensitivity, while those with small side chains or amide groups conferred resistance. Molecular dynamics simulation study revealed a structural basis for the differential TRIM5α sensitivities. The mutations at position 120 in the loop between helices 6 and 7 (L6/7 affected conformation of the neighboring loop between helices 4 and 5 (L4/5, and sensitive viruses had a common L4/5 conformation. In addition, the common L4/5 structures of the sensitive viruses were associated with a decreased probability of hydrogen bond formation between the 97th aspartic acid in L4/5 and the 119th arginine in L6/7. When we introduced aspartic acid-to-alanine substitution at position 97 (D97A of the resistant virus carrying glutamine at position 120 to disrupt hydrogen bond formation, the resultant virus became moderately sensitive. Interestingly, the virus carrying glutamic acid at position 120 showed resistance, while its predicted L4/5 conformation was similar to those of sensitive viruses. The D97A substitution failed to alter the resistance of this particular virus, indicating that the 120th amino acid residue itself is also involved in sensitivity regardless of the L4/5 conformation. These results suggested that a hydrogen bond between the L4/5 and L6/7 modulates the overall structure of the exposed surface of the CA, but the amino acid residue at position 120 is also directly involved in CM TRIM5

  1. High capsid-genome correlation facilitates creation of AAV libraries for directed evolution.

    Science.gov (United States)

    Nonnenmacher, Mathieu; van Bakel, Harm; Hajjar, Roger J; Weber, Thomas

    2015-04-01

    Directed evolution of adeno-associated virus (AAV) through successive rounds of phenotypic selection is a powerful method to isolate variants with improved properties from large libraries of capsid mutants. Importantly, AAV libraries used for directed evolution are based on the "natural" AAV genome organization where the capsid proteins are encoded in cis from replicating genomes. This is necessary to allow the recovery of the capsid DNA after each step of phenotypic selection. For directed evolution to be used successfully, it is essential to minimize the random mixing of capsomers and the encapsidation of nonmatching viral genomes during the production of the viral libraries. Here, we demonstrate that multiple AAV capsid variants expressed from Rep/Cap containing viral genomes result in near-homogeneous capsids that display an unexpectedly high capsid-DNA correlation. Next-generation sequencing of AAV progeny generated by bulk transfection of a semi-random peptide library showed a strong counter-selection of capsid variants encoding premature stop codons, which further supports a strong capsid-genome identity correlation. Overall, our observations demonstrate that production of "natural" AAVs results in low capsid mosaicism and high capsid-genome correlation. These unique properties allow the production of highly diverse AAV libraries in a one-step procedure with a minimal loss in phenotype-genotype correlation.

  2. Proteolytic Disassembly of Viral Outer Capsid Proteins Is Crucial for Reovirus-Mediated Type-I Interferon Induction in Both Reovirus-Susceptible and Reovirus-Refractory Tumor Cells

    Directory of Open Access Journals (Sweden)

    Yuki Katayama

    2015-01-01

    Full Text Available Oncolytic reovirus induces innate immune responses, which contribute to the antitumor activity of reovirus, following in vivo application. Reovirus-induced innate immune responses have been relatively well characterized in immune cells and mouse embryonic fibroblasts cells; however, the mechanisms and profiles of reovirus-induced innate immune responses in human tumor cells have not been well understood. In particular, differences in reovirus-induced innate immune responses between reovirus-susceptible and reovirus-refractory tumor cells remain unknown, although the intracellular trafficking of reovirus differs between these tumor cells. In this study, we examined reovirus-induced upregulation of interferon- (IFN- β and of the proapoptotic gene, Noxa, in reovirus-susceptible and -refractory tumor cells. IFN-β and Noxa were significantly induced by reovirus via the IFN-β promoter stimulator-1 (IPS-1 signaling in both types of tumor cells. Inhibition of cathepsins B and L, which are important for disassembly of reovirus outer capsid proteins and escape into cytoplasm, largely suppressed reovirus-induced upregulation of IFN-β and Noxa expression in not only reovirus-susceptible but also reovirus-refractory tumor cells. These results indicated that in both reovirus-susceptible and reovirus-refractory tumor cells, disassembly of the outer capsid proteins by cathepsins and the escape into the cytoplasm were crucial steps for reovirus-induced innate immunity.

  3. A minimal representation of the self-assembly of virus capsids

    CERN Document Server

    Llorente, J M Gomez; Breton, J

    2013-01-01

    Viruses are biological nanosystems with a capsid of protein-made capsomer units that encloses and protects the genetic material responsible for their replication. Here we show how the geometrical constraints of the capsomer-capsomer interaction in icosahedral capsids fix the form of the shortest and universal truncated multipolar expansion of the two-body interaction between capsomers. The structures of many of the icosahedral and related virus capsids are located as single lowest energy states of this potential energy surface. Our approach unveils relevant features of the natural design of the capsids and can be of interest in fields of nanoscience and nanotechnology where similar hollow convex structures are relevant.

  4. Varicella-zoster virus induces the formation of dynamic nuclear capsid aggregates

    Energy Technology Data Exchange (ETDEWEB)

    Lebrun, Marielle [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Thelen, Nicolas; Thiry, Marc [University of Liege (ULg), GIGA-Neurosciences, Laboratory of Cellular and Tissular Biology, Liege (Belgium); Riva, Laura; Ote, Isabelle; Condé, Claude; Vandevenne, Patricia [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Di Valentin, Emmanuel [University of Liege (ULg), GIGA-Viral Vectors Platform, Liege (Belgium); Bontems, Sébastien [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Sadzot-Delvaux, Catherine, E-mail: csadzot@ulg.ac.be [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium)

    2014-04-15

    The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond to capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane. - Highlights: • We created a recombinant VZV expressing the small capsid protein fused to the eGFP. • We identified nuclear dense structures containing capsid and procapsid proteins. • Correlative microscopy showed that the structures correspond to capsid aggregates. • Procapsids and partial capsids are found within the aggregates of WT and eGFP-23 VZV. • FRAP and FLIP experiments demonstrated that they are dynamic structures.

  5. Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

    Science.gov (United States)

    Cai, X.; Chang, D.; Rottinghaus, S.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

  6. Epitope-distal effects accompany the binding of two distinct antibodies to hepatitis B virus capsids

    NARCIS (Netherlands)

    Bereszczak, J.Z.; Rose, R.J.; Duijn, E. van; Watts, N.R.; Wingfield, P.T.; Steven, A.C.; Heck, A.J.R.

    2013-01-01

    Infection of humans by hepatitis B virus (HBV) induces the copious production of antibodies directed against the capsid protein (Cp). A large variety of anticapsid antibodies have been identified that differ in their epitopes. These data, and the status of the capsid as a major clinical antigen, mot

  7. Identification and Analyses of Vp-1B Allelic Variation in Triticum spelta L%斯卑尔脱小麦Vp-1B基因的等位变异鉴定和序列分析

    Institute of Scientific and Technical Information of China (English)

    曲若端; 杨燕; 孟建宇; 李淑芬; 赵宏斌

    2013-01-01

    Triticum spelta L is one of the first level genetic resources of wheat.Among many genes that can affect the Pre-harvest sprouting,the viviparous-1 (Vp-1) gene is the main important regulator that can promotes mature and dormancy of embryo in bread wheat.In this study,by the method of homologous cloning,the PCR fragments in Spelta 80,Spelta 225,Spelta 217 and Spelta 220 which had different PHS tolerance,Were sub-cloned and the sequence alignment showed that many variations exist in B3 domain,especially,in the third intron and the fifth intron,including long fragment deletions.In addition,there also exist many SNPs located in the first and the sixth exons,which changed the types of the amino acid.Based on the overlapping parts of the forward and reverse sequencing data,the partial sequences of the Vp-1B was joined into a whole length after their accuracy was judged,and named as SVp-1Bc,SVp-1Bd and SVp-1Be,respectively.Sequence analysis showed that SVp-1Bc has a deletions of 83 bp in the third intron,SVp-1Bd has a deletion of 25 bp fragments in the same intron,and SVp-1Be has a deletion of 10 bp which located in the fifth intron region of the Vp-1B gene.In view of the relationship between Triticum aestivum L and Triticum spelta L,these allelic variations of the Vp-1B in Triticum spelta L may associate with seed dormancy.%斯卑尔脱小麦是小麦的一级基因源.在影响小麦穗发芽的众多基因中,Vp-1是调节胚发育,促进胚成熟和休眠的主要转录调节因子.本研究通过同源克隆测序的方法分析了4种不同基因型斯卑尔脱小麦品种Vp-1基因在3B染色体上的等位变异,进一步分析了Vp-1基因在斯卑尔脱小麦和六倍体普通小麦中的差异.研究表明不同休眠特性的4份斯卑尔脱小麦Spelta80,Spelta220,Spelta217和Spelta225在3B染色体的Vp-1B基因上存在大量的等位变异,主要集中在Vp-1B基因的第三和第五内含子中,表现为一些大片段的序列缺失,另外的变异位于Vp

  8. 肠道病毒71型VP1蛋白在昆虫杆状病毒表达系统的表达

    Institute of Scientific and Technical Information of China (English)

    赵培培; 孔枕枕; 刘海冰; 茅凌翔; 苏兆亮; 仝佳; 王胜军; 陈建国

    2015-01-01

    目的 利用昆虫杆状病毒表达系统表达肠道病毒71型VP1蛋白并对其进行纯化.方法 首先通过化学合成法合成EV71 VP1基因,然后将EV71 VP1基因插入到供体质粒pFastBac1中,构成重组质粒pFastBac1-VP1,再转入JM190感受态细菌扩增,然后提取重组质粒pFastBac1-VP1转入DH10BacTM感受态细胞,通过转座从而获得重组质粒bacmid-VP1,采用脂质体Cellfectin Reagent将重组质粒bacmid-VP1转染Sf9细胞.通过SDS-PAGE、Western blot法检测目的蛋白的水平,经镍离子亲和层析,纯化VP1蛋白.结果 SDS-PAGE及Western blot法检测获得蛋白的相对分子质量与预期结果一致.Bradford分析纯化后蛋白的浓度为70 μg/mL,纯化度达90%.结论 利用昆虫杆状病毒表达系统成功表达EV71 VP1蛋白.

  9. Active cAMP-dependent protein kinase incorporated within highly purified HIV-1 particles is required for viral infectivity and interacts with viral capsid protein.

    Science.gov (United States)

    Cartier, Christine; Hemonnot, Bénédicte; Gay, Bernard; Bardy, Martine; Sanchiz, Céline; Devaux, Christian; Briant, Laurence

    2003-09-12

    Host cell components, including protein kinases such as ERK-2/mitogen-activated protein kinase, incorporated within human immunodeficiency virus type 1 (HIV-1) virions play a pivotal role in the ability of HIV to infect and replicate in permissive cells. The present work provides evidence that the catalytic subunit of cAMP-dependent protein kinase (C-PKA) is packaged within HIV-1 virions as demonstrated using purified subtilisin-digested viral particles. Virus-associated C-PKA was shown to be enzymatically active and able to phosphorylate synthetic substrate in vitro. Suppression of virion-associated C-PKA activity by specific synthetic inhibitor had no apparent effect on viral precursor maturation and virus assembly. However, virus-associated C-PKA activity was demonstrated to regulate HIV-1 infectivity as assessed by single round infection assays performed by using viruses produced from cells expressing an inactive form of C-PKA. In addition, virus-associated C-PKA was found to co-precipitate with and to phosphorylate the CAp24gag protein. Altogether our results indicate that virus-associated C-PKA regulates HIV-1 infectivity, possibly by catalyzing phosphorylation of the viral CAp24gag protein.

  10. SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption.

    Science.gov (United States)

    Ho, Michelle L; Adler, Benjamin A; Torre, Michael L; Silberg, Jonathan J; Suh, Junghae

    2013-12-20

    Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.

  11. In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite.

    Science.gov (United States)

    Erban, Tomas; Harant, Karel; Hubalek, Martin; Vitamvas, Pavel; Kamler, Martin; Poltronieri, Palmiro; Tyl, Jan; Markovic, Martin; Titera, Dalibor

    2015-09-11

    We investigated pathogens in the parasitic honeybee mite Varroa destructor using nanoLC-MS/MS (TripleTOF) and 2D-E-MS/MS proteomics approaches supplemented with affinity-chromatography to concentrate trace target proteins. Peptides were detected from the currently uncharacterized Varroa destructor Macula-like virus (VdMLV), the deformed wing virus (DWV)-complex and the acute bee paralysis virus (ABPV). Peptide alignments revealed detection of complete structural DWV-complex block VP2-VP1-VP3, VDV-1 helicase and single-amino-acid substitution A/K/Q in VP1, the ABPV structural block VP1-VP4-VP2-VP3 including uncleaved VP4/VP2, and VdMLV coat protein. Isoforms of viral structural proteins of highest abundance were localized via 2D-E. The presence of all types of capsid/coat proteins of a particular virus suggested the presence of virions in Varroa. Also, matches between the MWs of viral structural proteins on 2D-E and their theoretical MWs indicated that viruses were not digested. The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding. Hemolymph feeding also resulted in the detection of a variety of honeybee proteins. The advantages of MS-based proteomics for pathogen detection, false-positive pathogen detection, virus replication, posttranslational modifications, and the presence of honeybee proteins in Varroa are discussed.

  12. The capsid protein p38 of turnip crinkle virus is associated with the suppression of cucumber mosaic virus in Arabidopsis thaliana co-infected with cucumber mosaic virus and turnip crinkle virus.

    Science.gov (United States)

    Chen, Ying-Juan; Zhang, Jing; Liu, Jian; Deng, Xing-Guang; Zhang, Ping; Zhu, Tong; Chen, Li-Juan; Bao, Wei-Kai; Xi, De-Hui; Lin, Hong-Hui

    2014-08-01

    Infection of plants by multiple viruses is common in nature. Cucumber mosaic virus (CMV) and Turnip crinkle virus (TCV) belong to different families, but Arabidopsis thaliana and Nicotiana benthamiana are commonly shared hosts for both viruses. In this study, we found that TCV provides effective resistance to infection by CMV in Arabidopsis plants co-infected by both viruses, and this antagonistic effect is much weaker when the two viruses are inoculated into different leaves of the same plant. However, similar antagonism is not observed in N. benthamiana plants. We further demonstrate that disrupting the RNA silencing-mediated defense of the Arabidopsis host does not affect this antagonism, but capsid protein (CP or p38)-defective mutant TCV loses the ability to repress CMV, suggesting that TCV CP plays an important role in the antagonistic effect of TCV toward CMV in Arabidopsis plants co-infected with both viruses.

  13. Diminished reovirus capsid stability alters disease pathogenesis and littermate transmission.

    Directory of Open Access Journals (Sweden)

    Joshua D Doyle

    2015-03-01

    Full Text Available Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses.

  14. Development of a One-Step Duplex RT-PCR Method for the Simultaneous Detection of VP3/VP1 and VP1/P2B Regions of the Hepatitis A Virus.

    Science.gov (United States)

    Kim, Mi-Ju; Lee, Shin-Young; Kim, Hyun-Joong; Lee, Jeong Su; Joo, In Sun; Kwak, Hyo Sun; Kim, Hae-Yeong

    2016-08-28

    The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10(1) copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10(2) copies/20 g fresh lettuce, 9.7 × 10(3) copies/20 g frozen strawberries, and 4.1 × 10(3) copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR.

  15. Serological detection and analysis of anti-VP1 responses against various enteroviruses (EV) (EV-A, EV-B and EV-C) in Chinese individuals

    Science.gov (United States)

    Gao, Caixia; Ding, Yingying; Zhou, Peng; Feng, Jiaojiao; Qian, Baohua; Lin, Ziyu; Wang, Lili; Wang, Jinhong; Zhao, Chunyan; Li, Xiangyu; Cao, Mingmei; Peng, Heng; Rui, Bing; Pan, Wei

    2016-01-01

    The overall serological prevalence of EV infections based on ELISA remains unknown. In the present study, the antibody responses against VP1 of the EV-A species (enterovirus 71 (EV71), Coxsackievirus A16 (CA16), Coxsackievirus A5 (CA5) and Coxsackievirus A6 (CA6)), of the EV-B species (Coxsackievirus B3 (CB3)), and of the EV-C species (Poliovirus 1 (PV1)) were detected and analyzed by a NEIBM (novel evolved immunoglobulin-binding molecule)-based ELISA in Shanghai blood donors. The serological prevalence of anti-CB3 VP1 antibodies was demonstrated to show the highest level, with anti-PV1 VP1 antibodies at the second highest level, and anti-CA5, CA6, CA16 and EV71 VP1 antibodies at a comparatively low level. All reactions were significantly correlated at different levels, which were approximately proportional to their sequence similarities. Antibody responses against EV71 VP1 showed obvious differences with responses against other EV-A viruses. Obvious differences in antibody responses between August 2013 and May 2014 were revealed. These findings are the first to describe the detailed information of the serological prevalence of human antibody responses against the VP1 of EV-A, B and C viruses, and could be helpful for understanding of the ubiquity of EV infections and for identifying an effective approach for seroepidemiological surveillance based on ELISA. PMID:26917423

  16. Protection against Foot-and-Mouth Disease Virus in Guinea Pigs via Oral Administration of Recombinant Lactobacillus plantarum Expressing VP1.

    Directory of Open Access Journals (Sweden)

    Miao Wang

    Full Text Available Mucosal vaccination is an effective strategy for generating antigen-specific immune responses against mucosal infections of foot-and-mouth disease virus (FMDV. In this study, Lactobacillus plantarum strains NC8 and WCFS1 were used as oral delivery vehicles containing a pSIP411-VP1 recombinant plasmid to initiate mucosal and systemic immune responses in guinea pigs. Guinea pigs were orally vaccinated (three doses with NC8-pSIP411, NC8-pSIP411-VP1, WCFS1-pSIP411, WCFS1-pSIP411-VP1 or milk. Animals immunized with NC8-pSIP411-VP1 and WCFS1-pSIP411-VP1 developed high levels of antigen-specific serum IgG, IgA, IgM, mucosal secretory IgA (sIgA and neutralizing antibodies, and revealed stronger cell-mediated immune responses and enhanced protection against FMDV challenge compared with control groups. The recombinant pSIP411-VP1 effectively improved immunoprotection against FMDV in guinea pigs.

  17. Capsid modification of adeno-associated virus and tumor targeting gene therapy

    Institute of Scientific and Technical Information of China (English)

    XU ZengHu; ZHOU XiuMei; SHI WenFang; QIAN QiJun

    2008-01-01

    Targeting is critical for successful tumor gene therapy. The adeno-associated virus (AAV) has aroused wide concern due to its excellent advantages over other viral vectors in gene therapy. AAV has a broad infection spectrum, which also results in poor specificity towards tissues or cells and low transduction efficiency. Therefore, it is imperative to improve target and transduction efficiency in AAV-mediated gene therapy. Up to now, researchers have developed many strategies to modify AAV capsids for improving targeting or retargeting only desired cells. These strategies include not only traditional chemical modification, phage display technology, modification of AAV capsid genome, chimeric vectors and so on, but also many novel strategies involved in marker rescue strategy, direct evolution of capsid proteins, direct display random peptides on AAV capsid, AAVP (AAV-Phage), and etc. This review will summarize the advances of researches on the capsid modification of AAV to target malignant cells.

  18. Modeling of the rotavirus group C capsid predicts a surface topology distinct from other rotavirus species.

    Science.gov (United States)

    Eren, Elif; Zamuda, Kimberly; Patton, John T

    2016-01-01

    Rotavirus C (RVC) causes sporadic gastroenteritis in adults and is an established enteric pathogen of swine. Because RVC strains grow poorly in cell culture, which hinders generation of virion-derived RVC triple-layered-particle (TLP) structures, we used the known Rotavirus A (RVA) capsid structure to model the human RVC (Bristol) capsid. Comparative analysis of RVA and RVC capsid proteins showed major differences at the VP7 layer, an important target region for vaccine development due to its antigenic properties. Our model predicted the presence of a surface extended loop in RVC, which could form a major antigenic site on the capsid. We analyzed variations in the glycosylation patterns among RV capsids and identified group specific conserved sites. In addition, our results showed a smaller RVC VP4 foot, which protrudes toward the intermediate VP6 layer, in comparison to that of RVA. Finally, our results showed major structural differences at the VP8* glycan recognition sites.

  19. Capsid proteins from field strains of foot-and-mouth disease virus confer a pathogenic phenotype in cattle on an attenuated, cell-culture-adapted virus

    DEFF Research Database (Denmark)

    Bøtner, Anette; Kakker, Naresh K.; Barbezange, Cyril

    2011-01-01

    Chimeric foot-and-mouth disease viruses (FMDVs) have been generated from plasmids containing full-length FMDV cDNAs and characterized. The parental virus cDNA was derived from the cell-culture-adapted O1Kaufbeuren B64 (O1K B64) strain. Chimeric viruses, containing capsid coding sequences derived...... cells than the rescued parental O1K B64 virus. The two chimeric viruses displayed the expected antigenicity in serotype-specific antigen ELISAs. Following inoculation of each virus into cattle, the rescued O1K B64 strain proved to be attenuated whereas, with each chimeric virus, typical clinical signs...... from the O/UKG/34/2001 or A/Turkey 2/2006 field viruses, were constructed using the backbone from the O1K B64 cDNA, and viable viruses (O1K/O-UKG and O1K/A-Tur, respectively) were successfully rescued in each case. These viruses grew well in primary bovine thyroid cells but grew less efficiently in BHK...

  20. Assessment of stress tolerance, productivity, and forage quality in T1 transgenic alfalfa co-overexpressing ZxNHX and ZxVP1-1 from Zygophyllum xanthoxylum

    Directory of Open Access Journals (Sweden)

    Peng Kang

    2016-10-01

    Full Text Available Salinization, desertification, and soil nutrient deprivation are threatening the production of alfalfa (Medicago sativa L. in northern China. We have previously generated T0 transgenic alfalfa co-overexpressing Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 genes with enhanced salt and drought tolerance. To further develop this excellent breeding material into the new forage cultivar, stress tolerance, productivity, and forage quality of T1 transgenic alfalfa (GM were assessed in this study. The GM inherited the traits of salt and drought tolerance from T0 generation. Most importantly, co-overexpression of ZxNHX and ZxVP1-1 enhanced the tolerance to Pi deficiency in GM, which was associated with more Pi accumulation in plants. Meanwhile, T1 transgenic alfalfa developed a larger root system with increased root size, root dry weight and root/shoot ratio, which may be one important reason for the improvement of phosphorus nutrition and high biomass accumulation in GM under various conditions. GM also accumulated more crude protein, crude fibre, crude fat, and crude ash than wild-type (WT plants, especially under stress conditions and in the field. More interestingly, the crude fat contents sharply dropped in WT (by 66%-74%, whereas showed no change or decreased less in GM, when subjected to salinity, drought or low-Pi. Our results indicate that T1 transgenic alfalfa co-overexpressing ZxNHX and ZxVP1-1 shows stronger stress tolerance, higher productivity and better forage quality. This study provides a solid foundation for creating the alfalfa cultivars with high yield, good quality and wide adaptability on saline, dry and nutrient-deprived marginal lands of northern China.

  1. Structure of the capsid of Kilham rat virus from small-angle neutron scattering

    Energy Technology Data Exchange (ETDEWEB)

    Wobbe, C.R.; Mitra, S.; Ramakrishnan, V.

    1984-12-18

    The structure of empty capsids of Kilham rat virus, an autonomous parvovirus with icosahedral symmetry, was investigated by small-angle neutron scattering. From the forward scatter, the molecular weight was determined to be 4.0 x 10(6), and from the Guinier region, the radius of gyration was found to be 105 A in D2O and 104 A in H/sub 2/O. On the basis of the capsid molecular weight and the molecular weights and relative abundances of the capsid proteins, the authors propose that the capsid has a triangulation number of 1. Extended scattering curves and mathematical modeling revealed that the capsid consists of two shells of protein, the inner shell extending from 58 to 91 A in D2O and from 50 to 91 A in H/sub 2/O and containing 11% of the capsid scattering mass, and the outer shell extending to 121 A in H/sub 2/O and D2O. The inner shell appears to have a higher content of basic amino acids than the outer shell, based on its lower scattering density in D2O than in H/sub 2/O. The authors propose that all three capsid proteins contribute to the inner shell and that this basic region serves DNA binding and partial charge neutralization functions.

  2. VP1 crystal structure-guided exploration and optimization of 4,5-dimethoxybenzene-based inhibitors of rhinovirus 14 infection.

    Science.gov (United States)

    Da Costa, Laurène; Roche, Manon; Scheers, Els; Coluccia, Antonio; Neyts, Johan; Terme, Thierry; Leyssen, Pieter; Silvestri, Romano; Vanelle, Patrice

    2016-06-10

    Human rhinoviruses (HRV) are the predominant cause of common colds and flu-like illnesses, but are also responsible for virus-induced exacerbations of asthma and chronic obstructive pulmonary disease. However, to date, no drug has been approved yet for clinical use. In this study, we present the results of the structure-based lead optimization of a class of new small-molecule inhibitors that we previously reported to bind into the pocket beneath the canyon of the VP1 protein. A small series of analogues that we designed based on the available structure and interaction data were synthesized and evaluated for their potency to inhibit the replication of HRV serotype 14. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4-(pyridin-4-yl)phenyl)ethanol (3v) was found to be a potent inhibitor exhibiting micromolar activity (EC50 = 3.4 ± 1.0 μM) with a toxicity for HeLa cells that was significantly lower than that of our previous hit (LPCRW_0005, CC50 = 104.0 ± 22.2 μM; 3v, CC50 > 263 μM).

  3. Packaging Double-Helical DNA into Viral Capsids: Structures, Forces, and Energetics

    OpenAIRE

    Petrov, Anton S.; Harvey, Stephen C.

    2008-01-01

    Small, icosahedral double-stranded DNA bacteriophage pack their genomes tightly into preformed protein capsids using an ATP-driven motor. Coarse-grain molecular-mechanics models provide a detailed picture of DNA packaging in bacteriophage, revealing how conformation depends on capsid size and shape, and the presence or absence of a protein core. The forces that oppose packaging have large contributions from both electrostatic repulsions and the entropic penalty of confining the DNA into the c...

  4. Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1.

    Science.gov (United States)

    Liu, Lin; Ma, Yongping; Zhou, Huicong; Wu, Mingjun

    2016-01-01

    The nontoxic heat-labile toxin (LT) B subunit (LTB) was used as mucosal adjuvant experimentally. However, the mechanism of LTB adjuvant was still unclear. The LTB and enterovirus 71 (EV71) VP1 subunit (EVP1) were constructed in pET32 and expressed in E. coli BL21, respectively. The immunogenicity of purified EVP1 and the adjuvanticity of LTB were evaluated via intranasal immunization EVP1 plus LTB in Balb/c mice. In order to elucidate the proteome change triggered by the adjuvant of LTB, the proteomic profiles of LTB, EVP1, and LTB plus EVP1 were quantitatively analyzed by iTRAQ-LC-MS/MS (isobaric tags for relative and absolute quantitation; liquid chromatography-tandem mass spectrometry) in murine macrophage RAW264.7. The proteomic data were analyzed by bioinformatics and validated by western blot analysis. The predicted protein interactions were confirmed using LTB pull-down and the LTB processing pathway was validated by confocal microscopy. The results showed that LTB significantly boosted EVP1 specific systematic and mucosal antibodies. A total of 3666 differential proteins were identified in the three groups. Pathway enrichment of proteomic data predicted that LTB upregulated the specific and dominant MAPK (mitogen-activated protein kinase) signaling pathway and the protein processing in endoplasmic reticulum (PPER) pathway, whereas LTB or EVP1 did not significantly upregulate these two signaling pathways. Confocal microscopy and LTB pull-down assays confirmed that the LTB adjuvant was endocytosed and processed through endocytosis (ENS)-lysosomal-endoplasmic reticulum (ER) system.

  5. Genetic Analysis of the VP1 Region of Human Enterovirus 71 Strains Isolated in Fuyang, China, During 2008

    Institute of Scientific and Technical Information of China (English)

    Shao-hui MA; Jian-sheng LIU; Jing-jing WANG; Hai-jing SHI; Hui-juan YANG; Jun-ying CHEN; Long-ding LIU; Qi-han LI

    2009-01-01

    Enterovirus 71 (EV71) is a common cause of Hand, foot, and mouth disease (HFMD) and may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity of EV71, we determined and analyzed the complete VP1 sequences (891 nucleotides) from nine EV71 strains isolated in Fuyang, China. We found that nine EV71 strains isolated were over 98% homologous at the nucleotide level and 93%-100% homologous to members of the C4 subgenogroup. At the amino acid level, these Fuyang strains were 99% -100% homologous to one another, 97%-100% homologous to members of the C4 subgenogroup, and the histidine(H) at amino acid position 22 was conserved among the Fuyang strains. The results indicate that Fuyang isolates belong to genotype C4, and an H at position 22 appears to be a marker for the Fuyang strains.

  6. [Hand-foot-mouth disease pathogen separation and EV71 VP1 gene analysis in Sanmenxia City, Henan Province, China].

    Science.gov (United States)

    Wu, Shu-xing; Wu, Jing-fu; Yang, Jie; Wei, Hai-yan; Xu, Yu-ling; Huang, Xue-yong

    2014-11-01

    The aim of this study was to understand the enterovirus types and biological features of pediatric cases of HFMD in Sanmenxia City during 2011, and compare the latter to a cohort of healthy children. Stool samples of 55 cases of HFMD and 60 healthy children were collected for the isolation and identification of enteroviruses using RNA extraction and real-time RT-PCR assays. EV71 and CA16 were identified by nucleotide sequencing using virus-specific VP1 primers; for the other enteroviruses, 012/011 and 008/013 primers were used for amplification and sequencing. The results were analysed by sequence alignment with known sequences, and the characteristics of the EV71 VP1 gene were also analyzed. The detection rates for enteroviruses in cases of HFMD and healthy children were 52.73% (29/55) and 18.33% (11/60), respectively. Among these, there were 22 cases of EV71, four cases of CA16 and three cases of other enteroviruses in the cases with HFMD. Eleven healthy children had intestinal viruses, of which nine were Coxsackie B virus strains (81.82%, 9/11). Gene sequencing of the 19 EV71 strains illustrated that they were all subgenotype C4a, but the evolutionary tree showed an obvious clustering between cases from Lingbao City and Lushi County. This study demonstrates that the EV71 subgenotype C4a and CA16 strains were the most common cause of HFMD in Sanmenxia City in 2011, and that Coxsackie B strains were prevalent in healthy children. This finding may indicate that there is a widespread source of recessive infection in the community.

  7. Capsid coding region diversity of re-emerging lineage C foot-and-mouth disease virus serotype Asia1 from India.

    Science.gov (United States)

    Subramaniam, Saravanan; Mohapatra, Jajati K; Das, Biswajit; Sharma, Gaurav K; Biswal, Jitendra K; Mahajan, Sonalika; Misri, Jyoti; Dash, Bana B; Pattnaik, Bramhadev

    2015-07-01

    Foot-and-mouth disease virus (FMDV) serotype Asia1 was first reported in India in 1951, where three major genetic lineages (B, C and D) of this serotype have been described until now. In this study, the capsid protein coding region of serotype Asia1 viruses (n = 99) from India were analyzed, giving importance to the viruses circulating since 2007. All of the isolates (n = 50) recovered during 2007-2013 were found to group within the re-emerging cluster of lineage C (designated as sublineage C(R)). The evolutionary rate of sublineage C(R) was estimated to be slightly higher than that of the serotype as a whole, and the time of the most recent common ancestor for this cluster was estimated to be approximately 2001. In comparison to the older isolates of lineage C (1993-2001), the re-emerging viruses showed variation at eight amino acid positions, including substitutions at the antigenically critical residues VP279 and VP2131. However, no direct correlation was found between sequence variations and antigenic relationships. The number of codons under positive selection and the nature of the selection pressure varied widely among the structural proteins, implying a heterogeneous pattern of evolution in serotype Asia1. While episodic diversifying selection appears to play a major role in shaping the evolution of VP1 and VP3, selection pressure acting on codons of VP2 is largely pervasive. Further, episodic positive selection appears to be responsible for the early diversification of lineage C. Recombination events identified in the structural protein coding region indicates its probable role in adaptive evolution of serotype Asia1 viruses.

  8. Structural rigidity in the capsid assembly of cowpea chlorotic mottle virus

    Science.gov (United States)

    Hespenheide, B. M.; Jacobs, D. J.; Thorpe, M. F.

    2004-11-01

    The cowpea chlorotic mottle virus (CCMV) has a protein cage, or capsid, which encloses its genetic material. The structure of the capsid consists of 180 copies of a single protein that self-assemble inside a cell to form a complete capsid with icosahedral symmetry. The icosahedral surface can be naturally divided into pentagonal and hexagonal faces, and the formation of either of these faces has been proposed to be the first step in the capsid assembly process. We have used the software FIRST to analyse the rigidity of pentameric and hexameric substructures of the complete capsid to explore the viability of certain capsid assembly pathways. FIRST uses the 3D pebble game to determine structural rigidity, and a brief description of this algorithm, as applied to body-bar networks, is given here. We find that the pentameric substructure, which corresponds to a pentagonal face on the icosahedral surface, provides the best structural properties for nucleating the capsid assembly process, consistent with experimental observations.

  9. Structural rigidity in the capsid assembly of cowpea chlorotic mottle virus

    Energy Technology Data Exchange (ETDEWEB)

    Hespenheide, B M [Department of Physics and Astronomy, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504 (United States); Jacobs, D J [Department of Physics and Astronomy, California State University, 18111 Nordhoff Street, Northridge, CA 91330-8268 (United States); Thorpe, M F [Department of Physics and Astronomy, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504 (United States)

    2004-11-10

    The cowpea chlorotic mottle virus (CCMV) has a protein cage, or capsid, which encloses its genetic material. The structure of the capsid consists of 180 copies of a single protein that self-assemble inside a cell to form a complete capsid with icosahedral symmetry. The icosahedral surface can be naturally divided into pentagonal and hexagonal faces, and the formation of either of these faces has been proposed to be the first step in the capsid assembly process. We have used the software FIRST to analyse the rigidity of pentameric and hexameric substructures of the complete capsid to explore the viability of certain capsid assembly pathways. FIRST uses the 3D pebble game to determine structural rigidity, and a brief description of this algorithm, as applied to body-bar networks, is given here. We find that the pentameric substructure, which corresponds to a pentagonal face on the icosahedral surface, provides the best structural properties for nucleating the capsid assembly process, consistent with experimental observations.

  10. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

    Energy Technology Data Exchange (ETDEWEB)

    Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo [College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China); Wang, Junwei, E-mail: jwwang@neau.edu.cn [College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China)

    2011-05-27

    Highlights: {yields} All three capsid proteins can be expressed in insect cells in baculovirus expression system. {yields} All three recombinant proteins were spontaneously self-assemble into virus-like particles whose size and appearance were similar to those of native purified GPV virions. {yields} The immunogenicity of GPV-VLPs was better than commercial inactivated vaccine and attenuated vaccine. -- Abstract: Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.

  11. An alphavirus temperature-sensitive capsid mutant reveals stages of nucleocapsid assembly

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yan, E-mail: yzheng15@students.kgi.edu; Kielian, Margaret, E-mail: margaret.kielian@einstein.yu.edu

    2015-10-15

    Alphaviruses have a nucleocapsid core composed of the RNA genome surrounded by an icosahedral lattice of capsid protein. An insertion after position 186 in the capsid protein produced a strongly temperature-sensitive growth phenotype. Even when the structural proteins were synthesized at the permissive temperature (28 °C), subsequent incubation of the cells at the non-permissive temperature (37 °C) dramatically decreased mutant capsid protein stability and particle assembly. Electron microscopy confirmed the presence of cytoplasmic nucleocapsids in mutant-infected cells cultured at the permissive temperature, but these nucleocapsids were not stable to sucrose gradient separation. In contrast, nucleocapsids isolated from mutant virus particles had similar stability to that of wildtype virus. Our data support a model in which cytoplasmic nucleocapsids go through a maturation step during packaging into virus particles. The insertion site lies in the interface between capsid proteins in the assembled nucleocapsid, suggesting the region where such a stabilizing transition occurs. - Highlights: • We characterize an alphavirus capsid insertion mutation. • These capsid mutants are highly temperature sensitive for growth. • The insertion affects nucleocapsid stability. • Results suggest that the nucleocapsid is stabilized during virus budding.

  12. Assembly of bacteriophage T7. Dimensions of the bacteriophage and its capsids

    Energy Technology Data Exchange (ETDEWEB)

    Stroud, R.M.; Serwer, P.; Ross, M.J.

    1981-12-01

    The dimensions of bacteriophage T7 and T7 capsids have been investigated by small-angle x-ray scattering. Phage T7 behaves like a sphere of uniform density with an outer radius of 301 +/- 2 A (excluding the phage tail) and a calculated volume for protein plus nucleic acid of 1.14 +/- 0.05 x 10/sup -16/ ml. The outer radius determined of T7 phage in solution is approx.30% greater than the radius measured from electron micrographs, which indicates that considerable shrinkage occurs during preparation for electron microscopy. Capsids that have a phagelike envelope and do not contain DNA were obtained from lysates of T7-infected Escherichia coli (capsid II) and by separating the capsid component of T7 phage from the phage DNA by means of temperature shock (capsid IV). In both cases the peak protein density is at a radius of 275 A; the outer radius is 286 +/- 4 A, approx.5% smaller than the envelope of T7 phage. The thickness of the envelope of capsid II is 22 +/- 4 A, consistent with the thickness of protein estimated to be 23 +/- 5 A in whole T7 phage, as seen on electron micrographs in which the internal DNA is positively stained. The volume in T7 phage available to package DNA is estimated to be 9.2 +/- 0.4 x 10/sup -17/ ml. The packaged DNA adopts a regular packing with 23.6 A interplanar spacing between DNA strands. The angular width of the 23.6 A reflection shows that the mean DNA-DNA spacing throughout the phage head is 27.5 +/- <2.2 A. A T7 precursor capsid (capsid I) expands when pelleted for x-ray scattering in the ultracentrifuge to essentially the same outer dimensions as for capsids II and IV. This expansion of capsid I can be prevented by fixing with glutaraldehyde; fixed capsid I has peak density at a radius of 247 A, 10% less than capsid II or IV.

  13. The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition.

    Science.gov (United States)

    Goh, Lucas Y H; Hobson-Peters, Jody; Prow, Natalie A; Baker, Kelly; Piyasena, Thisun B H; Taylor, Carmel T; Rana, Ashok; Hastie, Marcus L; Gorman, Jeff J; Hall, Roy A

    2015-06-08

    Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1-35 and 140-210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

  14. The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition

    Directory of Open Access Journals (Sweden)

    Lucas Y. H. Goh

    2015-06-01

    Full Text Available Chikungunya virus (CHIKV is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs previously generated towards the capsid protein (CP of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1–35 and 140–210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

  15. Human polyoma JC virus minor capsid proteins, VP2 and VP3, enhance large T antigen binding to the origin of viral DNA replication: evidence for their involvement in regulation of the viral DNA replication.

    Science.gov (United States)

    Saribas, A Sami; Mun, Sarah; Johnson, Jaslyn; El-Hajmoussa, Mohammad; White, Martyn K; Safak, Mahmut

    2014-01-20

    JC virus (JCV) lytically infects the oligodendrocytes in the central nervous system in a subset of immunocompromized patients and causes the demyelinating disease, progressive multifocal leukoencephalopathy. JCV replicates and assembles into infectious virions in the nucleus. However, understanding the molecular mechanisms of its virion biogenesis remains elusive. In this report, we have attempted to shed more light on this process by investigating molecular interactions between large T antigen (LT-Ag), Hsp70 and minor capsid proteins, VP2/VP3. We demonstrated that Hsp70 interacts with VP2/VP3 and LT-Ag; and accumulates heavily in the nucleus of the infected cells. We also showed that VP2/VP3 associates with LT-Ag through their DNA binding domains resulting in enhancement in LT-Ag DNA binding to Ori and induction in viral DNA replication. Altogether, our results suggest that VP2/VP3 and Hsp70 actively participate in JCV DNA replication and may play critical roles in coupling of viral DNA replication to virion encapsidation.

  16. Expression and Purification of Major Capsid Protein of Orange-spotted Grouper Nervous Necrosis Virus%斜带石斑鱼神经坏死病毒主衣壳蛋白的原核表达与纯化

    Institute of Scientific and Technical Information of China (English)

    陈晓艳; 翁少萍; 殷志新; 何建国

    2005-01-01

    将含有斜带石斑鱼Epinephelus coioides神经坏死病毒(orange-spotted grouper nervous necrosis virus,OGNNV)主衣壳蛋白(major capsid protein, MCP)基因的重组表达质粒载体pET32a-MCP转化到大肠杆菌BL21(DE3)中进行融合表达,经SDS-PAGE分析和Western-blot鉴定,证实了重组大肠杆菌融合表达了斜带石斑鱼神经坏死病毒主衣壳蛋白.表达的融合蛋白主要以不可溶的包涵体形式存在,提取的包涵体中融合蛋白含量占60%以上,经柱层析纯化蛋白,纯化度达90%以上.

  17. Assembly of recombinant Israeli Acute Paralysis Virus capsids.

    Directory of Open Access Journals (Sweden)

    Junyuan Ren

    Full Text Available The dicistrovirus Israeli Acute Paralysis Virus (IAPV has been implicated in the worldwide decline of honey bees. Studies of IAPV and many other bee viruses in pure culture are restricted by available isolates and permissive cell culture. Here we show that coupling the IAPV major structural precursor protein ORF2 to its cognate 3C-like processing enzyme results in processing of the precursor to the individual structural proteins in a number of insect cell lines following expression by a recombinant baculovirus. The efficiency of expression is influenced by the level of IAPV 3C protein and moderation of its activity is required for optimal expression. The mature IAPV structural proteins assembled into empty capsids that migrated as particles on sucrose velocity gradients and showed typical dicistrovirus like morphology when examined by electron microscopy. Monoclonal antibodies raised to recombinant capsids were configured into a diagnostic test specific for the presence of IAPV. Recombinant capsids for each of the many bee viruses within the picornavirus family may provide virus specific reagents for the on-going investigation of the causes of honeybee loss.

  18. Characteristics of a foot-and-mouth disease virus with a partial VP1 G-H loop deletion in experimentally infected cattle

    DEFF Research Database (Denmark)

    Fowler, Veronica; Bashiruddin, John B.; Belsham, Graham;

    2014-01-01

    Previous work in cattle illustrated the protective efficacy and negative marker potential of a A serotype foot-and-mouth disease virus (FMDV) vaccine prepared from a virus lacking a significant portion of the VP1 G-H loop (termed A(−)). Since this deletion also includes the arginine...

  19. Co-expression of xerophyte Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 confers enhanced salinity tolerance in chimeric sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Wu, Guo-Qiang; Feng, Rui-Jun; Wang, Suo-Min; Wang, Chun-Mei; Bao, Ai-Ke; Wei, Li; Yuan, Hui-Jun

    2015-01-01

    Salinity is one of the major abiotic stresses that limit the growth and productivity of sugar beet (Beta vulgaris L.). To improve sugar beet's salinity tolerance, the ZxNHX and ZxVP1-1 genes encoding tonoplast Na(+)/H(+) antiporter and H(+)-PPase from xerophyte Zygophyllum xanthoxylum were co-expressed by Agrobacterium tumefaciens-mediated transformation. It is showed here that co-expression of ZxNHX and ZxVP1-1 confers enhanced salinity tolerance to the transformed sugar beet plants compared with the wild-type (WT) plants. The chimeric plants grew well in the presence of high salinity (400 mM NaCl), whereas WT plants displayed chlorosis and died within 8 days. Compared to WT plants, the chimeric plants co-expressing ZxNHX and ZxVP1-1 accumulated more proline, Na(+) and K(+) in their leaves and petioles when exposed to high salinity, which caused lower solute potential, retained more water and thus subjected to lesser cell membrane damage. Interestingly, the chimeric plants accumulated higher sucrose, glucose and fructose contents in their storage roots than WT plants in the absence or presence of high salinity. Our results suggested that co-expression of ZxNHX and ZxVP1-1 improved the osmoregulatory capacity in chimeric sugar beet through increased compartmentalization of ions into the vacuoles by enhancing the activity of proton pumps and thus mitigated Na(+)-toxicity for plants.

  20. Structural Transitions and Energy Landscape for Cowpea Chlorotic Mottle Virus Capsid Mechanics from Nanomanipulation in Vitro and in Silico

    Science.gov (United States)

    Kononova, Olga; Snijder, Joost; Brasch, Melanie; Cornelissen, Jeroen; Dima, Ruxandra I.; Marx, Kenneth A.; Wuite, Gijs J. L.; Roos, Wouter H.; Barsegov, Valeri

    2013-10-01

    Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Virus shells can have applications as nanocontainers and delivery vehicles in biotechnology and medicine. Combined AFM experiments and computational modeling on sub-second timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus (CCMV) capsid show that the capsid's physical properties are dynamic and local characteristics of the structure, which depend on the magnitude and geometry of mechanical input. Surprisingly, under large deformations the CCMV capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state dH = 11.5 - 12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending, and the entropy change TdS = 5.1 - 5.8 MJ/mol is mostly due to coherent in-plane rearrangements of protein chains, which result in the capsid stiffening. Dynamic coupling of these modes defines the extent of elasticity and reversibility of capsid mechanical deformation. This emerging picture illuminates how unique physico-chemical properties of protein nanoshells help define their structure and morphology, and determine their viruses' biological function.

  1. Magic-angle spinning NMR of intact bacteriophages: Insights into the capsid, DNA and their interface

    Science.gov (United States)

    Abramov, Gili; Morag, Omry; Goldbourt, Amir

    2015-04-01

    Bacteriophages are viruses that infect bacteria. They are complex macromolecular assemblies, which are composed of multiple protein subunits that protect genomic material and deliver it to specific hosts. Various biophysical techniques have been used to characterize their structure in order to unravel phage morphogenesis. Yet, most bacteriophages are non-crystalline and have very high molecular weights, in the order of tens of MegaDaltons. Therefore, complete atomic-resolution characterization on such systems that encompass both capsid and DNA is scarce. In this perspective article we demonstrate how magic-angle spinning solid-state NMR has and is used to characterize in detail bacteriophage viruses, including filamentous and icosahedral phage. We discuss the process of sample preparation, spectral assignment of both capsid and DNA and the use of chemical shifts and dipolar couplings to probe the capsid-DNA interface, describe capsid structure and dynamics and extract structural differences between viruses.

  2. Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with Altered Tropism

    OpenAIRE

    Wu, Pei; Xiao, Wu; Conlon, Thomas; Hughes, Jeffrey; Agbandje-McKenna, Mavis; FERKOL, THOMAS; Flotte, Terence; Muzyczka, Nicholas

    2000-01-01

    Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mut...

  3. Refinement of herpesvirus B-capsid structure on parallel supercomputers.

    Science.gov (United States)

    Zhou, Z H; Chiu, W; Haskell, K; Spears, H; Jakana, J; Rixon, F J; Scott, L R

    1998-01-01

    Electron cryomicroscopy and icosahedral reconstruction are used to obtain the three-dimensional structure of the 1250-A-diameter herpesvirus B-capsid. The centers and orientations of particles in focal pairs of 400-kV, spot-scan micrographs are determined and iteratively refined by common-lines-based local and global refinement procedures. We describe the rationale behind choosing shared-memory multiprocessor computers for executing the global refinement, which is the most computationally intensive step in the reconstruction procedure. This refinement has been implemented on three different shared-memory supercomputers. The speedup and efficiency are evaluated by using test data sets with different numbers of particles and processors. Using this parallel refinement program, we refine the herpesvirus B-capsid from 355-particle images to 13-A resolution. The map shows new structural features and interactions of the protein subunits in the three distinct morphological units: penton, hexon, and triplex of this T = 16 icosahedral particle.

  4. Conformational Changes in the Capsid of a Calicivirus upon Interaction with Its Functional Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Ossiboff, Robert J.; Zhou, Yi; Lightfoot, Patrick J.; Prasad, B.V. Venkataram; Parker, John S.L. (Baylor); (Cornell)

    2010-07-19

    Nonenveloped viral capsids are metastable structures that undergo conformational changes during virus entry that lead to interactions of the capsid or capsid fragments with the cell membrane. For members of the Caliciviridae, neither the nature of these structural changes in the capsid nor the factor(s) responsible for inducing these changes is known. Feline functional adhesion molecule A (fJAM-A) mediates the attachment and infectious viral entry of feline calicivirus (FCV). Here, we show that the infectivity of some FCV isolates is neutralized following incubation with the soluble receptor at 37 C. We used this property to select mutants resistant to preincubation with the soluble receptor. We isolated and sequenced 24 soluble receptor-resistant (srr) mutants and characterized the growth properties and receptor-binding activities of eight mutants. The location of the mutations within the capsid structure of FCV was mapped using a new 3.6-{angstrom} structure of native FCV. The srr mutations mapped to the surface of the P2 domain were buried at the protruding domain dimer interface or were present in inaccessible regions of the capsid protein. Coupled with data showing that both the parental FCV and the srr mutants underwent increases in hydrophobicity upon incubation with the soluble receptor at 37 C, these findings indicate that FCV likely undergoes conformational change upon interaction with its receptor. Changes in FCV capsid conformation following its interaction with fJAM-A may be important for subsequent interactions of the capsid with cellular membranes, membrane penetration, and genome delivery.

  5. Genetic diversity of the VP1/VP2 gene of canine parvovirus type 2b amplified from clinical specimens in Brazil Diversidade genética no gene VP1/VP2 do parvovirus canino tipo 2b amplificado de material clínico no Brasil

    OpenAIRE

    Cesar A. D. Pereira; Edison Luiz Durigon

    2000-01-01

    We evaluated the genetic diversity in the VP1/VP2 gene of CPV type 2b isolates from symptomatic dogs in Brazil. A total of 21 isolates collected from 1990 through 1995 previously typed as CPV2b by PCR assay were studied. Overall we found a high degree of similarity among sequences from different CPV clinical isolates collected. Genetic analysis of this selected region gave no indication of a specific Brazilian parvovirus lineage.Neste estudo foi avaliada a diversidade genética no gene VP1/VP2...

  6. 轻或重症手足口病患儿HEV71分离鉴定及其VP1基因型分析%Isolation and identification of HEV71 strains from children with hand-foot-and-mouth disease in different conditions and analysis of their VP1 genotypes

    Institute of Scientific and Technical Information of China (English)

    蒋卓婧; 何婷婷; 陈金堃; 严杰

    2016-01-01

    目的 从不同病情手足口病(HFMD)患儿中分离鉴定人肠道病毒71型(HEV71)并分析其VP1基因型及药物结合位点突变情况.方法 从2014年浙江省绍兴地区轻、重症各4例HFMD患儿标本中分离HEV71并用RT-PCR进行鉴定.采用RT-PCR扩增病毒分离株全长VP1基因并测序.采用专业生物信息学软件,对HEV71分离株进行VP1基因分型、确定VP1基因中药物结合位点突变情况及构建HEV71分离株系统进化树.结果 8例HFMD患儿标本中均分离出HEV71.HEV71分离株与文献报道的VP1-C4a基因亚型的核苷酸和氨基酸序列相似性分别高达97.2%~98.8%和99.3%~99.7%.2例重症患儿HEV71分离株分别在VP1基因序列第179位药物结合位点发生V179L点突变.5株HEV71与安徽和湖南分离株、3株与上海和山东分离株亲缘关系较近.结论 本地区不同病情HFMD患儿分离的HEV71均为VP1-C4a基因亚型,重症患儿HEV71分离株VP1基因第179位药物结合位点突变可能与病情严重程度有关.

  7. Risk Assessment of Cervical Lesion by Combined Detection of Papillomavirus L1 Capsid Protein and Human Papillomavirus Genotyping, Thinprep-cytology Test%HPV L1壳蛋白联合HPV分型、TCT检测技术对子宫颈病变进展风险的评估

    Institute of Scientific and Technical Information of China (English)

    宋晓霞; 刘玉玲; 杨晓; 王丽丽

    2013-01-01

    Objective:To explore risk assessment of cervical lesion and guidance of the best clinical triage management and treatment by combined detection of human papillomavirus LI capsid protein and human papillomavirus(HPV) genotyping, thinprep cytology test(TCT). Methods:Retrospective analysis of 1 593 women of cervical cancer screening in the gynecological clinic of the Second Affiliated Hospital of Zhengzhou University from September 2010 to December 2011 ,and TCT and genotyping of HPV-DNA testing at the same time,in which 592 patients who was HPV-positive or TCT positive or both abnormal were sent to colposcopic biopsy for pathological examination and who was HPV-DNA typing-positive were detection of HPV L1 capsid protein expression. Results:TCT combined with HPV-DNA detection in cervical intraepithelial neoplasia HI (CIN III ) and the above cases had the highest rate and reached 100% positivity in squamous cell carcinoma (SCC) ;the positive rate of HPV LI capsid protein expression show a decreased trend with the increasing level of cervical lesions,the expression of HPV LI capsid protein and SCC was O. The positive expression rate of HPVL1 capsid protein in each group was significantly different (P<0.05). Conclusions:It is an essential indicator that TCT combined HPV genotyping in cervical lesions screening,and HPVL1 capsid protein detection had important guiding value on risk assessment of cervical lesions. Three factors effective combination can be timely and accurate diversion and treatment of patients with cervical lesions.%目的:探讨人乳头瘤病毒L1 (human papilloma virus L1,HPV L1)壳蛋白联合HPV分型、液基薄层细胞学(thinprap cytology test,TCT)技术对预测子宫颈病变进展的风险评估以及指导临床最佳的分流管理与治疗.方法:回顾性分析2010年9月-2011年12月在郑州大学第二附属医院妇科门诊因宫颈病变就诊的1 593例有性生活的妇女,即同时进行TCT和HPV-DNA分型检测,对其中HPV阳性

  8. Enhancing immune responses of EV71 VP1 DNA vaccine by co-inoculating plasmid IL-12 or GM-CSF expressing vector in mice.

    Science.gov (United States)

    Peng, X; Fang, X; Li, J; Kong, L; Li, B; Ding, X

    2016-01-01

    Enterovirus 71 (EV71) is a major causative viral agent for large outbreaks of hand, foot, and mouth disease in children and infants, yet there is no vaccine or effective antiviral treatment for severe EV71 infection. The immunogenicity of EV71 VP1 DNA vaccine and the immunoregulatory activity of interleukin-12 (IL-12) or granulocyte-monocyte colony stimulating factor (GM-CSF) were investigated. DNA vaccine plasmids, pcDNA-VP1, pcDNA-IL-12 and pcDNA-GM-CSF were constructed and inoculated into BALB/c mice with or without pcDNA-IL-12 or pcDNA-GM-CSF by intramuscular injection. Cellular and humoral immune responses were assessed by indirect ELISA, lymphocyte proliferation assays, cytokine release assay and FACS. The VP1 DNA vaccine had good immunogenicity and can induce specific humoral and cellular immunity in BALB/c mice, while IL-2 or GM-CSF plays an immunoadjuvant role and enhances specific immune responses. This study provides a frame of reference for the design of DNA vaccines against EV71.

  9. Hidden symmetry of small spherical viruses and organization principles in "anomalous" and double-shelled capsid nanoassemblies.

    Science.gov (United States)

    Rochal, S B; Konevtsova, O V; Myasnikova, A E; Lorman, V L

    2016-09-29

    We propose the principles of structural organization in spherical nanoassemblies with icosahedral symmetry constituted by asymmetric protein molecules. The approach modifies the paradigmatic geometrical Caspar and Klug (CK) model of icosahedral viral capsids and demonstrates the common origin of both the "anomalous" and conventional capsid structures. In contrast to all previous models of "anomalous" viral capsids the proposed modified model conserves the basic structural principles of the CK approach and reveals the common hidden symmetry underlying all small viral shells. We demonstrate the common genesis of the "anomalous" and conventional capsids and explain their structures in the same frame. The organization principles are derived from the group theory analysis of the positional order on the spherical surface. The relationship between the modified CK geometrical model and the theory of two-dimensional spherical crystallization is discussed. We also apply the proposed approach to complex double-shelled capsids and capsids with protruding knob-like proteins. The introduced notion of commensurability for the concentric nanoshells explains the peculiarities of their organization and helps to predict analogous, but yet undiscovered, double-shelled viral capsid nanostructures.

  10. Dynamic pathways for viral capsid assembly

    OpenAIRE

    Hagan, Michael F.; Chandler, David

    2006-01-01

    We develop a class of models with which we simulate the assembly of particles into T1 capsid-like objects using Newtonian dynamics. By simulating assembly for many different values of system parameters, we vary the forces that drive assembly. For some ranges of parameters, assembly is facile, while for others, assembly is dynamically frustrated by kinetic traps corresponding to malformed or incompletely formed capsids. Our simulations sample many independent trajectories at various capsomer c...

  11. Clone and sequence analysis of 5’RACE of PvVP1 in Paspalumvaginatum%海滨雀稗液泡膜H+-PPase(PvVP1)5′端的克隆和序列分析

    Institute of Scientific and Technical Information of China (English)

    宋辉; 南志标; 蔡小宁; 钟小仙; 顾洪如

    2014-01-01

    根据已公布液泡膜 H+-PPase基因家族同源序列保守区设计简并引物,克隆出海滨雀稗中PvVP1基因的中间序列,然后用快速扩增cDNA末端(rapid amplification of cDNA end,RACE)方法,从海滨雀稗中克隆到PvVP15′cDNA序列。该序列ORF长1605 bp,编码535个氨基酸,其编码序列、氨基酸序列与玉米相应基因的同源性分别为93%和99%。%A pair of degenerated primers were designed based on previously cloned homologous cDNA seg-ments.The middle segment of PvVP1 was cloned,then the PvVP1 5 ’cDNA sequence also obtained using 5 ’ RACE.The open reading frame (ORF)of this sequence was 1605 bp,encoding 534 amino acids.The codon and amino acid sequences of the 5’ends of thePvVP1 gene shared 93% and 99% similarity,respectively,with those of Zeamays.

  12. The interaction of the HSV-1 tegument proteins pUL36 and pUL37 is essential for secondary envelopment during viral egress.

    Science.gov (United States)

    Kelly, Barbara J; Bauerfeind, Rudolf; Binz, Anne; Sodeik, Beate; Laimbacher, Andrea S; Fraefel, Cornel; Diefenbach, Russell J

    2014-04-01

    The herpes simplex virus type 1 (HSV-1) tegument proteins pUL36 (VP1/2) and pUL37 are essential for viral egress. We previously defined a minimal domain in HSV-1 pUL36, residues 548-572, as important for binding pUL37. Here, we investigated the role of this region in binding to pUL37 and facilitating viral replication. We deleted residues 548-572 in frame in a virus containing a mRFP tag at the N-terminus of the capsid protein VP26 and an eGFP tag at the C-terminus of pUL37 (HSV-1pUL36∆548-572). This mutant virus was unable to generate plaques in Vero cells, indicating that deletion of this region of pUL36 blocks viral replication. Imaging of HSV-1pUL36∆548-572-infected Vero cells, in comparison to parental and resucant, revealed a block in secondary envelopment of cytoplasmic capsids. In addition, immunoblot analysis suggested that failure to bind pUL37 affected the stability of pUL36. This study provides further insight into the role of this essential interaction.

  13. Selective Inhibitor of Nuclear Export (SINE) Compounds Alter New World Alphavirus Capsid Localization and Reduce Viral Replication in Mammalian Cells.

    Science.gov (United States)

    Lundberg, Lindsay; Pinkham, Chelsea; de la Fuente, Cynthia; Brahms, Ashwini; Shafagati, Nazly; Wagstaff, Kylie M; Jans, David A; Tamir, Sharon; Kehn-Hall, Kylene

    2016-11-01

    The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/β1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host's primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus.

  14. Breaking a virus: Identifying molecular level failure modes of a viral capsid by multiscale modeling

    Science.gov (United States)

    Krishnamani, V.; Globisch, C.; Peter, C.; Deserno, M.

    2016-07-01

    We use coarse-grained (CG) simulations to study the deformation of empty Cowpea Chlorotic Mottle Virus (CCMV) capsids under uniaxial compression, from the initial elastic response up to capsid breakage. Our CG model is based on the MARTINI force field and has been amended by a stabilizing elastic network, acting only within individual proteins, that was tuned to capture the fluctuation spectrum of capsid protein dimers, obtained from all atom simulations. We have previously shown that this model predicts force-compression curves that match AFM indentation experiments on empty CCMV capsids. Here we investigate details of the actual breaking events when the CCMV capsid finally fails. We present a symmetry classification of all relevant protein contacts and show that they differ significantly in terms of stability. Specifically, we show that interfaces which break readily are precisely those which are believed to form last during assembly, even though some of them might share the same contacts as other non-breaking interfaces. In particular, the interfaces that form pentamers of dimers never break, while the virtually identical interfaces within hexamers of dimers readily do. Since these units differ in the large-scale geometry and, most noticeably, the cone-angle at the center of the 5- or 6-fold vertex, we propose that the hexameric unit fails because it is pre-stressed. This not only suggests that hexamers of dimers form less frequently during the early stages of assembly; it also offers a natural explanation for the well-known β-barrel motif at the hexameric center as a post-aggregation stabilization mechanism. Finally, we identify those amino acid contacts within all key protein interfaces that are most persistent during compressive deformation of the capsid, thereby providing potential targets for mutation studies aiming to elucidate the key contacts upon which overall stability rests.

  15. Breaking a virus: Identifying molecular level failure modes of a viral capsid by multiscale modeling

    Science.gov (United States)

    Krishnamani, V.; Globisch, C.; Peter, C.; Deserno, M.

    2016-10-01

    We use coarse-grained (CG) simulations to study the deformation of empty Cowpea Chlorotic Mottle Virus (CCMV) capsids under uniaxial compression, from the initial elastic response up to capsid breakage. Our CG model is based on the MARTINI force field and has been amended by a stabilizing elastic network, acting only within individual proteins, that was tuned to capture the fluctuation spectrum of capsid protein dimers, obtained from all atom simulations. We have previously shown that this model predicts force-compression curves that match AFM indentation experiments on empty CCMV capsids. Here we investigate details of the actual breaking events when the CCMV capsid finally fails. We present a symmetry classification of all relevant protein contacts and show that they differ significantly in terms of stability. Specifically, we show that interfaces which break readily are precisely those which are believed to form last during assembly, even though some of them might share the same contacts as other non-breaking interfaces. In particular, the interfaces that form pentamers of dimers never break, while the virtually identical interfaces within hexamers of dimers readily do. Since these units differ in the large-scale geometry and, most noticeably, the cone-angle at the center of the 5- or 6-fold vertex, we propose that the hexameric unit fails because it is pre-stressed. This not only suggests that hexamers of dimers form less frequently during the early stages of assembly; it also offers a natural explanation for the well-known β-barrel motif at the hexameric center as a post-aggregation stabilization mechanism. Finally, we identify those amino acid contacts within all key protein interfaces that are most persistent during compressive deformation of the capsid, thereby providing potential targets for mutation studies aiming to elucidate the key contacts upon which overall stability rests.

  16. Structure of a Human Astrovirus Capsid-Antibody Complex and Mechanistic Insights into Virus Neutralization

    Energy Technology Data Exchange (ETDEWEB)

    Bogdanoff, Walter A.; Campos, Jocelyn; Perez, Edmundo I.; Yin, Lu; Alexander, David L.; DuBois, Rebecca M. (UCSC)

    2016-11-02

    ABSTRACT

    Human astroviruses (HAstVs) are a leading cause of viral diarrhea in young children, the immunocompromised, and the elderly. There are no vaccines or antiviral therapies against HAstV disease. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against reinfection by HAstV. However, development of anti-HAstV therapies is hampered by the gap in knowledge of protective antibody epitopes on the HAstV capsid surface. Here, we report the structure of the HAstV capsid spike domain bound to the neutralizing monoclonal antibody PL-2. The antibody uses all six complementarity-determining regions to bind to a quaternary epitope on each side of the dimeric capsid spike. We provide evidence that the HAstV capsid spike is a receptor-binding domain and that the antibody neutralizes HAstV by blocking virus attachment to cells. We identify patches of conserved amino acids that overlap the antibody epitope and may comprise a receptor-binding site. Our studies provide a foundation for the development of therapies to prevent and treat HAstV diarrheal disease.

    IMPORTANCEHuman astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies in healthy adults protect them against reinfection. Here, we determined the crystal structure of a complex of the HAstV capsid protein and a virus-neutralizing antibody. We show that the antibody binds to the outermost spike domain of the capsid, and we provide evidence that the antibody blocks virus attachment to human cells. Importantly, our findings suggest that a subunit-based vaccine focusing the immune system on the HAstV capsid spike domain could be effective in protecting children against HAstV disease.

  17. Construction of the recombinant human adenovirus type 3 expressing Norovirus capsid protein gene%诺如病毒衣壳蛋白重组人3型腺病毒的构建

    Institute of Scientific and Technical Information of China (English)

    田新贵; 周荣; 李海涛; 龚四堂; 张其威; 朱冰; 盛慧英; 钟家禹

    2008-01-01

    Objective To prepare recombinant human adenovirus type 3 expressing Norovirus cap-sid protein gene(Noro-orf2). Methods The cDNA for Noro-orf2 was amplifed by RT-PCR from stool of in-fantile gastroenteritis and cloned into the adenovirus shuttle vector pBSE3CMV-egfp. The vector pBSE3CMV-Nor was linearized with EeoR Ⅴ and Not Ⅰ, and transformed into E. coil BJ5183 with lined edenovirus ge-nomic DNA pLasmid pBRAdv3 by Rsr Ⅱ. The identification of recombinant adenovirus plasmid pBRAdv3E3dNor was performed by PCR, enzyme digestion and DNA sequencing. Then pBRAdv3E3dNor was digested with AsiS Ⅰ and transfeeted into Hep-2 cells with LipofectAMINETM 2000 to package recombi-nant adenovirus particles. Results Noro-orf2 was successfully inserted into the shuttle vector. The recombi-nant adenoviral plasmid pBRAdv3E3dNor was generated by homologous recombination in E. coil BJ5183 and confirmed by PCR and enzyme digestion. The recombinant adenovirus was successfully packaged and puri-fied. Norovirus eapsid protein gene expression was confirmed in Hep-2 cells by immunecytochemistry assay. Conclusion The recombinant type 3 adenovirus expressing Norovirus eapsid protein gene was successfully constructed. This study laid a foundation for developing vaccine against Norovirus.%目的 制备表达诺如病毒衣壳蛋白的重组人3型腺病毒.方法 将诺如病毒衣壳蛋白基因(Noro-orf2)克隆到腺病毒穿梭载体pBSE3CMV-egfp上,与线性化人3型腺病毒骨架质粒pBRAdv3共电转化感受态大肠杆菌BJ5183,使其在细菌内发生同源重组,带Noro-orf2基因的表达框置换腺病毒E3区,PCR及酶切筛选得到重组腺病毒质粒,将重组腺病毒质粒转染Hep-2细胞进行包装,获得感染性的重组腺病毒粒子,免疫组化分析重组腺病毒中诺如病毒衣壳蛋白的表达.结果 同源重组后经酶切和PCR鉴定证明插入Noro-orf2基因的重组腺病毒质粒pBRAdv3E3dNor成功构建,并经转染包装得到

  18. A molecular thermodynamic model for the stability of hepatitis B capsids

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jehoon; Wu, Jianzhong, E-mail: jwu@engr.ucr.edu [Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521 (United States)

    2014-06-21

    Self-assembly of capsid proteins and genome encapsidation are two critical steps in the life cycle of most plant and animal viruses. A theoretical description of such processes from a physiochemical perspective may help better understand viral replication and morphogenesis thus provide fresh insights into the experimental studies of antiviral strategies. In this work, we propose a molecular thermodynamic model for predicting the stability of Hepatitis B virus (HBV) capsids either with or without loading nucleic materials. With the key components represented by coarse-grained thermodynamic models, the theoretical predictions are in excellent agreement with experimental data for the formation free energies of empty T4 capsids over a broad range of temperature and ion concentrations. The theoretical model predicts T3/T4 dimorphism also in good agreement with the capsid formation at in vivo and in vitro conditions. In addition, we have studied the stability of the viral particles in response to physiological cellular conditions with the explicit consideration of the hydrophobic association of capsid subunits, electrostatic interactions, molecular excluded volume effects, entropy of mixing, and conformational changes of the biomolecular species. The course-grained model captures the essential features of the HBV nucleocapsid stability revealed by recent experiments.

  19. X-Ray Structures of the Hexameric Building Block of the HIV Capsid

    Energy Technology Data Exchange (ETDEWEB)

    Pornillos, Owen; Ganser-Pornillos, Barbie K.; Kelly, Brian N.; Hua, Yuanzi; Whitby, Frank G.; Stout, C. David; Sundquist, Wesley I.; Hill, Christopher P.; Yeager, Mark; (Scripps); (Utah)

    2009-09-11

    The mature capsids of HIV and other retroviruses organize and package the viral genome and its associated enzymes for delivery into host cells. The HIV capsid is a fullerene cone: a variably curved, closed shell composed of approximately 250 hexamers and exactly 12 pentamers of the viral CA protein. We devised methods for isolating soluble, assembly-competent CA hexamers and derived four crystallographically independent models that define the structure of this capsid assembly unit at atomic resolution. A ring of six CA N-terminal domains form an apparently rigid core, surrounded by an outer ring of C-terminal domains. Mobility of the outer ring appears to be an underlying mechanism for generating the variably curved lattice in authentic capsids. Hexamer-stabilizing interfaces are highly hydrated, and this property may be key to the formation of quasi-equivalent interactions within hexamers and pentamers. The structures also clarify the molecular basis for capsid assembly inhibition and should facilitate structure-based drug design strategies.

  20. Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site

    Science.gov (United States)

    Liu, Chuang; Perilla, Juan R.; Ning, Jiying; Lu, Manman; Hou, Guangjin; Ramalho, Ruben; Himes, Benjamin A.; Zhao, Gongpu; Bedwell, Gregory J.; Byeon, In-Ja; Ahn, Jinwoo; Gronenborn, Angela M.; Prevelige, Peter E.; Rousso, Itay; Aiken, Christopher; Polenova, Tatyana; Schulten, Klaus; Zhang, Peijun

    2016-03-01

    The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection.

  1. A molecular thermodynamic model for the stability of hepatitis B capsids

    Science.gov (United States)

    Kim, Jehoon; Wu, Jianzhong

    2014-06-01

    Self-assembly of capsid proteins and genome encapsidation are two critical steps in the life cycle of most plant and animal viruses. A theoretical description of such processes from a physiochemical perspective may help better understand viral replication and morphogenesis thus provide fresh insights into the experimental studies of antiviral strategies. In this work, we propose a molecular thermodynamic model for predicting the stability of Hepatitis B virus (HBV) capsids either with or without loading nucleic materials. With the key components represented by coarse-grained thermodynamic models, the theoretical predictions are in excellent agreement with experimental data for the formation free energies of empty T4 capsids over a broad range of temperature and ion concentrations. The theoretical model predicts T3/T4 dimorphism also in good agreement with the capsid formation at in vivo and in vitro conditions. In addition, we have studied the stability of the viral particles in response to physiological cellular conditions with the explicit consideration of the hydrophobic association of capsid subunits, electrostatic interactions, molecular excluded volume effects, entropy of mixing, and conformational changes of the biomolecular species. The course-grained model captures the essential features of the HBV nucleocapsid stability revealed by recent experiments.

  2. Dynamic pathways for viral capsid assembly

    Energy Technology Data Exchange (ETDEWEB)

    Hagan, Michael F.; Chandler, David

    2006-02-09

    We develop a class of models with which we simulate the assembly of particles into T1 capsid-like objects using Newtonian dynamics. By simulating assembly for many different values of system parameters, we vary the forces that drive assembly. For some ranges of parameters, assembly is facile, while for others, assembly is dynamically frustrated by kinetic traps corresponding to malformed or incompletely formed capsids. Our simulations sample many independent trajectories at various capsomer concentrations, allowing for statistically meaningful conclusions. Depending on subunit (i.e., capsomer) geometries, successful assembly proceeds by several mechanisms involving binding of intermediates of various sizes. We discuss the relationship between these mechanisms and experimental evaluations of capsid assembly processes.

  3. Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.

    OpenAIRE

    Lin, S F; Sun, R; Heston, L; Gradoville, L; Shedd, D; Haglund, K; Rigsby, M; Miller, G.

    1997-01-01

    We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV). The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate. The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene...

  4. Viral capsids: Mechanical characteristics, genome packaging and delivery mechanisms

    NARCIS (Netherlands)

    Roos, W.H.; Ivanovska, I.L.; Evilevitch, A.; Wuite, G.J.L.

    2007-01-01

    The main functions of viral capsids are to protect, transport and deliver their genome. The mechanical properties of capsids are supposed to be adapted to these tasks. Bacteriophage capsids also need to withstand the high pressures the DNA is exerting onto it as a result of the DNA packaging and its

  5. Immobilization and One-Dimensional Arrangement of Virus Capsids With Nanoscale Precision Using DNA Origami

    Science.gov (United States)

    Stephanopoulos, Nicholas; Liu, Minghui; Tong, Gary J.; Li, Zhe; Liu, Yan; Yan, Hao; Francis, Matthew B.

    2011-01-01

    Self-assembly has proven to be one of the most effective ways to arrange matter at the nanometer level. Biology, in particular, makes extensive use of self-assembly to position molecules over several length scales with a high degree of spatial control over structure. In recent years, one promising approach that takes advantage of biological self-assembly in order to build synthetic materials employs virus capsids, the protein shells that encapsulate the genetic material of viruses.1 Capsids are composed of multiple protein subunits that can assemble (either spontaneously or under an external stimulus) into a monodisperse structure with different geometries depending on the virus. By appropriately functionalizing the proteins that comprise the capsid, multiple copies of a molecule or other entity can be positioned with a predictable arrangement. A wide variety of components have been attached to and arranged by virus capsids, including chromophores,2 catalysts,3 nanoparticles and quantum dots,4 polymers,5 drug molecules,6 and imaging agents.7 PMID:20575574

  6. Genetic characteristics of the VP1 gene of enterovirus 71 strains in the City of Xiangyang, Hubei Province during a HFMD epidemic in 2011%2011年湖北省襄阳市手足口病主要病原EV71型病毒VP1基因特征分析

    Institute of Scientific and Technical Information of China (English)

    戴莹; 李静; 雷亚克; 杨朝晖; 霍细香

    2013-01-01

    Objective The genetic characteristics of the YP1 gene and phylogeny of enterovirus type 71 (EV71) strains were analyzed to investigate the genetic background of EY71 that caused a HFMD epidemic in the City of Xiangyang. Hubei Province. Methods VP 1 of EV71 strains from Xiangyang in 2011 was sequenced and its homology and phylogeny were analyzed. Results The full-length VP 1 region of Xiangyang EV71 strains was 891 bp. Nuclcotide and amino acid identity were 96. 5% -99.1% and 98. 1%-100%. respectively. Phylogenetic analysis based on the complete VP1 sequence also indicated that the Xiangyang EV71 strains belonged to the same subgenotype, C4a. Conclusion The genotype of Xiangyang EV71 strains belonged to the CM a subgenotype. There was no obvious antigenic drift or variation.%目的 调查2011年襄阳市手足口病主要病原EV71的基因型特征,分析和探讨该型病毒的VP1基因变异和分子进化特点. 方法 对2011年襄阳市流行株EV71进行VP1区核苷酸序列测定和同源性比较及遗传进化分析.结果 2011年襄阳市手足口病EV71病毒的VPI区核苷酸序列全长均为891 bp,与对照EV71毒株核苷酸序列同源性为96.5%~99.1%,编码蛋白氨基酸序列同源性为98.1%~100%.VP l区基因遗传进化分析显示,该中EV71病毒属于C4a基因亚型. 结论 2011年襄阳市手足口病疫情主要病原EV71病毒均属于C4a基因亚型,未产生明显的抗原漂移及变异.

  7. Virus capsid dissolution studied by microsecond molecular dynamics simulations.

    Science.gov (United States)

    Larsson, Daniel S D; Liljas, Lars; van der Spoel, David

    2012-01-01

    Dissolution of many plant viruses is thought to start with swelling of the capsid caused by calcium removal following infection, but no high-resolution structures of swollen capsids exist. Here we have used microsecond all-atom molecular simulations to describe the dynamics of the capsid of satellite tobacco necrosis virus with and without the 92 structural calcium ions. The capsid expanded 2.5% upon removal of the calcium, in good agreement with experimental estimates. The water permeability of the native capsid was similar to that of a phospholipid membrane, but the permeability increased 10-fold after removing the calcium, predominantly between the 2-fold and 3-fold related subunits. The two calcium binding sites close to the icosahedral 3-fold symmetry axis were pivotal in the expansion and capsid-opening process, while the binding site on the 5-fold axis changed little structurally. These findings suggest that the dissociation of the capsid is initiated at the 3-fold axis.

  8. Microplate-based assay for identifying small molecules that bind a specific intersubunit interface within the assembled HIV-1 capsid.

    Science.gov (United States)

    Halambage, Upul D; Wong, Jason P; Melancon, Bruce J; Lindsley, Craig W; Aiken, Christopher

    2015-09-01

    Despite the availability of >30 effective drugs for managing HIV-1 infection, no current therapy is curative, and long-term management is challenging owing to the emergence and spread of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the current treatment options and help to control resistance. The highly conserved HIV-1 capsid protein represents an attractive target because of its multiple roles in replication of the virus. However, the low antiviral potencies of the reported HIV-1 capsid-targeting inhibitors render them unattractive for therapeutic development. To facilitate the identification of more-potent HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small molecules that target a biologically active and specific intersubunit interface in the HIV-1 capsid. The assay, which is based on competitive displacement of a known capsid-binding small-molecule inhibitor, exhibited a signal-to-noise ratio of >9 and a Z factor of >0.8. In a pilot screen of a chemical library containing 2,400 druglike compounds, we obtained a hit rate of 1.8%. This assay has properties that are suitable for screening large compound libraries to identify novel HIV-1 capsid ligands with antiviral activity.

  9. Modeling of the human rhinovirus C capsid suggests a novel topography with insights on receptor preference and immunogenicity.

    Science.gov (United States)

    Basta, Holly A; Sgro, Jean-Yves; Palmenberg, Ann C

    2014-01-05

    Features of human rhinovirus (RV)-C virions that allow them to use novel cell receptors and evade immune responses are unknown. Unlike the RV-A+B, these isolates cannot be propagated in typical culture systems or grown for structure studies. Comparative sequencing, I-TASSER, MODELLER, ROBETTA, and refined alignment techniques led to a structural approximation for C15 virions, based on the extensive, resolved RV-A+B datasets. The model predicts that all RV-C VP1 proteins are shorter by 21 residues relative to the RV-A, and 35 residues relative to the RV-B, effectively shaving the RV 5-fold plateau from the particle. There are major alterations in VP1 neutralizing epitopes and the structural determinants for ICAM-1 and LDLR receptors. The VP2 and VP3 elements are similar among all RV, but the loss of sequence "words" contributing Nim1ab has increased the apparent selective pressure among the RV-C to fix mutations elsewhere in the VP1, creating a possible compensatory epitope.

  10. Assessment of the cross-protective capability of recombinant capsid proteins derived from pig, rat, and avian hepatitis E viruses (HEV) against challenge with a genotype 3 HEV in pigs.

    Science.gov (United States)

    Sanford, Brenton J; Opriessnig, Tanja; Kenney, Scott P; Dryman, Barbara A; Córdoba, Laura; Meng, Xiang-Jin

    2012-09-28

    Hepatitis E virus (HEV), the causative agent of hepatitis E, is primarily transmitted via the fecal-oral route through contaminated water supplies, although many sporadic cases of hepatitis E are transmitted zoonotically via direct contact with infected animals or consumption of contaminated animal meats. Genotypes 3 and 4 HEV are zoonotic and infect humans and other animal species, whereas genotypes 1 and 2 HEV are restricted to humans. There exists a single serotype of HEV, although the cross-protective ability among the animal HEV strains is unknown. Thus, in this study we expressed and characterized N-terminal truncated ORF2 capsid antigens derived from swine, rat, and avian HEV strains and evaluated their cross-protective ability in a pig challenge model. Thirty, specific-pathogen-free, pigs were divided into 5 groups of 6 pigs each, and each group of pigs were vaccinated with 200 μg of swine HEV, rat HEV, or avian HEV ORF2 antigen or PBS buffer (2 groups) as positive and negative control groups. After a booster dose immunization at 2 weeks post-vaccination, the vaccinated animals all seroconverted to IgG anti-HEV. At 4 weeks post-vaccination, the animals were intravenously challenged with a genotype 3 mammalian HEV, and necropsied at 4 weeks post-challenge. Viremia, fecal virus shedding, and liver histological lesions were compared to assess the protective and cross-protective abilities of these antigens against HEV challenge in pigs. The results indicated that pigs vaccinated with truncated recombinant capsid antigens derived from three animal strains of HEV induced a strong IgG anti-HEV response in vaccinated pigs, but these antigens confer only partial cross-protection against a genotype 3 mammalian HEV. The results have important implications for the efficacy of current vaccines and for future vaccine development, especially against the novel zoonotic animal strains of HEV.

  11. Spectrum of VP1 region genetic variants in the foot-and-mouth disease virus serotype O populations derived from infected cattle tongue epithelium.

    Science.gov (United States)

    Sarangi, L N; Mohapatra, J K; Subramaniam, S; Pandey, L K; Das, B; Sanyal, A; Misri, J; Pattnaik, B

    2015-09-01

    RNA virus population exists as a complex distribution of non-identical but closely related sequences known as viral quasispecies. Variant strains are selected from this quasispecies population in response to changing environment. The quasispecies dynamics of a virus existing within an infected host differs from that in a cell culture-adapted population. This study was carried out to explore the genetic variations present in the VP1 coding region of the foot-and-mouth disease (FMD) virus serotype O derived directly from infected cattle tongue epithelium. Molecular clonal populations of two serotype O strains belonging to lineages Ind2001 (IND 30/2011) and PanAsia2 (IND 5/2011) were sequenced at VP1 coding region. For IND 30/2011, 19 clones were sequenced and analysis showed variations at 12 nucleotide positions (nt) resulting in 8 amino acid (aa) replacements. Similarly, for IND 5/2011 virus, 18 clones were sequenced, of which six showed nt variations leading to 3 aa replacements. Most of the variable positions mapped to the surface-exposed loops and some of them were found in the neutralizing antigenic sites (position 81, 149, 169, 186 and 202 of IND 30/2011 and 141 of IND 5/2011), which potentially could be beneficial in rapid adaptive evolution of the virus by giving rise to antigenic variants to overcome neutralizing antibodies. These findings encourage further research into the landscape of the viral quasispecies population in vivo and its implication for viral ecology.

  12. Structural Transitions and Energy Landscape for Cowpea Chlorotic Mottle Virus Capsid Mechanics from Nanomanipulation in Vitro and in Silico

    Science.gov (United States)

    Kononova, Olga; Snijder, Joost; Brasch, Melanie; Cornelissen, Jeroen; Dima, Ruxandra I.; Marx, Kenneth A.; Wuite, Gijs J.L.; Roos, Wouter H.; Barsegov, Valeri

    2013-01-01

    Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Combined AFM experiments and computational modeling on subsecond timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus capsid show that the capsid’s physical properties are dynamic and local characteristics of the structure, which change with the depth of indentation and depend on the magnitude and geometry of mechanical input. Under large deformations, the Cowpea Chlorotic Mottle Virus capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state ΔHind = 11.5–12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending; the entropy change TΔSind = 5.1–5.8 MJ/mol is due to coherent in-plane rearrangements of protein chains, which mediate the capsid stiffening. Direct coupling of these modes defines the extent of (ir)reversibility of capsid indentation dynamics correlated with its (in)elastic mechanical response to the compressive force. This emerging picture illuminates how unique physico-chemical properties of protein nanoshells help define their structure and morphology, and determine their viruses’ biological function. PMID:24138865

  13. 一株鼻病毒A22型的分离鉴定及其VP1基因遗传特征分析%Isolation and identification of a rhinovirus A22 strain and analysis of genetic characters of its VP1 gene

    Institute of Scientific and Technical Information of China (English)

    戴素萍; 潘玥; 邵聪文; 吉玛; 朱艳菊; 张云昆; 朱云; 马绍辉

    2014-01-01

    目的 分析2011年昆明市手足口病患儿粪便中分离获得的人鼻病毒(human rhinovirus,HRV)A22型分离株KM4/2011全长VP1基因的遗传特征.方法 采用KMB17细胞对手足口病患儿粪便样本中筛查出的1株HRV样品进行病毒分离,应用RT-PCR法扩增病毒VP1基因,并进行测序,采用BLAST软件对所测定的节段序列进行数据库比对;采用Geneious basic 5.6.5软件进行核苷酸和氨基酸同源性比对;采用Mega 5.05软件将HRV A22及部分HRV A、B和C群全长VP1基因构建系统进化树.结果 分离株为KM4/2011,经扩增、测序和序列拼接获得长度为867 bp的VP1基因;KM4/2011株与其他HRV分离株核苷酸和氨基酸的同源性分别为48.1% ~ 91.0%和38.2% ~97.6%,其中与HRV A22分离株的核苷酸和氨基酸同源性较高,分别为90.8% ~ 91.0%和97.3% ~ 97.6%;基因进化树分析显示,KM4/201l株与HRV A22基因型属于同一个进化分枝.结论 KM4/2011分离株为HRV A22型,存在地域差异.

  14. The conformation of double-stranded DNA inside bacteriophages depends on capsid size and shape.

    Science.gov (United States)

    Petrov, Anton S; Boz, Mustafa Burak; Harvey, Stephen C

    2007-11-01

    The packaging of double-stranded DNA into bacteriophages leads to the arrangement of the genetic material into highly-packed and ordered structures. Although modern experimental techniques reveal the most probable location of DNA inside viral capsids, the individual conformations of DNA are yet to be determined. In the current study we present the results of molecular dynamics simulations of the DNA packaging into several bacteriophages performed within the framework of a coarse-grained model. The final DNA conformations depend on the size and shape of the capsid, as well as the size of the protein portal, if any. In particular, isometric capsids with small or absent portals tend to form concentric spools, whereas the presence of a large portal favors coaxial spooling; slightly and highly elongated capsids result in folded and twisted toroidal conformations, respectively. The results of the simulations also suggest that the predominant factor in defining the global DNA arrangement inside bacteriophages is the minimization of the bending stress upon packaging.

  15. Classification and Evolutionary Trends of Icosahedral Viral Capsids

    Directory of Open Access Journals (Sweden)

    Richard Kerner

    2008-01-01

    Full Text Available A classification of icosahedral viral capsids is proposed. We show how the self-organization of capsids during their formation implies a definite composition of their elementary building blocks. The exact number of hexamers with three different admissible symmetries is related to capsids' sizes, labelled by their T-numbers. Simple rules determining these numbers for each value of T are deduced and certain consequences concerning the probabilities of mutations and evolution of viruses are discussed.

  16. Analysis of the VP1 Coding Nucleotide Sequence of YNAs1.1 Isolate of Foot-and-Mouth Disease Virus Type Asia1%亚洲一型口蹄疫病毒YNAs1.1株结构蛋白VP1基因的核苷酸序列分析

    Institute of Scientific and Technical Information of China (English)

    张震宇; 杨永钦; 张青; 李乐; 严维耀; 蒋文俊; 信爱国; 郑兆鑫

    2001-01-01

    提取BHK21细胞增殖的亚洲一型口蹄疫病毒(foot-and-mouth disease virus Ser otype Asia1)强毒株YNAs1.1的RNA,用一对引物P7,P13经反转录 (RT)-PCR法扩增了约674 bp的DNA片段.克隆目的基因后,采用双脱氧DNA链末端终止法测得了YNAs1.1的VP1基因36~633核苷酸序列.分析表明,病毒VP1基因的核苷酸序列与以色列以及印度已报道的Asia1型FMDV的同源性分别为82.11%与88.07%,对应的氨基酸序列同源性为87.94%与93.47%.该序列在GeneBank登陆号为AF241566.

  17. Genetic diversity of the VP1/VP2 gene of canine parvovirus type 2b amplified from clinical specimens in Brazil Diversidade genética no gene VP1/VP2 do parvovirus canino tipo 2b amplificado de material clínico no Brasil

    Directory of Open Access Journals (Sweden)

    Cesar A. D. Pereira

    2000-10-01

    Full Text Available We evaluated the genetic diversity in the VP1/VP2 gene of CPV type 2b isolates from symptomatic dogs in Brazil. A total of 21 isolates collected from 1990 through 1995 previously typed as CPV2b by PCR assay were studied. Overall we found a high degree of similarity among sequences from different CPV clinical isolates collected. Genetic analysis of this selected region gave no indication of a specific Brazilian parvovirus lineage.Neste estudo foi avaliada a diversidade genética no gene VP1/VP2 do parvovírus canino tipo 2b a partir de amostras isoladas de cães sintomáticos no Brasil. Foram estudadas 21 amostras coletadas no período de 1990 à 1995, previamente caracterizadas como CPV 2b pela técnica de PCR. Observou-se alto grau de similaridade entre as seqüências estudadas e a análise genética da região selecionada não indicou a presença de uma linhagem brasileira específica.

  18. Theory of morphological transformation of viral capsid shells during maturation process

    CERN Document Server

    Konevtsova, O V; Rochal, S B

    2015-01-01

    In the frame of the Landau-Ginzburg formalism we propose a minimal phenomenological model for a morphological transformation in viral capsid shells. The transformation takes place during virus maturation process which renders virus infectious. The theory is illustrated on the example of the HK97 bacteriophage and viruses with similar morphological changes in the protective protein shell. The transformation is shown to be a structural phase transition driven by two order parameters. The first order parameter describes the isotropic expansion of the protein shell while the second one is responsible for the shape symmetry breaking and the resulting shell faceting. The group theory analysis and the resulting thermodynamic model make it possible to choose the parameter which discriminates between the icosahedral shell faceting often observed in viral capsids and the dodecahedral one observed in viruses of the Parvovirus family. Calculated phase diagram illustrates the discontinuous character of the virus morpholog...

  19. PATHOGEN SPECTRUM ANALYSIS OF HAND, FOOT AND MOUTH DISEASE AND GENETIC ANALYSIS OF THE VP1 REGION OF HUMAN ENTEROVIRUS 71 ISOLATED STRAINS IN HANGZHOU%浙江省杭州地区手足口病病原谱分析和肠道病毒71型VP1基因分析

    Institute of Scientific and Technical Information of China (English)

    郑书发; 崔大伟; 余斐; 杨先知; 陈瑜

    2011-01-01

    [Objective] To understand the pathogen spectrum of HFMD in Hangzhou, analyze enterovirus (EV) 71- type VP1 gene nucleotide and amino acid sequence, and understand the genetic characteristics of EV71 in Hangzhou. [Meth ods] 98 confirmed cases of HFMD stool specimens were collected. All of the specimens were identified by reverse transcription polymerase chain reaction (RT-PCR) with EV71 and Coxsackie (Cox) A16-type-specific primers. Full length VP1 genes from these six EV71 strains were amplified by RT-PCR and sequenced. The sequences were compared with VP1 genes from EV71 in GenBank database. Sequence analysis and type identification were performed by bioinformatics. [ Results] Of the RT-PCR, enterovirus 71 (EV71) was diagnosed in 22.4% of the patients (22 of 98) , and COXA16 was diagnosed in 27.6% (27of 98). The nucleotide and amitto acid homogeneity between of these 6 Zhejiang strains ranged from 92.2% to 98.5%and 99.0% to 100% respectively, there are no significant difference between severe and mild patients in the amino acid sequence. These 6 Ev71 iso lated strains were within genotype C subgenogroup C4 in the phylogenetic tree. Compare with representative strains of all geno types and subtypes,there was difference in the BC loop region between the amino acid sequence differences, and the SP70 re gion amino acid sequence was identical elsewhere. [Conclusion] The hand, foot and mouth diseases were mainly caused by the EV71 infection increasing proportion. In addition, the separation of EV71 in Hangzhou was belonged to subtype C genotype of the C4. There is still no evidence that this separation in Hangzhou 6 EV71 strains have enhanced virulence.%[目的]分析杭州地区手足口病的病原谱构成,并对肠道病毒(EV)71型的VP1基因进行核苷酸和氨基酸的序列的比对分析,了解杭州地区EV71的基因特征.[方法]采集98份手足口病确诊病例的粪便标本,用EV71和柯萨奇病毒A组(CoxA)16型特异性引物进行逆

  20. Preparation High Titer Anti-serum of Porcine Circovirus Type Ⅱ Capsid Protein by Hydrodynamics Gentic Immunization%水流动力学基因免疫制备猪Ⅱ型圆环病毒核衣壳蛋白高效价抗血清

    Institute of Scientific and Technical Information of China (English)

    樊宝良; 张瑾; 代红星; 黄培华

    2012-01-01

    为了建立一个简捷有效的抗血清的制备方法,本研究选用猪Ⅱ型圆环病毒核衣壳蛋白基因,使用水流动力学基因免疫的方法制备高效价抗血清的可行性.应用无内提取试剂盒制备猪Ⅱ型圆环病毒核衣壳蛋白基因真核表达载体pcDNA-Cap的无内毒素质粒.将该质粒使用水流动力学尾静脉注射法对小鼠(Mus musculus)进行基因免疫,重复免疫5次后采血收集血清;以原核表达获得的N末端去除了核定位序列的猪圆环病毒核衣壳蛋白表达产物作为抗原蛋白,以制备的小鼠血清作为一抗进行酶联免疫吸附试验(ELISA)和Western blot检测.结果显示,应用水流动力学尾静脉注射法获得抗血清稀释5000倍通过Western blot至少能够检测到10 ng的抗原蛋白,ELISA分析表明,其效价可达到1∶1000000,说明获得的抗血清具有很好的效价水平.这一研究为猪Ⅱ型圆环病毒相关研究用抗体的制备提供了一个简洁有效的方法,也为猪Ⅱ型圆环病毒的防治方法的建立提供了一个值得尝试的策略.%In order to establish a simple and efficient anti-serum preparation method for molecular biology research, in this research, porcine circovirus type II capsid protein gene was selected to research on the possiblity of preparing high titer anti-serum by hydrodynamics gentic immunization. Endotoxin free plasmid of pcDNA-Cap, with would express porcine circovirus type II capsid protein in the exkaryotic cell, was prepared using endotoxin free plasmid preparing kit and was delivered by hydrodynamics tail vein injection method for genetic immunization of mice (Mus musculus). After 5 times continuous immunization, the blood serum was collected. Using porcine circovirus type II capsid protein which has been deleted its nuclear laction signal sequence at its N-terminal and expressed in Escherichia coli as antigen, the prepared anti-serum was tested by enzymeliked immuno sorbent assay(ELISA) and

  1. 浙江省湖州市柯萨奇病毒A组16型分离株VP1区序列测定及系统进化分析%Genetic characteristics of VP1 region of coxsakievirus A16 strains isolated in Huzhou, Zhejiang, 2011-2012

    Institute of Scientific and Technical Information of China (English)

    纪蕾; 吴晓芳; 陈莉萍; 徐德顺; 沈月华; 查赟峰; 朱晓娟

    2014-01-01

    目的 了解浙江省湖州市引起手足口病(HFMD)的柯萨奇病毒A组16型(Cox A16)分离株的VP1区基因特征.方法 采用荧光定量反转录聚合酶链反应方法,对2011-2012年湖州市538份HFMD临床标本进行肠道病毒核酸检测;采用RD、Hep2细胞对Cox A16核酸阳性的标本进行病毒分离.选取17株具有代表性的Cox A16分离株进行VP1区的序列测定和分析.结果 2011-2012年湖州市HFMD的主要病原为Cox A16和肠道病毒71型(EV71);湖州地区Cox A16分离株VP1区的核苷酸和氨基酸序列同源性分别为89.5%~100%和99.3%~100%.与Cox A16的B1基因亚型代表株核苷酸同源性最高,在系统进化树上与B1亚型代表株相聚在一起,并且同时存在B1a和Bib两个进化分支.结论 湖州市的Cox A16分离株与国内其他地区类似,同属于B1基因亚型,并且有B1a和Bib两个进化分支共同进化和循环.

  2. Rationally designed interfacial peptides are efficient in vitro inhibitors of HIV-1 capsid assembly with antiviral activity.

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    Rebeca Bocanegra

    Full Text Available Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8 were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization, or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid

  3. 西安市手足口病肠道病毒71型分离株VP1区基因特征分析

    Institute of Scientific and Technical Information of China (English)

    付建军; 王海峰; 马超峰; 雷彩虹; 黄嘉丽; 邓慧玲

    2014-01-01

    目的:了解西安市2012年手足口病肠道病毒71型分离株VP1区基因特征,探讨基因型与病情轻重间的关系。方法采集肠道病毒71型(EV71)患者咽拭子或粪便标本,按等概率法随机选择6例普通型和6例重型EV71分离株,用逆转录-聚合酶链反应(RT-PCR)对EV71分离株进行VP1区基因扩增及核酸测序,并将测序结果与EV7各基因型代表株进行同源性和遗传进化分析。结果12株EV71分离株的基因型为C4基因亚型,其与C4亚型代表株VP1区核苷酸和氨基酸同源性最高,分别91.8%~93.0%和98.3%~99.3%,6例普通型和6例重型EV71分离株在VP1基因型区无明显差异,对12株EV71分离株进行遗传进化分析显示,12株流行株与C4亚型代表株聚集于同一个分支。结论西安12株EV71流行株的基因型属于C4亚型,基因型与病情严重程度可能无关。%Objectiveto study the gene characteristics of VP1 region of enterovirus 71(EV71)epidemic strains and to investigate the relationship between the genotype and the disease severity in Xi’an,2012.MethodsThe clinical specimens were collected from the hospital, according to the method of equal probability, randomly selected sixes case of common and sixes sever case from the EV71 strain isolated. Using the method of reverse transcription -polymerase chain reaction to amplify and sequence the VP1 gene of EV71,the resultswere compared the homologous and genetic evolution to each genotype representive stains of EV71. ResultsThe genotypes of 12 strains was belonged to the subgenotype of C4,the VP1 gene of 12 strains shared the highest homology with subgenotpye C4 both at nucleotide level(91.8%~93.0%)and at amino acid level(98.3~99.3%),to analyzed the evolution of the VP1 gene of EV71 isolated, the epidemic strains were clustered with subgenotpye C4 representive strain,there was no significant difference in VP1 gene between the isolated from common case and severe case

  4. Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli

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    Zhou Bihong

    2011-02-01

    Full Text Available Abstract Background In recent years, it has been gradually realized that bacterial inclusion bodies (IBs could be biologically active. In particular, several proteins including green fluorescent protein, β-galactosidase, β-lactamase, alkaline phosphatase, D-amino acid oxidase, polyphosphate kinase 3, maltodextrin phosphorylase, and sialic acid aldolase have been successfully produced as active IBs when fused to an appropriate partner such as the foot-and-mouth disease virus capsid protein VP1, or the human β-amyloid peptide Aβ42(F19D. As active IBs may have many attractive advantages in enzyme production and industrial applications, it is of considerable interest to explore them further. Results In this paper, we report that an ionic self-assembling peptide ELK16 (LELELKLK2 was able to effectively induce the formation of cytoplasmic inclusion bodies in Escherichia coli (E. coli when attached to the carboxyl termini of four model proteins including lipase A, amadoriase II, β-xylosidase, and green fluorescent protein. These aggregates had a general appearance similar to the usually reported cytoplasmic inclusion bodies (IBs under transmission electron microscopy or fluorescence confocal microscopy. Except for lipase A-ELK16 fusion, the three other fusion protein aggregates retained comparable specific activities with the native counterparts. Conformational analyses by Fourier transform infrared spectroscopy revealed the existence of newly formed antiparallel beta-sheet structures in these ELK16 peptide-induced inclusion bodies, which is consistent with the reported assembly of the ELK16 peptide. Conclusions This has been the first report where a terminally attached self-assembling β peptide ELK16 can promote the formation of active inclusion bodies or active protein aggregates in E. coli. It has the potential to render E. coli and other recombinant hosts more efficient as microbial cell factories for protein production. Our observation might

  5. X-ray structure of Triatoma virus empty capsid: insights into the mechanism of uncoating and RNA release in dicistroviruses.

    Science.gov (United States)

    Sánchez-Eugenia, Rubén; Durana, Aritz; López-Marijuan, Ibai; Marti, Gerardo A; Guérin, Diego M A

    2016-10-01

    In viruses, uncoating and RNA release are two key steps of successfully infecting a target cell. During these steps, the capsid must undergo the necessary conformational changes to allow RNA egress. Despite their importance, these processes are poorly understood in the family Dicistroviridae. Here, we used X-ray crystallography to solve the atomic structure of a Triatoma virus(TrV) empty particle (Protein Data Bank ID 5L7O), which is the resulting capsid after RNA release. It is observed that the overall shape of the capsid and of the three individual proteins is maintained in comparison with the mature virion. Furthermore, no channels indicative of RNA release are formed in the TrV empty particle. However, the most prominent change in the empty particle when compared with the mature virion is the loss of order in the N-terminal domain of the VP2 protein. In mature virions, the VP2 N-terminal domain of one pentamer is swapped with its twofold related copy in an adjacent pentamer, thereby stabilizing the binding between the pentamers. The loss of these interactions allows us to propose that RNA release may take place through transient flipping-out of pentameric subunits. The lower number of stabilizing interactions between the pentamers and the lack of formation of new holes support this model. This model differs from the currently accepted model for rhinoviruses and enteroviruses, in which genome externalization occurs by extrusion of the RNA through capsid channels.

  6. Virus capsid dissolution studied by microsecond molecular dynamics simulations.

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    Daniel S D Larsson

    Full Text Available Dissolution of many plant viruses is thought to start with swelling of the capsid caused by calcium removal following infection, but no high-resolution structures of swollen capsids exist. Here we have used microsecond all-atom molecular simulations to describe the dynamics of the capsid of satellite tobacco necrosis virus with and without the 92 structural calcium ions. The capsid expanded 2.5% upon removal of the calcium, in good agreement with experimental estimates. The water permeability of the native capsid was similar to that of a phospholipid membrane, but the permeability increased 10-fold after removing the calcium, predominantly between the 2-fold and 3-fold related subunits. The two calcium binding sites close to the icosahedral 3-fold symmetry axis were pivotal in the expansion and capsid-opening process, while the binding site on the 5-fold axis changed little structurally. These findings suggest that the dissociation of the capsid is initiated at the 3-fold axis.

  7. A novel universal neutralizing monoclonal antibody against enterovirus 71 that targets the highly conserved "knob" region of VP3 protein.

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    Tanja K Kiener

    Full Text Available Hand, foot and mouth disease caused by enterovirus 71(EV71 leads to the majority of neurological complications and death in young children. While putative inactivated vaccines are only now undergoing clinical trials, no specific treatment options exist yet. Ideally, EV71 specific intravenous immunoglobulins could be developed for targeted treatment of severe cases. To date, only a single universally neutralizing monoclonal antibody against a conserved linear epitope of VP1 has been identified. Other enteroviruses have been shown to possess major conformational neutralizing epitopes on both the VP2 and VP3 capsid proteins. Hence, we attempted to isolate such neutralizing antibodies against conformational epitopes for their potential in the treatment of infection as well as differential diagnosis and vaccine optimization. Here we describe a universal neutralizing monoclonal antibody that recognizes a conserved conformational epitope of EV71 which was mapped using escape mutants. Eight escape mutants from different subgenogroups (A, B2, B4, C2, C4 were rescued; they harbored three essential mutations either at amino acid positions 59, 62 or 67 of the VP3 protein which are all situated in the "knob" region. The escape mutant phenotype could be mimicked by incorporating these mutations into reverse genetically engineered viruses showing that P59L, A62D, A62P and E67D abolish both monoclonal antibody binding and neutralization activity. This is the first conformational neutralization epitope mapped on VP3 for EV71.

  8. Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle.

    Science.gov (United States)

    Guo, Hui-Chen; Sun, Shi-Qi; Jin, Ye; Yang, Shun-Li; Wei, Yan-Quan; Sun, De-Hui; Yin, Shuang-Hui; Ma, Jun-Wu; Liu, Zai-Xin; Guo, Jian-Hong; Luo, Jian-Xun; Yin, Hong; Liu, Xiang-Tao; Liu, Ding Xiang

    2013-07-04

    Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.

  9. Effect of lipophilicity modulation on inhibition of human rhinovirus capsid binders.

    Science.gov (United States)

    Morley, Andrew; Tomkinson, Nicholas; Cook, Andrew; MacDonald, Catherine; Weaver, Richard; King, Sarah; Jenkinson, Lesley; Unitt, John; McCrae, Christopher; Phillips, Tim

    2011-10-15

    To try and generate broad spectrum human rhinovirus VP1 inhibitors with more attractive physicochemical, DMPK and safety profiles, we explored the current SAR of known VP1 compounds. This lead to the identification of specific structural regions where reduction in polarity can be achieved, so guiding chemistry to analogues with significantly superior profiles to previously reported inhibitors.

  10. VP2 capsid domain of the H-1 parvovirus determines susceptibility of human cancer cells to H-1 viral infection.

    Science.gov (United States)

    Cho, I-R; Kaowinn, S; Song, J; Kim, S; Koh, S S; Kang, H-Y; Ha, N-C; Lee, K H; Jun, H-S; Chung, Y-H

    2015-05-01

    Although H-1 parvovirus is used as an antitumor agent, not much is known about the relationship between its specific tropism and oncolytic activity. We hypothesize that VP2, a major capsid protein of H-1 virus, determines H-1-specific tropism. To assess this, we constructed chimeric H-1 viruses expressing Kilham rat virus (KRV) capsid proteins, in their complete or partial forms. Chimeric H-1 viruses (CH1, CH2 and CH3) containing the whole KRV VP2 domain could not induce cytolysis in HeLa, A549 and Panc-1 cells. However, the other chimeric H-1 viruses (CH4 and CH5) expressing a partial KRV VP2 domain induced cytolysis. Additionally, the significant cytopathic effect caused by CH4 and CH5 infection in HeLa cells resulted from preferential viral amplification via DNA replication, RNA transcription and protein synthesis. Modeling of VP2 capsid protein showed that two variable regions (VRs) (VR0 and VR2) of H-1 VP2 protein protrude outward, because of the insertion of extra amino-acid residues, as compared with those of KRV VP2 protein. This might explain the precedence of H-1 VP2 protein over KRV in determining oncolytic activity in human cancer cells. Taking these results together, we propose that the VP2 protein of oncolytic H-1 parvovirus determines its specific tropism in human cancer cells.

  11. Motion of an antiviral compound in a rhinovirus capsid under rotational symmetry boundary conditions.

    Science.gov (United States)

    Yoneda, Shigetaka; Yoneda, Teruyo; Kurihara, Youji; Umeyama, Hideaki

    2002-08-01

    A molecular dynamics (MD) simulation of a complex of a rhinovirus protein shell referred to as a "capsid" and an anti-rhinovirus drug, WIN52084s, was performed under the rotational symmetry boundary conditions. For the simulation, the energy parameters of WIN52084s in all-atom approximations were determined by ab initio calculations using a 6-31G* basis set and the two-conformational two-stage restricted electrostatic potential fit method. The motion of WIN52084s and the capsid was focused on in the analysis of the trajectory of the simulation. The root mean square deviations of WIN52084s from the X-ray structure were decomposed to conformational, translational, and rotational components. The translation was further decomposed to radial, longitudinal, and lateral components. The conformation of WIN52084s was rigid, but moving in the pocket. The easiest path of motion for WlN52084s was on the longitudinal line, providing a track for the binding process required of the anti-rhinovirus drug to enter the pocket. The conformation of the pocket was also preserved in the simulation, although the position of the pocket in the capsid fluctuated in the lateral and radial directions.

  12. In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

    Science.gov (United States)

    Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

    2016-12-01

    HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

  13. Assessment of stress tolerance, productivity, and forage quality in T1 transgenic alfalfa co-overexpressing ZxNHX and ZxVP1-1 from Zygophyllum xanthoxylum

    OpenAIRE

    Peng Kang; Ai-Ke Bao; Tanweer Kumar; Ya-Qing Pan; Zhulatai Bao; Fei Wang; Suo-Min Wang

    2016-01-01

    Salinization, desertification, and soil nutrient deprivation are threatening the production of alfalfa (Medicago sativa L.) in northern China. We have previously generated T0 transgenic alfalfa co-overexpressing Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 genes with enhanced salt and drought tolerance. To further develop this excellent breeding material into the new forage cultivar, stress tolerance, productivity, and forage quality of T1 transgenic alfalfa (GM) were assessed in this study. The...

  14. Polymorphism of DNA conformation inside the bacteriophage capsid

    OpenAIRE

    Leforestier, Amélie

    2013-01-01

    Double-stranded DNA bacteriophage genomes are packaged into their icosahedral capsids at the highest densities known so far (about 50 % w:v). How the molecule is folded at such density and how its conformation changes upon ejection or packaging are fascinating questions still largely open. We review cryo-TEM analyses of DNA conformation inside partially filled capsids as a function of the physico-chemical environment (ions, osmotic pressure, temperature). We show that there exists a wide vari...

  15. Identification of B-cell epitopes in the capsid protein of avian hepatitis E virus (avian HEV) that are common to human and swine HEVs or unique to avian HEV.

    Science.gov (United States)

    Guo, H; Zhou, E-M; Sun, Z F; Meng, X-J; Halbur, P G

    2006-01-01

    Avian hepatitis E virus (avian HEV) was recently discovered in chickens from the USA that had hepatitis-splenomegaly (HS) syndrome. The complete genomic sequence of avian HEV shares about 50 % nucleotide sequence identity with those of human and swine HEVs. The open reading frame 2 (ORF2) protein of avian HEV has been shown to cross-react with human and swine HEV ORF2 proteins, but the B-cell epitopes in the avian HEV ORF2 protein have not been identified. Nine synthetic peptides from the predicted four antigenic domains of the avian HEV ORF2 protein were synthesized and corresponding rabbit anti-peptide antisera were generated. Using recombinant ORF2 proteins, convalescent pig and chicken antisera, peptides and anti-peptide rabbit sera, at least one epitope at the C terminus of domain II (possibly between aa 477-492) that is unique to avian HEV, one epitope in domain I (aa 389-410) that is common to avian, human and swine HEVs, and one or more epitopes in domain IV (aa 583-600) that are shared between avian and human HEVs were identified. Despite the sequence difference in ORF2 proteins between avian and mammalian HEVs and similar ORF2 sequence between human and swine HEV ORF2 proteins, rabbit antiserum against peptide 6 (aa 389-399) recognized only human HEV ORF2 protein, suggesting complexity of the ORF2 antigenicity. The identification of these B-cell epitopes in avian HEV ORF2 protein may be useful for vaccine design and may lead to future development of immunoassays for differential diagnosis of avian, swine and human HEV infections.

  16. α-Defensin HD5 Inhibits Human Papillomavirus 16 Infection via Capsid Stabilization and Redirection to the Lysosome

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    Mayim E. Wiens

    2017-01-01

    Full Text Available α-Defensins are an important class of abundant innate immune effectors that are potently antiviral against a number of nonenveloped viral pathogens; however, a common mechanism to explain their ability to block infection by these unrelated viruses is lacking. We previously found that human defensin 5 (HD5 blocks a critical host-mediated proteolytic processing step required for human papillomavirus (HPV infection. Here, we show that bypassing the requirement for this cleavage failed to abrogate HD5 inhibition. Instead, HD5 altered HPV trafficking in the cell. In the presence of an inhibitory concentration of HD5, HPV was internalized and reached the early endosome. The internalized capsid became permeable to antibodies and proteases; however, HD5 prevented dissociation of the viral capsid from the genome, reduced viral trafficking to the trans-Golgi network, redirected the incoming viral particle to the lysosome, and accelerated the degradation of internalized capsid proteins. This mechanism is equivalent to the mechanism by which HD5 inhibits human adenovirus. Thus, our data support capsid stabilization and redirection to the lysosome during infection as a general antiviral mechanism of α-defensins against nonenveloped viruses.

  17. α-Defensin HD5 Inhibits Human Papillomavirus 16 Infection via Capsid Stabilization and Redirection to the Lysosome

    Science.gov (United States)

    Wiens, Mayim E.

    2017-01-01

    ABSTRACT α-Defensins are an important class of abundant innate immune effectors that are potently antiviral against a number of nonenveloped viral pathogens; however, a common mechanism to explain their ability to block infection by these unrelated viruses is lacking. We previously found that human defensin 5 (HD5) blocks a critical host-mediated proteolytic processing step required for human papillomavirus (HPV) infection. Here, we show that bypassing the requirement for this cleavage failed to abrogate HD5 inhibition. Instead, HD5 altered HPV trafficking in the cell. In the presence of an inhibitory concentration of HD5, HPV was internalized and reached the early endosome. The internalized capsid became permeable to antibodies and proteases; however, HD5 prevented dissociation of the viral capsid from the genome, reduced viral trafficking to the trans-Golgi network, redirected the incoming viral particle to the lysosome, and accelerated the degradation of internalized capsid proteins. This mechanism is equivalent to the mechanism by which HD5 inhibits human adenovirus. Thus, our data support capsid stabilization and redirection to the lysosome during infection as a general antiviral mechanism of α-defensins against nonenveloped viruses. PMID:28119475

  18. Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution

    Science.gov (United States)

    Schur, Florian K. M.; Hagen, Wim J. H.; Rumlová, Michaela; Ruml, Tomáš; Müller, Barbara; Kräusslich, Hans-Georg; Briggs, John A. G.

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) assembly proceeds in two stages. First, the 55 kilodalton viral Gag polyprotein assembles into a hexameric protein lattice at the plasma membrane of the infected cell, inducing budding and release of an immature particle. Second, Gag is cleaved by the viral protease, leading to internal rearrangement of the virus into the mature, infectious form. Immature and mature HIV-1 particles are heterogeneous in size and morphology, preventing high-resolution analysis of their protein arrangement in situ by conventional structural biology methods. Here we apply cryo-electron tomography and sub-tomogram averaging methods to resolve the structure of the capsid lattice within intact immature HIV-1 particles at subnanometre resolution, allowing unambiguous positioning of all α-helices. The resulting model reveals tertiary and quaternary structural interactions that mediate HIV-1 assembly. Strikingly, these interactions differ from those predicted by the current model based on in vitro-assembled arrays of Gag-derived proteins from Mason-Pfizer monkey virus. To validate this difference, we solve the structure of the capsid lattice within intact immature Mason-Pfizer monkey virus particles. Comparison with the immature HIV-1 structure reveals that retroviral capsid proteins, while having conserved tertiary structures, adopt different quaternary arrangements during virus assembly. The approach demonstrated here should be applicable to determine structures of other proteins at subnanometre resolution within heterogeneous environments.

  19. Potential recombinant vaccine against influenza A virus based on M2e displayed on nodaviral capsid nanoparticles

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    Yong CY

    2015-04-01

    Full Text Available Chean Yeah Yong,1 Swee Keong Yeap,2 Kok Lian Ho,3 Abdul Rahman Omar,2,4 Wen Siang Tan1,2 1Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, 2Institute of Bioscience, 3Department of Pathology, Faculty of Medicine and Health Sciences, 4Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Selangor, Malaysia Abstract: Influenza A virus poses a major threat to human health, causing outbreaks from time to time. Currently available vaccines employ inactivated viruses of different strains to provide protection against influenza virus infection. However, high mutation rates of influenza virus hemagglutinin (H and neuraminidase (N glycoproteins give rise to vaccine escape mutants. Thus, an effective vaccine providing protection against all strains of influenza virus would be a valuable asset. The ectodomain of matrix 2 protein (M2e was found to be highly conserved despite mutations of the H and N glycoproteins. Hence, one to five copies of M2e were fused to the carboxyl-terminal end of the recombinant nodavirus capsid protein derived from Macrobrachium rosenbergii. The chimeric proteins harboring up to five copies of M2e formed nanosized virus-like particles approximately 30 nm in diameter, which could be purified easily by immobilized metal affinity chromatography. BALB/c mice immunized subcutaneously with these chimeric proteins developed antibodies specifically against M2e, and the titer was proportional to the copy numbers of M2e displayed on the nodavirus capsid nanoparticles. The fusion proteins also induced a type 1 T helper immune response. Collectively, M2e displayed on the nodavirus capsid nanoparticles could provide an alternative solution to a possible influenza pandemic in the future. Keywords: matrix 2 ectodomain, nodavirus capsid, virus-like particle, fusion protein, subunit vaccine, immunogenicity

  20. Development of Viral Capsid DNA Aptamer Conjugates as Cell-Targeted Delivery Vehicles

    Science.gov (United States)

    Tong, Gary Jen-Wei

    The ability to generate semi-synthetic DNA-protein conjugates has become increasingly important in the fields of chemical biology and nanobiotechnology. As applications in these fields become more complex, there is also an increased need for methods of attaching synthetic DNA to protein substrates in a well-defined manner. This work outlines the development of new methods for site-specific DNA-protein bioconjugation, as well as the development of novel viral capsid DNA aptamer conjugates for cell-targeting purposes. In order to generate DNA-protein conjugates in a site-specific manner, chemistries orthogonal to native functional groups present on DNA and proteins were exploited. In one method, the attachment of DNA to proteins was achieved via oxime formation. This strategy involved the in situ deprotection of an allyloxycarbonyl-protected alkoxyamine-bearing DNA in the presence of a protein containing a single ketone group. The utility of this approach was demonstrated in the synthesis of a DNA-GFP conjugate. In addition to the oxime formation route, two oxidative coupling methods were also developed for DNA-protein bioconjugation. The first reaction coupled phenylenediamine-containing DNA to anilines, which had been site-specifically incorporated into proteins, in the presence of NaIO4. These reaction conditions were demonstrated on the proteins bacteriophage MS2 and GFP, and were mild enough for the components to retain both protein structure and DNA base-pairing capabilities. The second oxidative coupling reaction conjugated aniline-containing proteins to DNA bearing an o-aminophenol moiety. This reaction occurred under similarly mild conditions; however, higher coupling yields were achieved on MS2 at shorter reaction times by using this strategy. In all three of these methods, the generation of a singly-modified product was achieved. Using one of our oxidative coupling strategies, MS2-DNA aptamer conjugates were synthesized for the development of multivalent

  1. Dissociation of an antiviral compound from the internal pocket of human rhinovirus 14 capsid.

    Science.gov (United States)

    Li, Yumin; Zhou, Zhigang; Post, Carol Beth

    2005-05-24

    WIN antiviral compounds bind human rhinovirus, as well as enterovirus and parechovirus, in an internal cavity located within the viral protein capsid. Access to the buried pocket necessitates deviation from the average viral protein structure identified by crystallography. We investigated the dissociation of WIN 52084 from the pocket in human rhinovirus 14 by using an adiabatic, biased molecular dynamics simulation method. Multiple dissociation trajectories are used to characterize the pathway. WIN 52084 exits between the polypeptide chain near the ends of betaC and betaH in a series of steps. Small, transient packing defects in the protein are sufficient for dissociation. A number of torsion-angle transitions of the antiviral compound are involved, which suggests that flexibility in antiviral compounds is important for binding. It is interesting to note that dissociation is associated with an increase in the conformational fluctuations of residues never in direct contact with WIN 52084 over the course of dissociation. These residues are N-terminal residues in the viral proteins VP3 and VP4 and are located in the interior of the capsid near the icosahedral 5-fold axis. The observed changes in dynamics may be relevant to structural changes associated with virion uncoating and its inhibition by antiviral compounds.

  2. A pseudo-atomic model for the capsid shell of bacteriophage lambda using chemical cross-linking/mass spectrometry and molecular modeling.

    Science.gov (United States)

    Singh, Pragya; Nakatani, Eri; Goodlett, David R; Catalano, Carlos Enrique

    2013-09-23

    Bacteriophage lambda is one of the most exhaustively studied of the double-stranded DNA viruses. Its assembly pathway is highly conserved among the herpesviruses and many of the bacteriophages, making it an excellent model system. Despite extensive genetic and biophysical characterization of many of the lambda proteins and the assembly pathways in which they are implicated, there is a relative dearth of structural information on many of the most critical proteins involved in lambda assembly and maturation, including that of the lambda major capsid protein. Toward this end, we have utilized a combination of chemical cross-linking/mass spectrometry and computational modeling to construct a pseudo-atomic model of the lambda major capsid protein as a monomer, as well as in the context of the assembled procapsid shell. The approach described here is generalizable and can be used to provide structural models for any biological complex of interest. The procapsid structural model is in good agreement with published biochemical data indicating that procapsid expansion exposes hydrophobic surface area and that this serves to nucleate assembly of capsid decoration protein, gpD. The model further implicates additional molecular interactions that may be critical to the assembly of the capsid shell and for the stabilization of the structure by the gpD decoration protein.

  3. Reverse Genetics for Fusogenic Bat-Borne Orthoreovirus Associated with Acute Respiratory Tract Infections in Humans: Role of Outer Capsid Protein σC in Viral Replication and Pathogenesis.

    Directory of Open Access Journals (Sweden)

    Takahiro Kawagishi

    2016-02-01

    Full Text Available Nelson Bay orthoreoviruses (NBVs are members of the fusogenic orthoreoviruses and possess 10-segmented double-stranded RNA genomes. NBV was first isolated from a fruit bat in Australia more than 40 years ago, but it was not associated with any disease. However, several NBV strains have been recently identified as causative agents for respiratory tract infections in humans. Isolation of these pathogenic bat reoviruses from patients suggests that NBVs have evolved to propagate in humans in the form of zoonosis. To date, no strategy has been developed to rescue infectious viruses from cloned cDNA for any member of the fusogenic orthoreoviruses. In this study, we report the development of a plasmid-based reverse genetics system free of helper viruses and independent of any selection for NBV isolated from humans with acute respiratory infection. cDNAs corresponding to each of the 10 full-length RNA gene segments of NBV were cotransfected into culture cells expressing T7 RNA polymerase, and viable NBV was isolated using a plaque assay. The growth kinetics and cell-to-cell fusion activity of recombinant strains, rescued using the reverse genetics system, were indistinguishable from those of native strains. We used the reverse genetics system to generate viruses deficient in the cell attachment protein σC to define the biological function of this protein in the viral life cycle. Our results with σC-deficient viruses demonstrated that σC is dispensable for cell attachment in several cell lines, including murine fibroblast L929 cells but not in human lung epithelial A549 cells, and plays a critical role in viral pathogenesis. We also used the system to rescue a virus that expresses a yellow fluorescent protein. The reverse genetics system developed in this study can be applied to study the propagation and pathogenesis of pathogenic NBVs and in the generation of recombinant NBVs for future vaccines and therapeutics.

  4. 景德镇市两例重症EV71手足口病毒VP1区基因型分析%Analysis of the VP1 region genotype of enterovirus 71 isolated from two severe HFMD patients in Jingdezhen City

    Institute of Scientific and Technical Information of China (English)

    胡芹; 涂智杰; 吴集才; 魏建萍; 张敏; 曹健

    2013-01-01

    目的:分析2011年景德镇地区两例临床症状不同的EV71手足口重症病例病毒株的分子生物学特征。方法采集2例临床症状不同的手足口病重症患者咽拭子标本,进行病毒分离和逆转录-聚合酶链反应(RT-PCR),并将其与GenBank已上传的其他EV71病毒株的VP1区进行相关生物信息学分析。结果两例临床症状不同的重症病例都由EV71病毒引起,他们都同属于C4a亚型,通过氨基酸序列比对发现它们在四个氨基酸的位点上存在变异引起不同临床症状,其中有神经症状的患儿出现第19位丙氨酸由缬氨酸取代(A→V),第106位酪氨酸由苯丙氨酸取代(Y→F);无神经症状的患儿出现第19位丙氨酸由半胱氨酸取代(A→C),第38位精氨酸由谷氨酰胺取代(R→Q),第293位丙氨酸由丝氨酸取代(A→S)。结论 EV71病毒是引起手足口重症病例的主要病原,临床症状的差异性主要体现在有无神经系统症状,通过基因分析发现不同位点氨基酸的突变可能成为临床症状差异性形成的生物学基础。%Objective To analyze the molecular characterization of two enterovirus 71 (EV71) strains isolated from two severe hand foot mouth disease (HFMD) patients with different clinical symptoms in Jingdezhen City in 2011. Methods Throat swab specimens were collected from the two patients and EV71 were isolated for reverse transcription-polymerase chain reaction (RT-PCR) analysis. The differences of VP1 region genotype between the isolated virus and virus in GenBank were analyzed. Results Both patients with different severe clinical symptoms were caused by EV71 C4a subtype. Using amino acid sequence alignment, we found that there were four mutated amino acids and they would cause the different clinical symptoms. In EV71 isolated from pa-tient with neurologic symptoms, the 19th amino acid alanine was replaced by valine (A→V), the 106th amino acid tyrosine was re-placed by

  5. Amino Acid sequence analysis of the two major outer Capsid Proteins (VP7 and VP4 from human-derived canine G3P[3] Rotavirus Strain Detected in Brazil

    Directory of Open Access Journals (Sweden)

    Adriana Luchs

    2013-12-01

    Full Text Available Introduction: A close look at the rotavirus group A (RVA genotypes in Brazil revealed the detection of a rare G3P[3] strain close related to canine strains. The aim of this study was to add to the already known genetic analysis by the description of the G3P[3] (IAL-R2638 strain amino acid characteristics. Methods: Amino acid sequence analysis and protein based trees were conducted using BioEdit and MEGA 4.0. Results: The VP7 and VP4 protein of the IAL-R2638 strain displayed the highest amino acid identity to the canine-derived human strain HCR3A (99.2%, and to the canine strain RV52/96 (96.4%, respectively. IAL-R2638 strain did not possess an extra VP7 N-linked glycosylation site at amino acid 238 recently described for some G3 strains, as well as RotaTeqTM G3 vaccine strain. The topology exhibited by phylogenetic trees in previous analysis were maintained in the present amino acid-based trees, reinforcing a stable relationship between G3P[3] strains. Conclusions: Amino acid analysis data were consistent with the previous sequence of data obtained for the IAL-R2638 strain, supporting its possible canine origin. Theoretically, RotaTeqTM vaccine could efficiently protect against G3P[3] infections based on the lack of the extra VP7 N-linked glycosylation site at amino acid 238. Phylogenetic analysis hypothesizes that all features undergo evolution independently of each other; however, unfavorable effects of nucleotide substitutions may be compensated by substitutions in other positions. The present study raises the question as to whether the amino acid-based trees could be applied as an approach to the study of RVA evolution, avoiding incorrect phylogenetic reconstructions.

  6. Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens.

    Directory of Open Access Journals (Sweden)

    Sarah R Klein

    Full Text Available Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.

  7. Polymorphism of DNA conformation inside the bacteriophage capsid.

    Science.gov (United States)

    Leforestier, Amélie

    2013-03-01

    Double-stranded DNA bacteriophage genomes are packaged into their icosahedral capsids at the highest densities known so far (about 50 % w:v). How the molecule is folded at such density and how its conformation changes upon ejection or packaging are fascinating questions still largely open. We review cryo-TEM analyses of DNA conformation inside partially filled capsids as a function of the physico-chemical environment (ions, osmotic pressure, temperature). We show that there exists a wide variety of DNA conformations. Strikingly, the different observed structures can be described by some of the different models proposed over the years for DNA organisation inside bacteriophage capsids: either spool-like structures with axial or concentric symmetries, or liquid crystalline structures characterised by a DNA homogeneous density. The relevance of these conformations for the understanding of DNA folding and unfolding upon ejection and packaging in vivo is discussed.

  8. Vaccination of mice with a modified Vaccinia Ankara (MVA) virus expressing the African horse sickness virus (AHSV) capsid protein VP2 induces virus neutralising antibodies that confer protection against AHSV upon passive immunisation.

    Science.gov (United States)

    Calvo-Pinilla, Eva; de la Poza, Francisco; Gubbins, Simon; Mertens, Peter Paul Clement; Ortego, Javier; Castillo-Olivares, Javier

    2014-02-13

    In previous studies we showed that a recombinant Modified Vaccinia Ankara (MVA) virus expressing the protein VP2 of AHSV serotype 4 (MVA-VP2) induced virus neutralising antibodies in horses and protected interferon alpha receptor gene knock-out mice (IFNAR-/-) against challenge. We continued these studies and determined, in the IFNAR-/- mouse model, whether the antibody responses induced by MVA-VP2 vaccination play a key role in protection against AHSV. Thus, groups of mice were vaccinated with wild type MVA (MVA-wt) or MVA-VP2 and the antisera from these mice were used in a passive immunisation experiment. Donor antisera from (a) MVA-wt; (b) MVA-VP2 vaccinated; or (c) MVA-VP2 vaccinated and AHSV infected mice, were transferred to AHSV non-immune recipient mice. The recipients were challenged with virulent AHSV together with MVA-VP2 vaccinated and MVA-wt vaccinated control animals and the levels of protection against AHSV-4 were compared between all these groups. The results showed that following AHSV challenge, mice that were passively immunised with MVA-VP2 vaccinated antisera were highly protected against AHSV disease and had lower levels of viraemia than recipients of MVA-wt antisera. Our study indicates that MVA-VP2 vaccination induces a highly protective humoral immune response against AHSV.

  9. A Prime-Boost Vaccination Strategy in Cattle to Prevent Foot-and-Mouth Disease Using a "Single-Cycle" Alphavirus Vector and Empty Capsid Particles

    DEFF Research Database (Denmark)

    Gullberg, Maria; Lohse, Louise; Bøtner, Anette

    2016-01-01

    in large-scale mammalian cell culture under high containment conditions. Here, we have expressed the FMDV capsid protein precursor (P1-2A) of strain O1 Manisa alone or with the FMDV 3C protease (3Cpro) using a "single cycle" packaged alphavirus self-replicating RNA based on Semliki Forest virus (SFV). When...

  10. Predicting antigenic sites on the foot-and-mouth disease virus capsid of the South African Territories (SAT) types using virus neutralization data

    Science.gov (United States)

    Foot-and-mouth disease virus (FMDV) outer capsid proteins 1B, 1C and 1D contribute to the virus serotype distribution and antigenic variants that exist within each of the seven serotypes. This study presents a phylogenetic, genetic and antigenic analysis of the South African Territories (SAT) seroty...

  11. Structural determination of importin alpha in complex with beak and feather disease virus capsid nuclear localization signal

    Energy Technology Data Exchange (ETDEWEB)

    Patterson, Edward I. [Charles Sturt University, School of Animal and Veterinary Sciences, Boorooma St., Wagga Wagga, New South Wales 2678 (Australia); EH Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Boorooma St., Wagga Wagga, New South Wales 2678 (Australia); Dombrovski, Andrew K. [Charles Sturt University, School of Biomedical Sciences, Boorooma St., Wagga Wagga, New South Wales 2678 (Australia); Swarbrick, Crystall M.D. [Charles Sturt University, School of Biomedical Sciences, Boorooma St., Wagga Wagga, New South Wales 2678 (Australia); EH Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Boorooma St., Wagga Wagga, New South Wales 2678 (Australia); Raidal, Shane R. [Charles Sturt University, School of Animal and Veterinary Sciences, Boorooma St., Wagga Wagga, New South Wales 2678 (Australia); EH Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Boorooma St., Wagga Wagga, New South Wales 2678 (Australia); Forwood, Jade K., E-mail: jforwood@csu.edu.au [Charles Sturt University, School of Biomedical Sciences, Boorooma St., Wagga Wagga, New South Wales 2678 (Australia); EH Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Boorooma St., Wagga Wagga, New South Wales 2678 (Australia)

    2013-09-06

    Highlights: •Circovirus capsid proteins contain large nuclear localization signals (NLS). •A method of nuclear import has not been elucidated. •Beak and feather disease virus (BFDV) capsid NLS was crystallized with importin α. •The structure showed BFDV NLS binding to the major site of importin α. •Result shows implications for mechanism of nuclear transport for all circoviruses. -- Abstract: Circoviruses represent a rapidly increasing genus of viruses that infect a variety of vertebrates. Replication requires shuttling viral molecules into the host cell nucleus, a process facilitated by capsid-associated protein (Cap). Whilst a nuclear localization signal (NLS) has been shown to mediate nuclear translocation, the mode of nuclear transport remains to be elucidated. To better understand this process, beak and feather disease virus (BFDV) Cap NLS was crystallized with nuclear import receptor importin-α (Impα). Diffraction yielded structural data to 2.9 Å resolution, and the binding site on both Impα and BFDV Cap NLS were well resolved. The binding mechanism for the major site is likely conserved across circoviruses as supported by the similarity of NLSs in circovirus Caps. This finding illuminates a crucial step for infection of host cells by this viral family, and provides a platform for rational drug design against the binding interface.

  12. Structure of recombinant capsids formed by the beta-annulus deletion mutant -- rCP (Delta48-59) of Sesbania mosaic virus.

    Science.gov (United States)

    Pappachan, Anju; Subashchandrabose, Chinnathambi; Satheshkumar, P S; Savithri, H S; Murthy, M R N

    2008-05-25

    A unique feature of several T=3 icosahedral viruses is the presence of a structure called the beta-annulus formed by extensive hydrogen bonding between protein subunits related by icosahedral three-fold axis of symmetry. This unique structure has been suggested as a molecular switch that determines the T=3 capsid assembly. In order to examine the importance of the beta-annulus, a deletion mutant of Sesbania mosaic virus coat protein in which residues 48-59 involved in the formation of the beta-annulus were deleted retaining the rest of the residues in the amino terminal segment (rCP (Delta48-59)) was constructed. When expressed in Escherichia coli, the mutant protein assembled into virus like particles of sizes close to that of the wild type virus particles. The purified capsids were crystallized and their three dimensional structure was determined at 3.6 A resolution by X-ray crystallography. The mutant capsid structure closely resembled that of the native virus particles. However, surprisingly, the structure revealed that the assembly of the particles has proceeded without the formation of the beta-annulus. Therefore, the beta-annulus is not essential for T=3 capsid assembly as speculated earlier and may be formed as a consequence of the particle assembly. This is the first structural demonstration that the virus particle morphology with and without the beta-annulus could be closely similar.

  13. Modification of a loop sequence between α-helices 6 and 7 of virus capsid (CA protein in a human immunodeficiency virus type 1 (HIV-1 derivative that has simian immunodeficiency virus (SIVmac239 vif and CA α-helices 4 and 5 loop improves replication in cynomolgus monkey cells

    Directory of Open Access Journals (Sweden)

    Adachi Akio

    2009-08-01

    Full Text Available Abstract Background Human immunodeficiency virus type 1 (HIV-1 productively infects only humans and chimpanzees but not cynomolgus or rhesus monkeys while simian immunodeficiency virus isolated from macaque (SIVmac readily establishes infection in those monkeys. Several HIV-1 and SIVmac chimeric viruses have been constructed in order to develop an animal model for HIV-1 infection. Construction of an HIV-1 derivative which contains sequences of a SIVmac239 loop between α-helices 4 and 5 (L4/5 of capsid protein (CA and the entire SIVmac239 vif gene was previously reported. Although this chimeric virus could grow in cynomolgus monkey cells, it did so much more slowly than did SIVmac. It was also reported that intrinsic TRIM5α restricts the post-entry step of HIV-1 replication in rhesus and cynomolgus monkey cells, and we previously demonstrated that a single amino acid in a loop between α-helices 6 and 7 (L6/7 of HIV type 2 (HIV-2 CA determines the susceptibility of HIV-2 to cynomolgus monkey TRIM5α. Results In the study presented here, we replaced L6/7 of HIV-1 CA in addition to L4/5 and vif with the corresponding segments of SIVmac. The resultant HIV-1 derivatives showed enhanced replication capability in established T cell lines as well as in CD8+ cell-depleted primary peripheral blood mononuclear cells from cynomolgus monkey. Compared with the wild type HIV-1 particles, the viral particles produced from a chimeric HIV-1 genome with those two SIVmac loops were less able to saturate the intrinsic restriction in rhesus monkey cells. Conclusion We have succeeded in making the replication of simian-tropic HIV-1 in cynomolgus monkey cells more efficient by introducing into HIV-1 the L6/7 CA loop from SIVmac. It would be of interest to determine whether HIV-1 derivatives with SIVmac CA L4/5 and L6/7 can establish infection of cynomolgus monkeys in vivo.

  14. Engineering Virus Capsids Into Biomedical Delivery Vehicles: Structural Engineering Problems in Nanoscale.

    Science.gov (United States)

    Bajaj, Saumya; Banerjee, Manidipa

    2015-01-01

    Virus capsids have evolved to protect the genome sequestered in their interior from harsh environmental conditions, and to deliver it safely and precisely to the host cell of choice. This characteristic makes them naturally perfect containers for delivering therapeutic molecules to specific locations. Development of an ideal virus-based nano-container for medical usage requires that the capsid be converted into a targetable protein cage which retains the original stability, flexibility and host cell penetrating properties of the native particles, without the associated immunogenicity, and is able to encapsulate large quantities of therapeutic or diagnostic material. In the last few years, several icosahedral, non-enveloped viruses, with a diameter of 25-90 nm-a size which conveniently falls within the 10-100 nm range desirable for biomedical nanoparticles-have been chemically or genetically engineered towards partial fulfilment of the above criteria. This review summarizes the approaches taken towards engineering viruses into biomedical delivery devices and discusses the challenges involved in achieving this goal.

  15. Codon optimization of the rabbit hemorrhagic disease virus (RHDV) capsid gene leads to increased gene expression in Spodoptera frugiperda 9 (Sf9) cells.

    Science.gov (United States)

    Gao, Jingpeng; Meng, Chunchun; Chen, Zongyan; Li, Chuanfeng; Liu, Guangqing

    2013-01-01

    Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.

  16. Specificity of an anti-capsid antibody associated with Hepatitis B Virus-related acute liver failure.

    Science.gov (United States)

    Wu, Weimin; Chen, Zhaochun; Cheng, Naiqian; Watts, Norman R; Stahl, Stephen J; Farci, Patrizia; Purcell, Robert H; Wingfield, Paul T; Steven, Alasdair C

    2013-01-01

    Previously, the livers of patients suffering from acute liver failure (ALF), a potentially fatal syndrome arising from infection by Hepatitis B Virus (HBV), were found to contain massive amounts of an antibody specific for the core antigen (HBcAg) capsid. We have used cryo-electron microscopy and molecular modeling to define its epitope. HBV capsids are icosahedral shells with 25Å-long dimeric spikes, each a 4-helix bundle, protruding from the contiguous "floor". Of the anti-HBcAg antibodies previously characterized, most bind around the spike tip while one binds to the floor. The ALF-associated antibody binds tangentially to a novel site on the side of the spike. This epitope is conformational. The Fab binds with high affinity to its principal determinants but has lower affinities for quasi-equivalent variants. The highest occupancy site is on one side of a spike, with no detectable binding to the corresponding site on the other side. Binding of one Fab per dimer was also observed by analytical ultracentrifugation. The Fab did not bind to the e-antigen dimer, a non-assembling variant of capsid protein. These findings support the propositions that antibodies with particular specificities may correlate with different clinical expressions of HBV infection and that antibodies directed to particular HBcAg epitopes may be involved in ALF pathogenesis.

  17. Increasing Type 1 Poliovirus Capsid Stability by Thermal Selection

    Science.gov (United States)

    Adeyemi, Oluwapelumi O.; Nicol, Clare

    2016-01-01

    ABSTRACT Poliomyelitis is a highly infectious disease caused by poliovirus (PV). It can result in paralysis and may be fatal. Integrated global immunization programs using live-attenuated oral (OPV) and/or inactivated (IPV) PV vaccines have systematically reduced its spread and paved the way for eradication. Immunization will continue posteradication to ensure against reintroduction of the disease, but there are biosafety concerns for both OPV and IPV. They could be addressed by the production and use of virus-free virus-like particle (VLP) vaccines that mimic the “empty” capsids (ECs) normally produced in viral infection. Although ECs are antigenically indistinguishable from mature virus particles, they are less stable and readily convert into an alternative conformation unsuitable for vaccine purposes. Stabilized ECs, expressed recombinantly as VLPs, could be ideal candidate vaccines for a polio-free world. However, although genome-free PV ECs have been expressed as VLPs in a variety of systems, their inherent antigenic instability has proved a barrier to further development. In this study, we selected thermally stable ECs of type 1 PV (PV-1). The ECs are antigenically stable at temperatures above the conversion temperature of wild-type (wt) virions. We have identified mutations on the capsid surface and in internal networks that are responsible for EC stability. With reference to the capsid structure, we speculate on the roles of these residues in capsid stability and postulate that such stabilized VLPs could be used as novel vaccines. IMPORTANCE Poliomyelitis is a highly infectious disease caused by PV and is on the verge of eradication. There are biosafety concerns about reintroduction of the disease from current vaccines that require live virus for production. Recombinantly expressed virus-like particles (VLPs) could address these inherent problems. However, the genome-free capsids (ECs) of wt PV are unstable and readily change antigenicity to a form not

  18. Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design.

    Science.gov (United States)

    Kotecha, Abhay; Seago, Julian; Scott, Katherine; Burman, Alison; Loureiro, Silvia; Ren, Jingshan; Porta, Claudine; Ginn, Helen M; Jackson, Terry; Perez-Martin, Eva; Siebert, C Alistair; Paul, Guntram; Huiskonen, Juha T; Jones, Ian M; Esnouf, Robert M; Fry, Elizabeth E; Maree, Francois F; Charleston, Bryan; Stuart, David I

    2015-10-01

    Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.

  19. Useful scars: Physics of the capsids of archaeal viruses

    Science.gov (United States)

    Perotti, L. E.; Dharmavaram, S.; Klug, W. S.; Marian, J.; Rudnick, J.; Bruinsma, R. F.

    2016-07-01

    We propose a physical model for the capsids of tailed archaeal viruses as viscoelastic membranes under tension. The fluidity is generated by thermal motion of scarlike structures that are an intrinsic feature of the ground state of large particle arrays covering surfaces with nonzero Gauss curvature. The tension is generated by a combination of the osmotic pressure of the enclosed genome and an extension force generated by filamentous structure formation that drives the formation of the tails. In continuum theory, the capsid has the shape of a surface of constant mean curvature: an unduloid. Particle arrays covering unduloids are shown to exhibit pronounced subdiffusive and diffusive single-particle transport at temperatures that are well below the melting temperature of defect-free particle arrays on a surface with zero Gauss curvature.

  20. Three-dimensional structure determination of capsid of Aedes albopicus C6/36 cell densovirus

    Institute of Scientific and Technical Information of China (English)

    CHENG Lingpeng; CHEN Senxiong; Jenifer M.Brannan; Joanita Jakana; ZHANG Qinfen; Z.H.Zhou; ZHANG Jingqiang

    2004-01-01

    The three-dimensional structure of capsid of Aedes albopictus C6/36 densovirus was determined to 14-(A) resolution by electron cryomicroscopy and computer reconstruction. The triangulation number of the capsid is 1. There are 12 holes in each triangular face and a spike on each 5-fold vertex. The validity of the capsid and nucleic acid densities in the reconstructions was discussed.

  1. A mutation deleting sequences encoding the amino terminus of human cytomegalovirus UL84 impairs interaction with UL44 and capsid localization.

    Science.gov (United States)

    Strang, Blair L; Bender, Brian J; Sharma, Mayuri; Pesola, Jean M; Sanders, Rebecca L; Spector, Deborah H; Coen, Donald M

    2012-10-01

    Protein-protein interactions are required for many biological functions. Previous work has demonstrated an interaction between the human cytomegalovirus DNA polymerase subunit UL44 and the viral replication factor UL84. In this study, glutathione S-transferase pulldown assays indicated that residues 1 to 68 of UL84 are both necessary and sufficient for efficient interaction of UL84 with UL44 in vitro. We created a mutant virus in which sequences encoding these residues were deleted. This mutant displayed decreased virus replication compared to wild-type virus. Immunoprecipitation assays showed that the mutation decreased but did not abrogate association of UL84 with UL44 in infected cell lysate, suggesting that the association in the infected cell can involve other protein-protein interactions. Further immunoprecipitation assays indicated that IRS1, TRS1, and nucleolin are candidates for such interactions in infected cells. Quantitative real-time PCR analysis of viral DNA indicated that the absence of the UL84 amino terminus does not notably affect viral DNA synthesis. Western blotting experiments and pulse labeling of infected cells with [(35)S]methionine demonstrated a rather modest downregulation of levels of multiple proteins and particularly decreased levels of the minor capsid protein UL85. Electron microscopy demonstrated that viral capsids assemble but are mislocalized in nuclei of cells infected with the mutant virus, with fewer cytoplasmic capsids detected. In sum, deletion of the sequences encoding the amino terminus of UL84 affects interaction with UL44 and virus replication unexpectedly, not viral DNA synthesis. Mislocalization of viral capsids in infected cell nuclei likely contributes to the observed decrease in virus replication.

  2. Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.

    Science.gov (United States)

    Lin, S F; Sun, R; Heston, L; Gradoville, L; Shedd, D; Haglund, K; Rigsby, M; Miller, G

    1997-04-01

    We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV). The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate. The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene of herpesvirus saimiri. KSHV sVCA was expressed from a 0.85-kb mRNA present late in lytic KSHV replication in BC-1 cells. This transcript was sensitive to phosphonoacetic acid and phosphonoformic acid, inhibitors of herpesvirus DNA replication. KSHV sVCA expressed in mammalian cells or Escherichia coli or translated in vitro was recognized as an antigen by antisera from KS patients. Rabbit antisera raised to KSHV sVCA expressed in E. coli detected a 22-kDa protein in KSHV-infected human B cells. Overexpressed KSHV sVCA purified from E. coli and used as an antigen in immunoblot screening assay did not cross-react with EBV BFRF3. Antibodies to sVCA were present in 89% of 47 human immunodeficiency virus (HIV)-positive patients with KS, in 20% of 54 HIV-positive patients without KS, but in none of 122 other patients including children born to HIV-seropositive mothers and patients with hemophilia, autoimmune disease, or nasopharyngeal carcinoma. Low-titer antibody was detected in three sera from 28 healthy subjects. Antibodies to recombinant sVCA correlate with KS in high-risk populations. Recombinant sVCA can be used to examine the seroepidemiology of infection with KSHV in the general population.

  3. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes.

    Directory of Open Access Journals (Sweden)

    Bruno Hernáez

    2016-04-01

    Full Text Available African swine fever virus (ASFV is a nucleocytoplasmic large DNA virus (NCLDV that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs.

  4. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes

    Science.gov (United States)

    Hernáez, Bruno; Guerra, Milagros; Salas, María L.

    2016-01-01

    African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

  5. Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo.

    Science.gov (United States)

    Li, Chengwen; Hirsch, Matthew; Asokan, Aravind; Zeithaml, Brian; Ma, Hong; Kafri, Tal; Samulski, R Jude

    2007-07-01

    A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response.

  6. Adeno-Associated Virus Type 2 (AAV2) Capsid-Specific Cytotoxic T Lymphocytes Eliminate Only Vector-Transduced Cells Coexpressing the AAV2 Capsid In Vivo▿

    Science.gov (United States)

    Li, Chengwen; Hirsch, Matthew; Asokan, Aravind; Zeithaml, Brian; Ma, Hong; Kafri, Tal; Samulski, R. Jude

    2007-01-01

    A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response. PMID:17475652

  7. Production, purification, and capsid stability of rhinovirus C types.

    Science.gov (United States)

    Griggs, Theodor F; Bochkov, Yury A; Nakagome, Kazuyuki; Palmenberg, Ann C; Gern, James E

    2015-06-01

    The rhinovirus C (RV-C) were discovered in 2006 and these agents are an important cause of respiratory morbidity. Little is known about their biology. RV-C15 (C15) can be produced by transfection of recombinant viral RNA into cells and subsequent purification over a 30% sucrose cushion, even though yields and infectivity of other RV-C genotypes with this protocol are low. The goal of this study was to determine whether poor RV-C yields were due to capsid instability, and moreover, to develop a robust protocol suitable for the purification of many RV-C types. Capsid stability assays indicated that virions of RV-C41 (refractory to purification) have similar tolerance for osmotic and temperature stress as RV-A16 (purified readily), although C41 is more sensitive to low pH. Modification to the purification protocol by removing detergent increased the yield of RV-C. Addition of nonfat dry milk to the sucrose cushion increased the virus yield but sacrificed purity of the viral suspension. Analysis of virus distribution following centrifugation indicated that the majority of detectable viral RNA (vRNA) was found in pellets refractory to resuspension. Reduction of the centrifugal force with commiserate increase in spin-time improved the recovery of RV-C for both C41 and C2. Transfection of primary lung fibroblasts (WisL cells) followed by the modified purification protocol further improved yields of infectious C41 and C2. Described herein is a higher yield purification protocol suitable for RV-C types refractory to the standard purification procedure. The findings suggest that aggregation-adhesion problems rather than capsid instability influence RV-C yield during purification.

  8. Study of clinical application of HPV L1 capsid protein combined with HPV typing and TCT detec-tion in diagnosis and treatment of cervical lesion%HPV L1壳蛋白联合 HPV 分型、TCT 检测在宫颈病变诊治中的临床应用研究

    Institute of Scientific and Technical Information of China (English)

    沈姚琴; 赖娟; 贺俊霞

    2015-01-01

    Objective To investigate the expression of human papilloma virus L1 (HPV L1)capsid protein in cervical lesions and different human papillomavirus ( HPV) subtypes, and to guide clinical triage management and best individual treatment.Methods Retrospective analysis of 2012 January to 2014 Janu-ary in Jiaxing Hospital of Traditional Chinese Medicine gynecology clinic for HPV L1 protein combined with HPV type, and liquid-based cytology test ( TCT) of 176 patients data.Results The positive expression rate of HPV L1 protein with TCT examination in the negative for intraepithelial lesion or malignancy ( NILM) , atypical squamous cells of undetermined significance( ASCUS) , low-grade squamous intraepithe lial lesion ( LSIL) , atypical squamous cells not except high lesion ( ASC-H) , high-grade squamous intraep-ithelial lesion ( HSIL) , and squamous-cell carcinoma ( SCC) was 28.99%, 44.19%, 64.44%, 22.22%, 12.50%, and 0, respectively.No significant differences were found between the NILM and ASCUS groups ( P >0.05) .The positive expression rate of HPV L1 protein in LSIL group was the highest, and it was sta-tistically significantly different from ASC-H and HSIL groups (χ2 =3.88,5.50, P 0.05 ); and statistically significant difference was found between CIN Ⅰgroup and CINⅡ, CINⅢgroup (χ2 =4.53,5.56, P 0.05) .Conclusions Detection of HPV L1 protein is of clinical value to evaluate the risk of cervical lesions.HPV L1 protein combined with HPV type and TCT detection is helpful for traffic man-agement and personalized treatment, and benefit patients with cervical lesions.%目的:探讨人乳头瘤病毒L1(HPV L1)壳蛋白在不同宫颈病变及不同HPV亚型中的表达,以指导宫颈病变患者的个体化治疗及分流管理。方法回顾性分析本院妇科门诊行HPV L1壳蛋白联合HPV分型、液基薄层细胞学检测( TCT)的176例患者的临床资料。结果宫颈TCT结果中不同情况与HPV L1壳蛋白的阳性表达率分

  9. Alternative Polyadenylation of Human Bocavirus at Its 3′ End Is Regulated by Multiple Elements and Affects Capsid Expression

    Science.gov (United States)

    Hao, Sujuan; Zhang, Junmei; Chen, Zhen; Xu, Huanzhou; Wang, Hanzhong

    2016-01-01

    ABSTRACT Alternative processing of human bocavirus (HBoV) P5 promoter-transcribed RNA is critical for generating the structural and nonstructural protein-encoding mRNA transcripts. The regulatory mechanism by which HBoV RNA transcripts are polyadenylated at proximal [(pA)p] or distal [(pA)d] polyadenylation sites is still unclear. We constructed a recombinant HBoV infectious clone to study the alternative polyadenylation regulation of HBoV. Surprisingly, in addition to the reported distal polyadenylation site, (pA)d, a novel distal polyadenylation site, (pA)d2, which is located in the right-end hairpin (REH), was identified during infectious clone transfection or recombinant virus infection. (pA)d2 does not contain typical hexanucleotide polyadenylation signal, upstream elements (USE), or downstream elements (DSE) according to sequence analysis. Further study showed that HBoV nonstructural protein NS1, REH, and cis elements of (pA)d were necessary and sufficient for efficient polyadenylation at (pA)d2. The distance and sequences between (pA)d and (pA)d2 also played a key role in the regulation of polyadenylation at (pA)d2. Finally, we demonstrated that efficient polyadenylation at (pA)d2 resulted in increased HBoV capsid mRNA transcripts and protein translation. Thus, our study revealed that all the bocaviruses have distal poly(A) signals on the right-end palindromic terminus, and alternative polyadenylation at the HBoV 3′ end regulates its capsid expression. IMPORTANCE The distal polyadenylation site, (pA)d, of HBoV is located about 400 nucleotides (nt) from the right-end palindromic terminus, which is different from those of bovine parvovirus (BPV) and canine minute virus (MVC) in the same genus whose distal polyadenylation is located in the right-end stem-loop structure. A novel polyadenylation site, (pA)d2, was identified in the right-end hairpin of HBoV during infectious clone transfection or recombinant virus infection. Sequence analysis showed that (pA)d2

  10. Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung

    Directory of Open Access Journals (Sweden)

    Sabrina V. Martini

    2016-07-01

    Full Text Available Background/Aims: Adeno-associated virus (AAV vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Methods: Eighteen C57BL/6 mice were randomly assigned into three groups: (1 a control group (CTRL animals underwent intratracheal (i.t. instillation of saline, (2 the wild-type AAV9 group (WT-AAV9, 1010 vg, and (3 the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg, which received (i.t. self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP. Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. Results: No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30% compared with their wild-type counterparts, without eliciting an inflammatory response. Conclusion: Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy.

  11. Cloning and Functional Analysis of Viviparous-1 Promoter in Wheat%小麦穗发芽抗性相关Vp1基因启动子的分离及功能验证

    Institute of Scientific and Technical Information of China (English)

    孙永伟; 聂丽娜; 马有志; 徐兆师; 夏兰琴

    2011-01-01

    成熟期穗发芽严重影响小麦产量和品质.Vp1是调节胚发育,促进胚成熟和休眠的重要转录因子,对小麦种子休眠和穗发芽抗性具有重要作用.本研究分离了普通小麦B基因组Vp1基因的启动子,生物信息学预测结果表明,其含有9个脱落酸响应元件ABRE、2个DREB和6个MYB干旱响应元件、3个赤霉素响应元件GARE、1个水杨酸响应元件TCA-E、2个茉莉酸甲酯响应元件TGACG-motif、4个SKn-1和1个RYREPE胚乳特异表达元件.采用5′端缺失的方法,构建了系列含Vp1启动子不同区段融合GUS报告基因的瞬时表达载体和植物表达载体.通过基因枪转化小麦愈伤组织,瞬时表达结果显示,Vp1启动子在无诱导的情况下不能启动GUS基因表达,在低温、ABA、GA、PEG和NaC1诱导后可以启动GUS基因表达,表现诱导表达特性,且其诱导表达强度随启动子缺失片段长度变短而减弱.利用Gateway方法成功构建了6个启动子各缺失片段类型的植物表达载体,并通过农杆菌介导转化四倍体小麦Stewart,获得转基因植株.该启动子可有效启动GUS基因在转基因植株的花药、糊粉层、穗轴及根中表达,其他组织中没有表达.当启动子片段大于660 bp时,外源ABA可诱导启动子启动GUS基因在转基因植株茎节中的表达.%Viviparousl (Vpl) plays an important role in regulating embryo development, seed dormancy, and germination in higher plants. Although we previously demonstrated a close correlation between VplB allelic variation and the different levels of tolerance to pre-harvest sprouting (PHS) in common wheat, little is known about the potential cw-elements in VplB promoter region, which may be involved in the control of PHS by VplB. In this study, a 2 232 bp VplB upstream sequence was isolated. In silico analysis revealed the presence of nine ABRE, two DREB, six MYB, three GARE, one TCA-E, two TGACG-motif, four SKn-1, and one RYREPE cis-elements in the

  12. Single hepatitis-B virus core capsid binding to individual nuclear pore complexes in Hela cells.

    Science.gov (United States)

    Lill, Yoriko; Lill, Markus A; Fahrenkrog, Birthe; Schwarz-Herion, Kyrill; Paulillo, Sara; Aebi, Ueli; Hecht, Bert

    2006-10-15

    We investigate the interaction of hepatitis B virus capsids lacking a nuclear localization signal with nuclear pore complexes (NPCs) in permeabilized HeLa cells. Confocal and wide-field optical images of the nuclear envelope show well-spaced individual NPCs. Specific interactions of capsids with single NPCs are characterized by extended residence times of capsids in the focal volume which are characterized by fluorescence correlation spectroscopy. In addition, single-capsid-tracking experiments using fast wide-field fluorescence microscopy at 50 frames/s allow us to directly observe specific binding via a dual-color colocalization of capsids and NPCs. We find that binding occurs with high probability on the nuclear-pore ring moiety, at 44 +/- 9 nm radial distance from the central axis.

  13. Detection of Aichi virus with antibody targeting of conserved viral protein 1 epitope.

    Science.gov (United States)

    Chen, Yao-Shen; Chen, Bao-Chen; Lin, You-Sheng; Chang, Jenn-Tzong; Huang, Tsi-Shu; Chen, Jih-Jung; Chang, Tsung-Hsien

    2013-10-01

    Aichi virus (AiV) is an emerging single-stranded, positive-sense, non-enveloped RNA virus in the Picornaviridae that causes acute gastroenteritis in humans. The first case of AiV infection in Taiwan was diagnosed in a human neonate with enterovirus-associated symptoms; the virus was successfully isolated and propagated. To establish a method to detect AiV, we analyzed the antigen epitope and generated a polyclonal antibody against AiV viral protein 1 (VP1). This peptide-purified anti-AiV VP1 antibody showed high specificity against AiV VP1 without cross-reaction to nine other tested strains of Picornaviruses. The anti-AiV VP1 antibody was used in immunofluorescence analysis, immunoblotting, and enzyme-linked immunosorbent assay to elucidate the cell tropism and replication kinetics of AiV. Use of the anti-AiV VP1 antibody also revealed AiV infection restriction with interferon type I and polyI/C antiviral treatment. The AiV infection and detection system may provide an in vitro platform for AiV virology study.

  14. The VPS4 component of the ESCRT machinery plays an essential role in HPV infectious entry and capsid disassembly

    Science.gov (United States)

    Broniarczyk, Justyna; Pim, David; Massimi, Paola; Bergant, Martina; Goździcka-Józefiak, Anna; Crump, Colin; Banks, Lawrence

    2017-01-01

    Human Papillomavirus (HPV) infection involves multiple steps, from cell attachment, through endocytic trafficking towards the trans-Golgi network, and, ultimately, the entry into the nucleus during mitosis. An essential viral protein in infectious entry is the minor capsid protein L2, which engages different components of the endocytic sorting machinery during this process. The ESCRT machinery is one such component that seems to play an important role in the early stages of infection. Here we have analysed the role of specific ESCRT components in HPV infection, and we find an essential role for VPS4. Loss of VPS4 blocks infection with multiple PV types, suggesting an evolutionarily conserved critical step in infectious entry. Intriguingly, both L1 and L2 can interact with VPS4, and appear to be in complex with VPS4 during the early stages of virus infection. By using cell lines stably expressing a dominant-negative mutant form of VPS4, we also show that loss of VPS4 ATPase activity results in a marked delay in capsid uncoating, resulting in a defect in the endocytic transport of incoming PsVs. These results demonstrate that the ESCRT machinery, and in particular VPS4, plays a critical role in the early stages of PV infection.

  15. Construction of a novel coarse grain model for simulations of HIV capsid assembly to capture the backbone structure and inter-domain motions in solution

    Directory of Open Access Journals (Sweden)

    Xin Qiao

    2015-12-01

    Full Text Available We show the construction of a novel coarse grain model for simulations of HIV capsid assembly based on four structural models of HIV capsid proteins: isolated hexamer 3H47.pdb, tubular assembly 3J34.pdb, isolated pentamer 3P05.pdb and C-terminus dimer 2KOD.pdb. The data demonstrates the derivation of inter-domain motions from all atom Molecular Dynamics simulations and comparison with the motions derived from the analysis of solution NMR results defined in 2M8L.pdb. Snapshots from a representative Monte Carlo simulation with 128 dimeric subunit proteins based on 3J34.pdb are shown in addition to the quantitative analysis of its assembly pathway. Movies of the assembly process are compiled with snapshots of representative simulations of four structural models. The methods and data in this article were utilized in Qiao et al. (in press [1] to probe the mechanism of polymorphism and curvature control of HIV capsid assembly.

  16. Immobilization and One-Dimensional Arrangement of Virus Capsids with Nanoscale Precision Using DNA Origami

    Energy Technology Data Exchange (ETDEWEB)

    Stephanopoulos, Nicholas [Univ. of California, Berkeley, CA (United States); Liu, Minghui [Arizona State Univ., Tempe, AZ (United States); Tong, Gary J [Univ. of California, Berkeley, CA (United States); Li, Zhe [Arizona State Univ., Tempe, AZ (United States); Liu, Yan [Arizona State Univ., Tempe, AZ (United States); Yan, Hao [Arizona State Univ., Tempe, AZ (United States); Francis, Matthew B [Univ. of California, Berkeley, CA (United States)

    2010-06-24

    DNA origami was used as a scaffold to arrange spherical virus capsids into one-dimensional arrays with precise nanoscale positioning. To do this, we first modified the interior surface of bacteriophage MS2 capsids with fluorescent dyes as a model cargo. An unnatural amino acid on the external surface was then coupled to DNA strands that were complementary to those extending from origami tiles. Two different geometries of DNA tiles (rectangular and triangular) were used. The capsids associated with tiles of both geometries with virtually 100% efficiency under mild annealing conditions, and the location of capsid immobilization on the tile could be controlled by the position of the probe strands. The rectangular tiles and capsids could then be arranged into one-dimensional arrays by adding DNA strands linking the corners of the tiles. The resulting structures consisted of multiple capsids with even spacing (~100 nm). We also used a second set of tiles that had probe strands at both ends, resulting in a one-dimensional array of alternating capsids and tiles. This hierarchical self-assembly allows us to position the virus particles with unprecedented control and allows the future construction of integrated multicomponent systems from biological scaffolds using the power of rationally engineered DNA nanostructures.

  17. Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids

    Science.gov (United States)

    Castle, Michael J.; Turunen, Heikki T.; Vandenberghe, Luk H.; Wolfe, John H.

    2016-01-01

    More than one hundred naturally occurring variants of adeno-associated virus (AAV) have been identified, and this library has been further expanded by an array of techniques for modification of the viral capsid. AAV capsid variants possess unique antigenic profiles and demonstrate distinct cellular tropisms driven by differences in receptor binding. AAV capsids can be chemically modified to alter tropism, can be produced as hybrid vectors that combine the properties of multiple serotypes, and can carry peptide insertions that introduce novel receptor-binding activity. Furthermore, directed evolution of shuffled genome libraries can identify engineered variants with unique properties, and rational modification of the viral capsid can alter tropism, reduce blockage by neutralizing antibodies, or enhance transduction efficiency. This large number of AAV variants and engineered capsids provides a varied toolkit for gene delivery to the CNS and retina, with specialized vectors available for many applications, but selecting a capsid variant from the array of available vectors can be difficult. This chapter describes the unique properties of a range of AAV variants and engineered capsids, and provides a guide for selecting the appropriate vector for specific applications in the CNS and retina. PMID:26611584

  18. Role of the disaggregase ClpB in processing of proteins aggregated as inclusion bodies.

    Science.gov (United States)

    Zblewska, Kamila; Krajewska, Joanna; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina

    2014-08-01

    Overproduction of heterologous proteins in bacterial systems often results in the formation of insoluble inclusion bodies (IBs), which is a major impediment in biochemical research and biotechnology. In principle, the activity of molecular chaperones could be employed to gain control over the IB formation and to improve the recombinant protein yields, but the potential of each of the major bacterial chaperones (DnaK/J, GroEL/ES, and ClpB) to process IBs has not been fully established yet. We investigated the formation of inclusion bodies (IBs) of two aggregation-prone proteins, VP1LAC and VP1GFP, overproduced in Escherichiacoli in the presence and absence of the chaperone ClpB. We found that both ClpB isoforms, ClpB95 and ClpB80 accumulated in E. coli cells during the production of IBs. The amount of IB proteins increased in the absence of ClpB. ClpB supported the resolubilization and reactivation of the aggregated VP1LAC and VP1GFP in E. coli cells. The IB disaggregation was optimal in the presence of both ClpB95 and ClpB80. Our results indicate an essential role of ClpB in controlling protein aggregation and inclusion body formation in bacteria.

  19. On the geometry of regular icosahedral capsids containing disymmetrons

    CERN Document Server

    Ang, Kai-Siang

    2016-01-01

    Icosahedral virus capsids are composed of symmetrons, organized arrangements of capsomers. There are three types of symmetrons: disymmetrons, trisymmetrons, and pentasymmetrons, which have different shapes and are centered on the icosahedral 2-fold, 3-fold and 5-fold axes of symmetry, respectively. In 2010 [Sinkovits & Baker] gave a classification of all possible ways of building an icosahedral structure solely from trisymmetrons and pentasymmetrons, which requires the triangulation number T to be odd. In the present paper we incorporate disymmetrons to obtain a geometric classification of icosahedral viruses formed by regular penta-, tri-, and disymmetrons. For every class of solutions, we further provide formulas for symmetron sizes and parity restrictions on h, k, and T numbers. We also present several methods in which invariants may be used to classify a given configuration.

  20. AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

    Energy Technology Data Exchange (ETDEWEB)

    Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M., E-mail: wilsonjm@mail.med.upenn.edu

    2014-04-15

    Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8.