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Sample records for sarcoma-associated herpesvirus-encoded orf11

  1. A negative element involved in Kaposi's sarcoma-associated herpesvirus-encoded ORF11 gene expression

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    Chen, Lei [Los Alamos National Laboratory

    2009-01-01

    The ORF11 of the Kaposi's sarcoma-associated herpesvirus (KSHV) is a lytic viral gene with delayed-early expression kinetics. How the ORF11 gene expression is regulated in the KSHV lytic cascade is largely unknown. Here we report that the deletion of the KSHV viral IL-6 gene from the viral genome leads to deregulated ORF11 gene expression. The KSHV-encoded viral IL-6 protein was found not to be essentially involved in the regulation of ORF11, suggesting a potential transcriptional cis-regulation. A negative element was identified downstream of the ORF11 gene, which suppresses the ORF11 basal promoter activity in a position-independent manner.

  2. Kaposi's sarcoma-associated herpesvirus-encoded LANA associates with glucocorticoid receptor and enhances its transcriptional activities

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    Togi, Sumihito; Nakasuji, Misa; Muromoto, Ryuta; Ikeda, Osamu; Okabe, Kanako; Kitai, Yuichi; Kon, Shigeyuki [Department of Immunology, Graduate School of Pharmaceutical Sciences Hokkaido University, Sapporo 060-0812 (Japan); Oritani, Kenji [Department of Hematology and Oncology, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Matsuda, Tadashi, E-mail: tmatsuda@pharm.hokudai.ac.jp [Department of Immunology, Graduate School of Pharmaceutical Sciences Hokkaido University, Sapporo 060-0812 (Japan)

    2015-07-31

    Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA), which interacts with cellular proteins, plays a central role in modification of viral and/or cellular gene expression. Here, we show that LANA associates with glucocorticoid receptor (GR), and that LANA enhances the transcriptional activity of GR. Co-immunoprecipitation revealed a physical interaction between LANA and GR in transiently transfected 293T and HeLa cells. In human B-lymphoma cells, LANA overexpression enhanced GR activity and cell growth suppression following glucocorticoid stimulation. Furthermore, confocal microscopy showed that activated GR was bound to LANA and accumulated in the nucleus, leading to an increase in binding of activated GR to the glucocorticoid response element of target genes. Taken together, KSHV-derived LANA acts as a transcriptional co-activator of GR. Our results might suggest a careful use of glucocorticoids in the treatment of patients with KSHV-related malignancies such as Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. - Highlights: • KSHV-LANA enhances the transcriptional activity of GR in 293T and HeLa cells. • KSHV-LANA physically associates with GR. • KSHV-LANA enhances GR activation and cell growth suppression in human B-lymphocytes. • KSHV-LANA influences the nuclear retention and DNA binding activity of GR.

  3. Kaposi's sarcoma associated herpesvirus encoded viral FLICE inhibitory protein K13 activates NF-κB pathway independent of TRAF6, TAK1 and LUBAC.

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    Hittu Matta

    Full Text Available Kaposi's sarcoma associated herpesvirus encoded viral FLICE inhibitory protein (vFLIP K13 activates the NF-κB pathway by binding to the NEMO/IKKγ subunit of the IκB kinase (IKK complex. However, it has remained enigmatic how K13-NEMO interaction results in the activation of the IKK complex. Recent studies have implicated TRAF6, TAK1 and linear ubiquitin chains assembled by a linear ubiquitin chain assembly complex (LUBAC consisting of HOIL-1, HOIP and SHARPIN in IKK activation by proinflammatory cytokines.Here we demonstrate that K13-induced NF-κB DNA binding and transcriptional activities are not impaired in cells derived from mice with targeted disruption of TRAF6, TAK1 and HOIL-1 genes and in cells derived from mice with chronic proliferative dermatitis (cpdm, which have mutation in the Sharpin gene (Sharpin(cpdm/cpdm. Furthermore, reconstitution of NEMO-deficient murine embryonic fibroblast cells with NEMO mutants that are incapable of binding to linear ubiquitin chains supported K13-induced NF-κB activity. K13-induced NF-κB activity was not blocked by CYLD, a deubiquitylating enzyme that can cleave linear and Lys63-linked ubiquitin chains. On the other hand, NEMO was required for interaction of K13 with IKK1/IKKα and IKK2/IKKβ, which resulted in their activation by "T Loop" phosphorylation.Our results demonstrate that K13 activates the NF-κB pathway by binding to NEMO which results in the recruitment of IKK1/IKKα and IKK2/IKKβ and their subsequent activation by phosphorylation. Thus, K13 activates NF-κB via a mechanism distinct from that utilized by inflammatory cytokines. These results have important implications for the development of therapeutic agents targeting K13-induced NF-κB for the treatment of KSHV-associated malignancies.

  4. p53 Tumor Suppressor Protein Stability and Transcriptional Activity Are Targeted by Kaposi's Sarcoma-Associated Herpesvirus-Encoded Viral Interferon Regulatory Factor 3

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    Baresova, Petra; Musilova, Jana; Pitha, Paula M.

    2014-01-01

    Viruses have developed numerous strategies to counteract the host cell defense. Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus linked to the development of Kaposi's sarcoma, Castleman's disease, and primary effusion lymphoma (PEL). The virus-encoded viral interferon regulatory factor 3 (vIRF-3) gene is a latent gene which is involved in the regulation of apoptosis, cell cycle, antiviral immunity, and tumorigenesis. vIRF-3 was shown to interact with p53 and inhibit p53-mediated apoptosis. However, the molecular mechanism underlying this phenomenon has not been established. Here, we show that vIRF-3 associates with the DNA-binding domain of p53, inhibits p53 phosphorylation on serine residues S15 and S20, and antagonizes p53 oligomerization and the DNA-binding affinity. Furthermore, vIRF-3 destabilizes p53 protein by increasing the levels of p53 polyubiquitination and targeting p53 for proteasome-mediated degradation. Consequently, vIRF-3 attenuates p53-mediated transcription of the growth-regulatory p21 gene. These effects of vIRF-3 are of biological relevance since the knockdown of vIRF-3 expression in KSHV-positive BC-3 cells, derived from PEL, leads to an increase in p53 phosphorylation, enhancement of p53 stability, and activation of p21 gene transcription. Collectively, these data suggest that KSHV evolved an efficient mechanism to downregulate p53 function and thus facilitate uncontrolled cell proliferation and tumor growth. PMID:24248600

  5. NEMO is essential for Kaposi's sarcoma-associated herpesvirus-encoded vFLIP K13-induced gene expression and protection against death receptor-induced cell death, and its N-terminal 251 residues are sufficient for this process.

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    Tolani, Bhairavi; Matta, Hittu; Gopalakrishnan, Ramakrishnan; Punj, Vasu; Chaudhary, Preet M

    2014-06-01

    Kaposi's sarcoma-associated herpesvirus-encoded viral FLICE inhibitory protein (vFLIP) K13 was originally believed to protect virally infected cells against death receptor-induced apoptosis by interfering with caspase 8/FLICE activation. Subsequent studies revealed that K13 also activates the NF-κB pathway by binding to the NEMO/inhibitor of NF-κB (IκB) kinase gamma (IKKγ) subunit of an IKK complex and uses this pathway to modulate the expression of genes involved in cellular survival, proliferation, and the inflammatory response. However, it is not clear if K13 can also induce gene expression independently of NEMO/IKKγ. The minimum region of NEMO that is sufficient for supporting K13-induced NF-κB has not been delineated. Furthermore, the contribution of NEMO and NF-κB to the protective effect of K13 against death receptor-induced apoptosis remains to be determined. In this study, we used microarray analysis on K13-expressing wild-type and NEMO-deficient cells to demonstrate that NEMO is required for modulation of K13-induced genes. Reconstitution of NEMO-null cells revealed that the N-terminal 251 amino acid residues of NEMO are sufficient for supporting K13-induced NF-κB but fail to support tumor necrosis factor alpha (TNF-α)-induced NF-κB. K13 failed to protect NEMO-null cells against TNF-α-induced cell death but protected those reconstituted with the NEMO mutant truncated to include only the N-terminal 251 amino acid residues [the NEMO(1-251) mutant]. Taken collectively, our results demonstrate that NEMO is required for modulation of K13-induced genes and the N-terminal 251 amino acids of NEMO are sufficient for supporting K13-induced NF-κB. Finally, the ability of K13 to protect against TNF-α-induced cell death is critically dependent on its ability to interact with NEMO and activate NF-κB. Kaposi's sarcoma-associated herpesvirus-encoded vFLIP K13 is believed to protect virally infected cells against death receptor-induced apoptosis and to

  6. Identification and characterization of Kaposi's sarcoma-associated herpesvirus open reading frame 11 promotor activation

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    Chen, Lei [Los Alamos National Laboratory

    2008-01-01

    Open reading frame 11 (ORF11) of Kaposi's sarcoma-associated herpesvirus belongs to a herpesviral homologous protein family shared by some members of the gamma- herpesvirus subfamily. Little is known about this ORF11 homologous protein family. We have characterized an unknown open reading frame, ORF11, located adjacent and in the opposite orientation to a well-characterized viral IL-6 gene. Northern blot analysis reveals that ORF11 is expressed during the KSHV lytic cycle with delayed-early transcription kinetics. We have determined the 5{prime} and 3{prime} untranslated region of the unspliced ORF11 transcript and identified both the transcription start site and the transcription termination site. Core promoter region, representing ORF11 promoter activity, was mapped to a 159nt fragment 5{prime} most proximal to the transcription start site. A functional TATA box was identified in the core promoter region. Interestingly, we found that ORF11 transcriptional activation is not responsive to Rta, the KSHV lytic switch protein. We also discovered that part of the ORF11 promoter region, the 209nt fragment upstream of the transcription start site, was repressed by phorbol esters. Our data help to understand transcription regulation of ORF11 and to elucidate roles of ORF11 in KSHV pathogenesis and life cycle.

  7. Molecular piracy of Kaposi's sarcoma associated herpesvirus.

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    Choi, J; Means, R E; Damania, B; Jung, J U

    2001-01-01

    Kaposi's Sarcoma associated Herpesvirus (KSHV) is the most recently discovered human tumor virus and is associated with the pathogenesis of Kaposi's sarcoma, primary effusion lymphoma, and Multicentric Casttleman's disease. KSHV contains numerous open reading frames with striking homology to cellular genes. These viral gene products play a variety of roles in KSHV-associated pathogenesis by disrupting cellular signal transduction pathways, which include interferon-mediated anti-viral responses, cytokine-regulated cell growth, apoptosis, and cell cycle control. In this review, we will attempt to cover our understanding of how viral proteins deregulate cellular signaling pathways, which ultimately contribute to the conversion of normal cells to cancerous cells.

  8. Mutations in c10orf11, a melanocyte-differentiation gene, cause autosomal-recessive albinism

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    Grønskov, Karen; Dooley, Christopher M; Østergaard, Elsebet

    2013-01-01

    , we identified a 3.5 Mb homozygous region (10q22.2-q22.3) on chromosome 10. The region contains five protein-coding genes, and sequencing of one of these, C10orf11, revealed a nonsense mutation that segregated with the disease and showed a recessive inheritance pattern. Investigation of additional...... albinism-affected individuals from the Faroe Islands revealed that five out of eight unrelated affected persons had the nonsense mutation in C10orf11. Screening of a cohort of autosomal-recessive-albinism-affected individuals residing in Denmark showed a homozygous 1 bp duplication in C10orf11...... in substantially decreased pigmentation and a reduction of the apparent number of pigmented melanocytes. The morphant phenotype was rescued by wild-type C10orf11, but not by mutant C10orf11. In conclusion, we have identified a melanocyte-differentiation gene, C10orf11, which when mutated causes autosomal...

  9. Critical analysis of an oncolytic herpesvirus encoding granulocyte-macrophage colony stimulating factor for the treatment of malignant melanoma.

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    Hughes, Tasha; Coffin, Robert S; Lilley, Caroline E; Ponce, Rafael; Kaufman, Howard L

    2014-01-01

    Oncolytic viruses that selectively lyse tumor cells with minimal damage to normal cells are a new area of therapeutic development in oncology. An attenuated herpesvirus encoding the granulocyte-macrophage colony stimulating factor (GM-CSF), known as talimogene laherparepvec (T-VEC), has been identified as an attractive oncolytic virus for cancer therapy based on preclinical tumor studies and results from early-phase clinical trials and a large randomized Phase III study in melanoma. In this review, we discuss the basic biology of T-VEC, describe the role of GM-CSF as an immune adjuvant, summarize the preclinical data, and report the outcomes of published clinical trials using T-VEC. The emerging data suggest that T-VEC is a safe and potentially effective antitumor therapy in malignant melanoma and represents the first oncolytic virus to demonstrate therapeutic activity against human cancer in a randomized, controlled Phase III study.

  10. Mutations in c10orf11, a melanocyte-differentiation gene, cause autosomal-recessive albinism.

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    Grønskov, Karen; Dooley, Christopher M; Østergaard, Elsebet; Kelsh, Robert N; Hansen, Lars; Levesque, Mitchell P; Vilhelmsen, Kaj; Møllgård, Kjeld; Stemple, Derek L; Rosenberg, Thomas

    2013-03-07

    Autosomal-recessive albinism is a hypopigmentation disorder with a broad phenotypic range. A substantial fraction of individuals with albinism remain genetically unresolved, and it has been hypothesized that more genes are to be identified. By using homozygosity mapping of an inbred Faroese family, we identified a 3.5 Mb homozygous region (10q22.2-q22.3) on chromosome 10. The region contains five protein-coding genes, and sequencing of one of these, C10orf11, revealed a nonsense mutation that segregated with the disease and showed a recessive inheritance pattern. Investigation of additional albinism-affected individuals from the Faroe Islands revealed that five out of eight unrelated affected persons had the nonsense mutation in C10orf11. Screening of a cohort of autosomal-recessive-albinism-affected individuals residing in Denmark showed a homozygous 1 bp duplication in C10orf11 in an individual originating from Lithuania. Immunohistochemistry showed localization of C10orf11 in melanoblasts and melanocytes in human fetal tissue, but no localization was seen in retinal pigment epithelial cells. Knockdown of the zebrafish (Danio rerio) homolog with the use of morpholinos resulted in substantially decreased pigmentation and a reduction of the apparent number of pigmented melanocytes. The morphant phenotype was rescued by wild-type C10orf11, but not by mutant C10orf11. In conclusion, we have identified a melanocyte-differentiation gene, C10orf11, which when mutated causes autosomal-recessive albinism in humans. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  11. ORF11 Protein Interacts with the ORF9 Essential Tegument Protein in Varicella-Zoster Virus Infection

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    Oliver, Stefan L.; Reichelt, Mike; Sommer, Marvin H.; Haas, Jürgen; Roviš, Tihana L.; Arvin, Ann M.

    2013-01-01

    The tegument proteins encoded by ORF11 and ORF9 of varicella-zoster virus (VZV) are conserved among all alphaherpesvirus. We previously demonstrated that the ORF9 gene is essential, whereas ORF11 is dispensable in vitro but its deletion severely impairs VZV infection of skin xenografts in the SCID mouse model in vivo. Here we report that ORF11 protein interacts with ORF9 protein in infected cells as well as in the absence of other viral proteins, and we have mapped the ORF11 protein domain involved in their interaction. Although ORF11 is an RNA binding protein, the interaction between ORF11 and ORF9 proteins was not mediated by RNA or DNA bridging. VZV recombinants with mutations preventing ORF11 protein binding to ORF9 protein had no effect on 6-day growth kinetics based on plaque numbers, but plaque sizes were reduced in vitro. However, disruption of the ORF11 and ORF9 protein interaction was associated with failure to replicate in skin xenografts in vivo. Further, we demonstrate that in the absence of their interaction, the ORF9 protein displays an identical cellular localization, accumulating in the trans-Golgi region, whereas the ORF11 protein exhibits aberrant localization, dispersing throughout the cytoplasm. Overall, our observations suggest that while complete tegument assembly may not be necessary for VZV replication in vitro, the interaction between the ORF11 and ORF9 proteins appears to be critical for the proper localization of ORF11 protein to the assembly complex and for production of infectious virus during VZV pathogenesis in skin. PMID:23427162

  12. Critical analysis of an oncolytic herpesvirus encoding granulocyte-macrophage colony stimulating factor for the treatment of malignant melanoma

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    Hughes T

    2014-01-01

    Full Text Available Tasha Hughes,1 Robert S Coffin,2 Caroline E Lilley,2 Rafael Ponce,2 Howard L Kaufman1 1Departments of General Surgery and Immunology and Microbiology, Rush University Medical Center, Chicago IL, 2BioVex, Inc, a subsidiary of Amgen, Inc, Sherman Oaks, CA, USA Abstract: Oncolytic viruses that selectively lyse tumor cells with minimal damage to normal cells are a new area of therapeutic development in oncology. An attenuated herpesvirus encoding the granulocyte-macrophage colony stimulating factor (GM-CSF, known as talimogene laherparepvec (T-VEC, has been identified as an attractive oncolytic virus for cancer therapy based on preclinical tumor studies and results from early-phase clinical trials and a large randomized Phase III study in melanoma. In this review, we discuss the basic biology of T-VEC, describe the role of GM-CSF as an immune adjuvant, summarize the preclinical data, and report the outcomes of published clinical trials using T-VEC. The emerging data suggest that T-VEC is a safe and potentially effective antitumor therapy in malignant melanoma and represents the first oncolytic virus to demonstrate therapeutic activity against human cancer in a randomized, controlled Phase III study. Keywords: granulocyte-macrophage colony stimulating factor, herpesvirus, melanoma, oncolytic virus, treatment

  13. Mechanisms of Kaposi's Sarcoma-Associated Herpesvirus Latency and Reactivation

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    Fengchun Ye

    2011-01-01

    Full Text Available The life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV consists of latent and lytic replication phases. During latent infection, only a limited number of KSHV genes are expressed. However, this phase of replication is essential for persistent infection, evasion of host immune response, and induction of KSHV-related malignancies. KSHV reactivation from latency produces a wide range of viral products and infectious virions. The resulting de novo infection and viral lytic products modulate diverse cellular pathways and stromal microenvironment, which promote the development of Kaposi's sarcoma (KS. The mechanisms controlling KSHV latency and reactivation are complex, involving both viral and host factors, and are modulated by diverse environmental factors. Here, we review the cellular and molecular basis of KSHV latency and reactivation with a focus on the most recent advancements in the field.

  14. Myalgic encephalomyelitis/chronic fatigue syndrome and gulf war illness patients exhibit increased humoral responses to the herpesviruses-encoded dUTPase: Implications in disease pathophysiology.

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    Halpin, Peter; Williams, Marshall Vance; Klimas, Nancy G; Fletcher, Mary Ann; Barnes, Zachary; Ariza, Maria Eugenia

    2017-09-01

    Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Gulf War Illness (GWI) are debilitating diseases with overlapping symptomology and there are currently no validated tests for definitive diagnosis of either syndrome. While there is evidence supporting the premise that some herpesviruses may act as possible triggers of ME/CFS, the involvement of herpesviruses in the pathophysiology of GWI has not been studied in spite of a higher prevalence of ME/CFS in these patients. We have previously demonstrated that the deoxyuridine triphosphate nucleotidohydrolases (dUTPase) encoded by Epstein-Barr virus (EBV), human herpesvirus-6 (HHV-6), and varicella-zoster virus (VZV) possess novel functions in innate and adaptive immunity. The results of this study demonstrate that a significant percentage of patients with ME/CFS (30.91-52.7%) and GWI (29.34%) are simultaneously producing antibodies against multiple human herpesviruses-encoded dUTPases and/or the human dUTPase when compared to controls (17.21%). GWI patients exhibited significantly higher levels of antibodies to the HHV-6 and human dUTPases than controls (P = 0.0053 and P = 0.0036, respectively), while the ME/CFS cohort had higher anti-EBV-dUTPase antibodies than in both GWI patients (P = 0.0008) and controls (P < 0.0001) as well as significantly higher anti-human dUTPase antibodies than in controls (P = 0.0241). These results suggest that screening of patients' sera for the presence of various combinations of anti-dUTPase antibodies could be used as potential biomarkers to help identify/distinguish patients with these syndromes and better direct treatment. © 2017 Wiley Periodicals, Inc.

  15. Kaposi's sarcoma associated herpesvirus (KSHV entry into target cells

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    Sayan eChakraborty

    2012-01-01

    Full Text Available Herpesvirus infection of target cells is a complex process involving multiple host cell surface molecules (receptors and multiple viral envelope glycoproteins. Kaposi’s sarcoma associated herpesvirus (KSHV or HHV-8 infects a variety of in vivo target cells such as endothelial cells, B cells, monocytes, epithelial cells, and keratinocytes. KSHV also infects a diversity of in vitro target cells and establishes in vitro latency in many of these cell types. KSHV interactions with the host cell surface molecules and its mode of entry in the various target cells are critical for the understanding of KSHV pathogenesis. KSHV is the first herpesvirus shown to interact with adherent target cell integrins and this interaction initiates the host cell pre-existing signal pathways that are utilized for successful infection. This chapter discusses the various aspects of the early stage of KSHV infection of target cells, receptors used and issues that need to be clarified and future directions. The various signaling events triggered by KSHV infection and the potential role of signaling events in the different stages of infection are summarized providing the framework and starting point for further detailed studies essential to fully comprehend the pathogenesis of KSHV.

  16. Chromosome aberrations involving 10q22: report of three overlapping interstitial deletions and a balanced translocation disrupting C10orf11

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    Tzschach, Andreas; Bisgaard, Anne-Marie; Kirchhoff, Maria

    2010-01-01

    and hypotonia. We also report on the results of breakpoint analysis by array painting in a mentally retarded patient with a balanced chromosome translocation 46,XY,t(10;13)(q22;p13)dn. The breakpoint in 10q22 was found to disrupt C10orf11, a brain-expressed gene in the common deleted interval of patients 1...

  17. The assembly domain of the small capsid protein of Kaposi's sarcoma-associated herpesvirus.

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    Kreitler, Dale; Capuano, Christopher M; Henson, Brandon W; Pryce, Erin N; Anacker, Daniel; McCaffery, J Michael; Desai, Prashant J

    2012-11-01

    Self-assembly of Kaposi's sarcoma-associated herpesvirus capsids occurs when six proteins are coexpressed in insect cells using recombinant baculoviruses; however, if the small capsid protein (SCP) is omitted from the coinfection, assembly does not occur. Herein we delineate and identify precisely the assembly domain and the residues of SCP required for assembly. Hence, six residues, R14, D18, V25, R46, G66, and R70 in the assembly domain, when changed to alanine, completely abolish or reduce capsid assembly.

  18. Association between malaria exposure and Kaposi's sarcoma-associated herpes virus seropositivity in Uganda.

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    Nalwoga, Angela; Cose, Stephen; Wakeham, Katie; Miley, Wendell; Ndibazza, Juliet; Drakeley, Christopher; Elliott, Alison; Whitby, Denise; Newton, Robert

    2015-05-01

    Unlike other herpes viruses, Kaposi's sarcoma-associated herpes virus (KSHV) is not ubiquitous worldwide and is most prevalent in sub-Saharan Africa. The reasons for this are unclear. As part of a wider investigation of factors that facilitate transmission in Uganda, a high prevalence country, we examined the association between antimalaria antibodies and seropositivity against KSHV. Antibodies against P. falciparum merozoite surface protein (PfMSP)-1, P. falciparum apical membrane antigen (PfAMA)-1 and KSHV antigens (ORF73 and K8.1) were measured in samples from 1164 mothers and 1227 children. Kaposi's sarcoma-associated herpes virus seroprevalence was 69% among mothers and 15% children. Among mothers, KSHV seroprevalence increased with malaria antibody titres: from 60% to 82% and from 54% to 77%, comparing those with the lowest and highest titres for PfMSP-1 and PfAMA-1, respectively (P < 0.0001). Among children, only antibodies to PfAMA-1 were significantly associated with KSHV seropositivity, (P < 0.0001). In both mothers and children, anti-ORF73 antibodies were more strongly associated with malaria antibodies than anti-K8.1 antibodies. The association between malaria exposure and KSHV seropositivity suggests that malaria is a cofactor for KSHV infection or reactivation. © 2015 The Authors. Tropical Medicine & International Health published by John Wiley & Sons Ltd.

  19. The T-Cell Immune Response against Kaposi's Sarcoma-Associated Herpesvirus.

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    Robey, Rebecca C; Mletzko, Salvinia; Gotch, Frances M

    2010-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the aetiological agent of Kaposi's sarcoma (KS), the most frequently arising malignancy in individuals with untreated HIV/AIDS. There are several lines of evidence to indicate that Kaposi's sarcoma oncogenesis is associated with loss of T-cell-mediated control of KSHV-infected cells. KSHV can establish life-long asymptomatic infection in immune-competent individuals. However, when T-cell immune control declines, for example, through AIDS or treatment with immunosuppressive drugs, both the prevalence of KSHV infection and the incidence of KS in KSHV carriers dramatically increase. Moreover, a dramatic and spontaneous improvement in KS is frequently seen when immunity is restored, for example, through antiretroviral therapy or the cessation of iatrogenic drugs. In this paper we describe the current state of knowledge on the T-cell immune responses against KSHV.

  20. The T-Cell Immune Response against Kaposi's Sarcoma-Associated Herpesvirus

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    Rebecca C. Robey

    2010-01-01

    Full Text Available Kaposi's sarcoma-associated herpesvirus (KSHV is the aetiological agent of Kaposi's sarcoma (KS, the most frequently arising malignancy in individuals with untreated HIV/AIDS. There are several lines of evidence to indicate that Kaposi's sarcoma oncogenesis is associated with loss of T-cell-mediated control of KSHV-infected cells. KSHV can establish life-long asymptomatic infection in immune-competent individuals. However, when T-cell immune control declines, for example, through AIDS or treatment with immunosuppressive drugs, both the prevalence of KSHV infection and the incidence of KS in KSHV carriers dramatically increase. Moreover, a dramatic and spontaneous improvement in KS is frequently seen when immunity is restored, for example, through antiretroviral therapy or the cessation of iatrogenic drugs. In this paper we describe the current state of knowledge on the T-cell immune responses against KSHV.

  1. Kaposi's Sarcoma-Associated Herpesvirus-Related Solid Lymphoma Involving the Heart and Brain

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    Jason R. Andrews

    2011-01-01

    Full Text Available Since its discovery in 1994, Kaposi's sarcoma-associated herpesvirus (KSHV has been associated with lymphoproliferative disorders, particularly in patients infected with human immunodeficiency virus (HIV. The disorders most strongly linked to KSHV are multicentric Castleman's Disease (MCD, primary effusion lymphoma, and diffuse large B-cell lymphomas. We report an unusual case of KSHV-associated lymphoma in an HIV-infected patient manifesting with myocardial and central nervous system involvement. We discuss this case in the context of increasing array of KSHV-associated lymphomas. In the HIV-infected patient with a mass lesion, a history of cutaneous Kaposi's sarcoma and prolonged immunosuppression should alert clinicians as to the possibility of KSHV-associated lymphoproliferative disorders, in order to establish a timely diagnosis.

  2. The Chromatin Landscape of Kaposi’s Sarcoma-Associated Herpesvirus

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    Zsolt Toth

    2013-05-01

    Full Text Available Kaposi’s sarcoma-associated herpesvirus is an oncogenic γ-herpesvirus that causes latent infection in humans. In cells, the viral genome adopts a highly organized chromatin structure, which is controlled by a wide variety of cellular and viral chromatin regulatory factors. In the past few years, interrogation of the chromatinized KSHV genome by whole genome-analyzing tools revealed that the complex chromatin landscape spanning the viral genome in infected cells has important regulatory roles during the viral life cycle. This review summarizes the most recent findings regarding the role of histone modifications, histone modifying enzymes, DNA methylation, microRNAs, non-coding RNAs and the nuclear organization of the KSHV epigenome in the regulation of latent and lytic viral gene expression programs as well as their connection to KSHV-associated pathogenesis.

  3. The C terminus of the synovial sarcoma-associated SSX proteins interacts with the LIM homeobox protein LHX4.

    NARCIS (Netherlands)

    Bruijn, D.R.H. de; Dijk, A.H.A.; Willemse, M.P.; Geurts van Kessel, A.H.M.

    2008-01-01

    As a result of the synovial sarcoma-associated t(X;18) translocation, the SS18 gene on chromosome 18 is fused to either one of the three closely related SSX genes on the X chromosome. The SS18 protein is thought to act as a transcriptional co-activator, whereas the SSX proteins are thought to act as

  4. Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory

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    Chen, Christopher Phillip; Lyu, Yuanzhi; Chuang, Frank; Nakano, Kazushi; Izumiya, Chie; Jin, Di; Campbell, Mel

    2017-01-01

    ABSTRACT Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional “factories,” which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of “viral transcriptional factories” decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an “all-in-one” factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells. IMPORTANCE B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the

  5. Structural Analysis of Thymidylate Synthase from Kaposi's Sarcoma-Associated Herpesvirus with the Anticancer Drug Raltitrexed.

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    Yong Mi Choi

    Full Text Available Kaposi's sarcoma-associated herpesvirus (KSHV is a highly infectious human herpesvirus that causes Kaposi's sarcoma. KSHV encodes functional thymidylate synthase, which is a target for anticancer drugs such as raltitrexed or 5-fluorouracil. Thymidylate synthase catalyzes the conversion of 2'-deoxyuridine-5'-monophosphate (dUMP to thymidine-5'-monophosphate (dTMP using 5,10-methylenetetrahydrofolate (mTHF as a co-substrate. The crystal structures of thymidylate synthase from KSHV (apo, complexes with dUMP (binary, and complexes with both dUMP and raltitrexed (ternary were determined at 1.7 Å, 2.0 Å, and 2.4 Å, respectively. While the ternary complex structures of human thymidylate synthase and E. coli thymidylate synthase had a closed conformation, the ternary complex structure of KSHV thymidylate synthase was observed in an open conformation, similar to that of rat thymidylate synthase. The complex structures of KSHV thymidylate synthase did not have a covalent bond between the sulfhydryl group of Cys219 and C6 atom of dUMP, unlike the human thymidylate synthase. The catalytic Cys residue demonstrated a dual conformation in the apo structure, and its sulfhydryl group was oriented toward the C6 atom of dUMP with no covalent bond upon ligand binding in the complex structures. These structural data provide the potential use of antifolates such as raltitrexed as a viral induced anticancer drug and structural basis to design drugs for targeting the thymidylate synthase of KSHV.

  6. Structural Analysis of Thymidylate Synthase from Kaposi's Sarcoma-Associated Herpesvirus with the Anticancer Drug Raltitrexed.

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    Choi, Yong Mi; Yeo, Hyun Ku; Park, Young Woo; Lee, Jae Young

    2016-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a highly infectious human herpesvirus that causes Kaposi's sarcoma. KSHV encodes functional thymidylate synthase, which is a target for anticancer drugs such as raltitrexed or 5-fluorouracil. Thymidylate synthase catalyzes the conversion of 2'-deoxyuridine-5'-monophosphate (dUMP) to thymidine-5'-monophosphate (dTMP) using 5,10-methylenetetrahydrofolate (mTHF) as a co-substrate. The crystal structures of thymidylate synthase from KSHV (apo), complexes with dUMP (binary), and complexes with both dUMP and raltitrexed (ternary) were determined at 1.7 Å, 2.0 Å, and 2.4 Å, respectively. While the ternary complex structures of human thymidylate synthase and E. coli thymidylate synthase had a closed conformation, the ternary complex structure of KSHV thymidylate synthase was observed in an open conformation, similar to that of rat thymidylate synthase. The complex structures of KSHV thymidylate synthase did not have a covalent bond between the sulfhydryl group of Cys219 and C6 atom of dUMP, unlike the human thymidylate synthase. The catalytic Cys residue demonstrated a dual conformation in the apo structure, and its sulfhydryl group was oriented toward the C6 atom of dUMP with no covalent bond upon ligand binding in the complex structures. These structural data provide the potential use of antifolates such as raltitrexed as a viral induced anticancer drug and structural basis to design drugs for targeting the thymidylate synthase of KSHV.

  7. Detection of Kaposi's sarcoma associated herpesvirus nucleic acids using a smartphone accessory.

    Science.gov (United States)

    Mancuso, Matthew; Cesarman, Ethel; Erickson, David

    2014-10-07

    Kaposi's sarcoma (KS) is an infectious cancer occurring in immune-compromised patients, caused by Kaposi's sarcoma associated herpesvirus (KSHV). Our vision is to simplify the process of KS diagnosis through the creation of a smartphone based point-of-care system capable of yielding an actionable diagnostic readout starting from a raw biopsy sample. In this work we develop the sensing mechanism for the overall system, a smartphone accessory capable of detecting KSHV nucleic acids. The accessory reads out microfluidic chips filled with a colorimetric nanoparticle assay targeted at KSHV. We calculate that our final device can read out gold nanoparticle solutions with an accuracy of 0.05 OD, and we demonstrate that it can detect DNA sequences from KSHV down to 1 nM. We believe that through integration with our previously developed components, a smartphone based system like the one studied here can provide accurate detection information, as well as a simple platform for field based clinical diagnosis and research.

  8. Kaposi's sarcoma-associated herpesvirus ORF6 gene is essential in viral lytic replication.

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    Can Peng

    Full Text Available Kaposi's sarcoma associated herpesvirus (KSHV is associated with Kaposis's sarcoma (KS, primary effusion lymphoma and multicentric Castleman's disease. KSHV encodes at least 8 open reading frames (ORFs that play important roles in its lytic DNA replication. Among which, ORF6 of KSHV encodes an ssDNA binding protein that has been proved to participate in origin-dependent DNA replication in transient assays. To define further the function of ORF6 in the virus life cycle, we constructed a recombinant virus genome with a large deletion within the ORF6 locus by using a bacterial artificial chromosome (BAC system. Stable 293T cells carrying the BAC36 (wild type and BACΔ6 genomes were generated. When monolayers of 293T-BAC36 and 293T-BACΔ6 cells were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA and sodium butyrate, infectious virus was detected from the 293T-BAC36 cell supernatants only and not from the 293T- BACΔ6 cell supernatants. DNA synthesis was defective in 293T-BACΔ6 cells. Expression of ORF6 in trans in BACΔ6-containing cells was able to rescue both defects. Our results provide genetic evidence that ORF6 is essential for KSHV lytic replication. The stable 293T cells carrying the BAC36 and BACΔ6 genomes could be used as tools to investigate the detailed functions of ORF6 in the lytic replication of KSHV.

  9. Seroprevalence of Kaposi's sarcoma-associated herpesvirus infection among blood donors from Texas.

    Science.gov (United States)

    Baillargeon, J; Deng, J H; Hettler, E; Harrison, C; Grady, J J; Korte, L G; Alexander, J; Montalvo, E; Jenson, H B; Gao, S J

    2001-10-01

    Kaposi's sarcoma-associated herpesvirus (KSHV), a gammaherpesvirus recently discovered among AIDS patients with Kaposi's sarcoma, is a potential candidate for screening in blood and plasma donors. While a number of studies have assessed KSHV infection among U.S. blood donors, larger-scale population-based studies would be necessary to develop more refined estimates of the magnitude and variation of KSHV infection across different geographic regions of the U.S. blood supply. The goal of the present study, therefore, was to determine the seroprevalence of KSHV infection and to assess demographic correlates of KSHV infection among south Texas blood donors. KSHV infection was determined using specific serologic assays that measure antibodies to KSHV latent and lytic antigens. The overall seroprevalence of KSHV in Texas blood donors (15.0%) is substantially higher than previously reported among blood donor and general population samples in the United States. This high rate of KSHV infection persisted across most of the sociodemographic subgroups under study but was particularly elevated among participants with less than a high school education. The infection rate also increased linearly with age. The elevated infection rate reported in the present study suggests that screening methods to detect KSHV infection in blood donors should be considered. In view of the etiologic role of KSHV for several malignancies, it would be important for future studies to directly assess the risk of KSHV transmission via blood transfusion.

  10. Viral Interleukin-6: Role in Kaposi's Sarcoma-Associated Herpesvirus–Associated Malignancies

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    Sakakibara, Shuhei

    2011-01-01

    Viral interleukin-6 (vIL-6) is a product of Kaposi's sarcoma-associated herpesvirus (KSHV) expressed in latently infected cells and to a higher degree during viral replication. A distinctive feature of vIL-6 is the ability to directly bind and activate gp130 signaling in the absence of other receptor subunits. Secretion of vIL-6 is generally poor, but vIL-6 can activate gp130 from inside the cell. Due to the wide cell distribution of gp130, vIL-6 has the potential to induce a wide range of biological effects. Expression of vIL-6 is variable in KSHV-associated Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman's disease (MCD), and in a newly described MCD-like systemic inflammatory syndrome observed in human immunodeficiency virus-positive patients. PEL effusions usually contain vIL-6 at high concentrations; since vIL-6 induces vascular endothelial growth factor, vIL-6 likely contributes to vascular permeability and formation of PEL effusions. Lymph nodes affected with MCD contain vIL-6-positive cells, and vIL-6 levels rise in conjunction with flares of the disease and likely contribute to symptoms of inflammation. The development of vIL-6 inhibitors is a potentially important advance in the treatment of KSHV-associated malignancies where vIL-6 is expressed. PMID:21767154

  11. Control of Kaposi's sarcoma-associated herpesvirus reactivation induced by multiple signals.

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    Fuqu Yu

    Full Text Available The ability to control cellular functions can bring about many developments in basic biological research and its applications. The presence of multiple signals, internal as well as externally imposed, introduces several challenges for controlling cellular functions. Additionally the lack of clear understanding of the cellular signaling network limits our ability to infer the responses to a number of signals. This work investigates the control of Kaposi's sarcoma-associated herpesvirus reactivation upon treatment with a combination of multiple signals. We utilize mathematical model-based as well as experiment-based approaches to achieve the desired goals of maximizing virus reactivation. The results show that appropriately selected control signals can induce virus lytic gene expression about ten folds higher than a single drug; these results were validated by comparing the results of the two approaches, and experimentally using multiple assays. Additionally, we have quantitatively analyzed potential interactions between the used combinations of drugs. Some of these interactions were consistent with existing literature, and new interactions emerged and warrant further studies. The work presents a general method that can be used to quantitatively and systematically study multi-signal induced responses. It enables optimization of combinations to achieve desired responses. It also allows identifying critical nodes mediating the multi-signal induced responses. The concept and the approach used in this work will be directly applicable to other diseases such as AIDS and cancer.

  12. Epidemiology of Kaposi's sarcoma-associated herpesvirus in Asia: Challenges and opportunities.

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    Zhang, Tiejun; Wang, Linding

    2017-04-01

    Kaposi's sarcoma-associated herpes virus (KSHV) also referred to as human herpesvirus-8 (HHV-8), is a gamma herpes virus and recently discovered human virus. Since its discovery, a myriad of studies has been conducted to explore its pathogenesis mechanisms. However, despite our consistently increasing understanding of KSHV biology and its clinical manifestations, only little progress has been made in understanding of its epidemiology characteristics which in turn hampered the management of KSHV-associated diseases and public health. Asia, the largest continent with a diversity of populations, has been thought to be with relative lower KSHV prevalence and diseases burden. The epidemiology of KSHV in this area is obscure either. The present review summarizes the current knowledge pertaining to the epidemiology of KSHV across Asian countries. Studies available in the literature have shown a substantial variation in this region indicating the virus is not ubiquitous in Asia countries as is the case with other human herpes viruses. Also, the MSM has been reconfirmed to be at the highest risk of KSHV infection in Asia highlighting the need for an increased focus on this previously marginalized population. Because of the paucity of data available, the epidemiologic characteristics of KSHV are difficult to determine in Asian countries. Future systematic collection of data to inform KSHV prevention strategies in Asia is urgently needed. J. Med. Virol. 89:563-570, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Non-human primate model of Kaposi's sarcoma-associated herpesvirus infection.

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    Heesoon Chang

    2009-10-01

    Full Text Available Since Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8 was first identified in Kaposi's sarcoma (KS lesions of HIV-infected individuals with AIDS, the basic biological understanding of KSHV has progressed remarkably. However, the absence of a proper animal model for KSHV continues to impede direct in vivo studies of viral replication, persistence, and pathogenesis. In response to this need for an animal model of KSHV infection, we have explored whether common marmosets can be experimentally infected with human KSHV. Here, we report the successful zoonotic transmission of KSHV into common marmosets (Callithrix jacchus, Cj, a New World primate. Marmosets infected with recombinant KSHV rapidly seroconverted and maintained a vigorous anti-KSHV antibody response. KSHV DNA and latent nuclear antigen (LANA were readily detected in the peripheral blood mononuclear cells (PBMCs and various tissues of infected marmosets. Remarkably, one orally infected marmoset developed a KS-like skin lesion with the characteristic infiltration of leukocytes by spindle cells positive for KSHV DNA and proteins. These results demonstrate that human KSHV infects common marmosets, establishes an efficient persistent infection, and occasionally leads to a KS-like skin lesion. This is the first animal model to significantly elaborate the important aspects of KSHV infection in humans and will aid in the future design of vaccines against KSHV and anti-viral therapies targeting KSHV coinfected tumor cells.

  14. The HIV protease inhibitor nelfinavir inhibits Kaposi's sarcoma-associated herpesvirus replication in vitro.

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    Gantt, Soren; Carlsson, Jacquelyn; Ikoma, Minako; Gachelet, Eliora; Gray, Matthew; Geballe, Adam P; Corey, Lawrence; Casper, Corey; Lagunoff, Michael; Vieira, Jeffrey

    2011-06-01

    Kaposi's sarcoma (KS) is the most common HIV-associated cancer worldwide and is associated with high levels of morbidity and mortality in some regions. Antiretroviral (ARV) combination regimens have had mixed results for KS progression and resolution. Anecdotal case reports suggest that protease inhibitors (PIs) may have effects against KS that are independent of their effect on HIV infection. As such, we evaluated whether PIs or other ARVs directly inhibit replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the gammaherpesvirus that causes KS. Among a broad panel of ARVs tested, only the PI nelfinavir consistently displayed potent inhibitory activity against KSHV in vitro as demonstrated by an efficient quantitative assay for infectious KSHV using a recombinant virus, rKSHV.294, which expresses the secreted alkaline phosphatase. This inhibitory activity of nelfinavir against KSHV replication was confirmed using virus derived from a second primary effusion lymphoma cell line. Nelfinavir was similarly found to inhibit in vitro replication of an alphaherpesvirus (herpes simplex virus) and a betaherpesvirus (human cytomegalovirus). No activity was observed with nelfinavir against vaccinia virus or adenovirus. Nelfinavir may provide unique benefits for the prevention or treatment of HIV-associated KS and potentially other human herpesviruses by direct inhibition of replication.

  15. Structural Analysis of Thymidylate Synthase from Kaposi?s Sarcoma-Associated Herpesvirus with the Anticancer Drug Raltitrexed

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    Choi, Yong Mi; Yeo, Hyun Ku; Park, Young Woo; Lee, Jae Young

    2016-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a highly infectious human herpesvirus that causes Kaposi's sarcoma. KSHV encodes functional thymidylate synthase, which is a target for anticancer drugs such as raltitrexed or 5-fluorouracil. Thymidylate synthase catalyzes the conversion of 2'-deoxyuridine-5'-monophosphate (dUMP) to thymidine-5'-monophosphate (dTMP) using 5,10-methylenetetrahydrofolate (mTHF) as a co-substrate. The crystal structures of thymidylate synthase from KSHV (apo), co...

  16. Kaposi's Sarcoma-Associated Herpesvirus ORF18 and ORF30 Are Essential for Late Gene Expression during Lytic Replication

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    Gong, Danyang; Wu, Nicholas C.; Xie, Yafang; Feng, Jun; Tong, Leming; Brulois, Kevin F.; Luan, Harding; Du, Yushen; Jung, Jae U.; Wang, Cun-Yu; Kang, Mo Kwan; Park, No-Hee; Sun, Ren; Wu, Ting-Ting

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several human malignances. As saliva is likely the major vehicle for KSHV transmission, we studied in vitro KSHV infection of oral epithelial cells. Through infection of two types of oral epithelial cells, normal human oral keratinocytes (NHOKs) and papilloma-immortalized human oral keratinocyte (HOK16B) cells, we found that KSHV can undergo robust lytic replication in oral epithelial cells. By employing de novo lytic infection...

  17. Antagonism of host antiviral responses by Kaposi's sarcoma-associated herpesvirus tegument protein ORF45.

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    Fan Xiu Zhu

    2010-05-01

    Full Text Available Virus infection of a cell generally evokes an immune response by the host to defeat the intruder in its effort. Many viruses have developed an array of strategies to evade or antagonize host antiviral responses. Kaposi's sarcoma-associated herpesvirus (KSHV is demonstrated in this report to be able to prevent activation of host antiviral defense mechanisms upon infection. Cells infected with wild-type KSHV were permissive for superinfection with vesicular stomatitis virus (VSV, suggesting that KSHV virions fail to induce host antiviral responses. We previously showed that ORF45, a KSHV immediate-early protein as well as a tegument protein of virions, interacts with IRF-7 and inhibits virus-mediated type I interferon induction by blocking IRF-7 phosphorylation and nuclear translocation (Zhu et al., Proc. Natl. Acad. Sci. USA. 99:5573-5578, 2002. Here, using an ORF45-null recombinant virus, we demonstrate a profound role of ORF45 in inhibiting host antiviral responses. Infection of cells with an ORF45-null mutant recombinant KSHV (BAC-stop45 triggered an immune response that resisted VSV super-infection, concomitantly associated with appreciable increases in transcription of type I IFN and downstream anti-viral effector genes. Gain-of-function analysis showed that ectopic expression of ORF45 in human fibroblast cells by a lentivirus vector decreased the antiviral responses of the cells. shRNA-mediated silencing of IRF-7, that predominantly regulates both the early and late phase induction of type I IFNs, clearly indicated its critical contribution to the innate antiviral responses generated against incoming KSHV particles. Thus ORF45 through its targeting of the crucial IRF-7 regulated type I IFN antiviral responses significantly contributes to the KSHV survival immediately following a primary infection allowing for progression onto subsequent stages in its life-cycle.

  18. The epigenetic landscape of latent Kaposi sarcoma-associated herpesvirus genomes.

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    Thomas Günther

    Full Text Available Herpesvirus latency is generally thought to be governed by epigenetic modifications, but the dynamics of viral chromatin at early timepoints of latent infection are poorly understood. Here, we report a comprehensive spatial and temporal analysis of DNA methylation and histone modifications during latent infection with Kaposi Sarcoma-associated herpesvirus (KSHV, the etiologic agent of Kaposi Sarcoma and primary effusion lymphoma (PEL. By use of high resolution tiling microarrays in conjunction with immunoprecipitation of methylated DNA (MeDIP or modified histones (chromatin IP, ChIP, our study revealed highly distinct landscapes of epigenetic modifications associated with latent KSHV infection in several tumor-derived cell lines as well as de novo infected endothelial cells. We find that KSHV genomes are subject to profound methylation at CpG dinucleotides, leading to the establishment of characteristic global DNA methylation patterns. However, such patterns evolve slowly and thus are unlikely to control early latency. In contrast, we observed that latency-specific histone modification patterns were rapidly established upon a de novo infection. Our analysis furthermore demonstrates that such patterns are not characterized by the absence of activating histone modifications, as H3K9/K14-ac and H3K4-me3 marks were prominently detected at several loci, including the promoter of the lytic cycle transactivator Rta. While these regions were furthermore largely devoid of the constitutive heterochromatin marker H3K9-me3, we observed rapid and widespread deposition of H3K27-me3 across latent KSHV genomes, a bivalent modification which is able to repress transcription in spite of the simultaneous presence of activating marks. Our findings suggest that the modification patterns identified here induce a poised state of repression during viral latency, which can be rapidly reversed once the lytic cycle is induced.

  19. Reactivation and Lytic Replication of Kaposi’s Sarcoma-Associated Herpesvirus: An Update

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    Yan Yuan

    2017-04-01

    Full Text Available The life cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV consists of two phases, latent and lytic. The virus establishes latency as a strategy for avoiding host immune surveillance and fusing symbiotically with the host for lifetime persistent infection. However, latency can be disrupted and KSHV is reactivated for entry into the lytic replication. Viral lytic replication is crucial for efficient dissemination from its long-term reservoir to the sites of disease and for the spread of the virus to new hosts. The balance of these two phases in the KSHV life cycle is important for both the virus and the host and control of the switch between these two phases is extremely complex. Various environmental factors such as oxidative stress, hypoxia, and certain chemicals have been shown to switch KSHV from latency to lytic reactivation. Immunosuppression, unbalanced inflammatory cytokines, and other viral co-infections also lead to the reactivation of KSHV. This review article summarizes the current understanding of the initiation and regulation of KSHV reactivation and the mechanisms underlying the process of viral lytic replication. In particular, the central role of an immediate-early gene product RTA in KSHV reactivation has been extensively investigated. These studies revealed multiple layers of regulation in activation of RTA as well as the multifunctional roles of RTA in the lytic replication cascade. Epigenetic regulation is known as a critical layer of control for the switch of KSHV between latency and lytic replication. The viral non-coding RNA, PAN, was demonstrated to play a central role in the epigenetic regulation by serving as a guide RNA that brought chromatin remodeling enzymes to the promoters of RTA and other lytic genes. In addition, a novel dimension of regulation by microPeptides emerged and has been shown to regulate RTA expression at the protein level. Overall, extensive investigation of KSHV reactivation and lytic

  20. A novel mechanism inducing genome instability in Kaposi's sarcoma-associated herpesvirus infected cells.

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    Brian R Jackson

    2014-05-01

    Full Text Available Kaposi's sarcoma-associated herpesvirus (KSHV is an oncogenic herpesvirus associated with multiple AIDS-related malignancies. Like other herpesviruses, KSHV has a biphasic life cycle and both the lytic and latent phases are required for tumorigenesis. Evidence suggests that KSHV lytic replication can cause genome instability in KSHV-infected cells, although no mechanism has thus far been described. A surprising link has recently been suggested between mRNA export, genome instability and cancer development. Notably, aberrations in the cellular transcription and export complex (hTREX proteins have been identified in high-grade tumours and these defects contribute to genome instability. We have previously shown that the lytically expressed KSHV ORF57 protein interacts with the complete hTREX complex; therefore, we investigated the possible intriguing link between ORF57, hTREX and KSHV-induced genome instability. Herein, we show that lytically active KSHV infected cells induce a DNA damage response and, importantly, we demonstrate directly that this is due to DNA strand breaks. Furthermore, we show that sequestration of the hTREX complex by the KSHV ORF57 protein leads to this double strand break response and significant DNA damage. Moreover, we describe a novel mechanism showing that the genetic instability observed is a consequence of R-loop formation. Importantly, the link between hTREX sequestration and DNA damage may be a common feature in herpesvirus infection, as a similar phenotype was observed with the herpes simplex virus 1 (HSV-1 ICP27 protein. Our data provide a model of R-loop induced DNA damage in KSHV infected cells and describes a novel system for studying genome instability caused by aberrant hTREX.

  1. Kaposi's Sarcoma Associated-Herpes Virus (KSHV Seroprevalence in Pregnant Women in South Africa

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    Malope-Kgokong Babatyi I

    2010-08-01

    Full Text Available Abstract Background Factors previously associated with Kaposi's sarcoma-associated herpesvirus (KSHV transmission in Africa include sexual, familial, and proximity to river water. We measured the seroprevalence of KSHV in relation to HIV, syphilis, and demographic factors among pregnant women attending public antenatal clinics in the Gauteng province of South Africa. Methods We tested for antibodies to KSHV lytic K8.1 and latent Orf73 antigens in 1740 pregnant women attending antenatal clinics who contributed blood to the "National HIV and Syphilis Sero-Prevalence Survey - South Africa, 2001". Information on HIV and syphilis serology, age, education, residential area, gravidity, and parity was anonymously linked to evaluate risk factors for KSHV seropositivity. Clinics were grouped by municipality regions and their proximity to the two main river catchments defined. Results KSHV seropositivity (reactive to either lytic K8.1 and latent Orf73 was nearly twice that of HIV (44.6% vs. 23.1%. HIV and syphilis seropositivity was 12.7% and 14.9% in women without KSHV, and 36.1% and 19.9% respectively in those with KSHV. Women who are KSHV seropositive were 4 times more likely to be HIV positive than those who were KSHV seronegative (AOR 4.1 95%CI: 3.4 - 5.7. Although, women with HIV infection were more likely to be syphilis seropositive (AOR 1.8 95%CI: 1.3 - 2.4, no association between KSHV and syphilis seropositivity was observed. Those with higher levels of education had lower levels of KSHV seropositivity compared to those with lower education levels. KSHV seropositivity showed a heterogeneous pattern of prevalence in some localities. Conclusions The association between KSHV and HIV seropositivity and a lack of common association with syphilis, suggests that KSHV transmission may involve geographical and cultural factors other than sexual transmission.

  2. Kaposi's sarcoma-associated herpesvirus infection and Kaposi's sarcoma in Brazil

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    S. Ramos-da-Silva

    2006-05-01

    Full Text Available Kaposi's sarcoma (KS became a critical health issue with the emergence of acquired immunodeficiency syndrome (AIDS in the 1980s. Four clinical-epidemiological forms of KS have been described: classical KS, endemic KS, iatrogenic KS, and AIDS-associated KS. In 1994, Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus type 8 was identified by Chang and colleagues, and has been detected worldwide at frequencies ranging from 80 to 100%. The aim of the present study was to evaluate the frequency of KSHV infection in KS lesions from HIV-positive and HIV-negative patients in Brazil, as well as to review the current knowledge about KS transmission and detection. For these purposes, DNA from 51 cases of KS was assessed by PCR: 20 (39.2% cases of classical KS, 29 (56.9% of AIDS-associated KS and 2 (3.9% of iatrogenic KS. Most patients were males (7.5:1, M/F, and mean age was 47.9 years (SD = ± 18.7 years. As expected, HIV-positive KS patients were younger than patients with classical KS. On the other hand, patients with AIDS-associated KS have early lesions (patch and plaque compared to classical KS patients (predominantly nodular lesions. This is assumed to be the result of the early diagnose of KS in the HIV-positive setting. KSHV infection was detected by PCR in almost all cases (48/51; 94.1%, irrespectively of the clinical-epidemiological form of KS. These results show that KSHV is associated with all forms of KS in Brazilian patients, a fact that supports the role of this virus in KS pathogenesis.

  3. Regulation of viral and cellular gene expression by Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA.

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    Rossetto, Cyprian C; Tarrant-Elorza, Margaret; Verma, Subhash; Purushothaman, Pravinkumar; Pari, Gregory S

    2013-05-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the cause of Kaposi's sarcoma and body cavity lymphoma. In cell culture, KSHV results in a latent infection, and lytic reactivation is usually induced with the expression of K-Rta or by treatment with phorbol 12-myristate 13-acetate (TPA) and/or n-butyrate. Lytic infection is marked by the activation of the entire viral genomic transcription cascade and the production of infectious virus. KSHV-infected cells express a highly abundant, long, noncoding transcript referred to as polyadenylated nuclear RNA (PAN RNA). PAN RNA interacts with specific demethylases and physically binds to the KSHV genome to mediate activation of viral gene expression. A recombinant BACmid lacking the PAN RNA locus fails to express K-Rta and does not produce virus. We now show that the lack of PAN RNA expression results in the failure of the initiation of the entire KSHV transcription program. In addition to previous findings of an interaction with demethylases, we show that PAN RNA binds to protein components of Polycomb repression complex 2 (PRC2). RNA-Seq analysis using cell lines that express PAN RNA shows that transcription involving the expression of proteins involved in cell cycle, immune response, and inflammation is dysregulated. Expression of PAN RNA in various cell types results in an enhanced growth phenotype, higher cell densities, and increased survival compared to control cells. Also, PAN RNA expression mediates a decrease in the production of inflammatory cytokines. These data support a role for PAN RNA as a major global regulator of viral and cellular gene expression.

  4. Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells

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    Cai, Xuezhong; Lu, Shihua; Zhang, Zhihong; Gonzalez, Carlos M.; Damania, Blossom; Cullen, Bryan R.

    2005-01-01

    MicroRNAs (miRNAs) are an endogenously encoded class of small RNAs that have been proposed to function as key posttranscriptional regulators of gene expression in a range of eukaryotic species, including humans. The small size of miRNA precursors makes them potentially ideal for use by viruses as inhibitors of host cell defense pathways. Here, we demonstrate that the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an array of 11 distinct miRNAs, all of whic...

  5. PAN's Labyrinth: Molecular biology of Kaposi's sarcoma-associated herpesvirus (KSHV) PAN RNA, a multifunctional long noncoding RNA.

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    Rossetto, Cyprian C; Pari, Gregory S

    2014-11-04

    Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesivrus, the causative agent of Kaposi's sarcoma and body cavity lymphomas. During infection KSHV produces a highly abundant long non-coding polyadenylated RNA that is retained in the nucleus known as PAN RNA. Long noncoding RNAs (lncRNA) are key regulators of gene expression and are known to interact with specific chromatin modification complexes, working in cis and trans to regulate gene expression. Data strongly supports a model where PAN RNA is a multifunctional regulatory transcript that controls KSHV gene expression by mediating the modification of chromatin by targeting the KSHV repressed genome.

  6. The Crystal Structure of PF-8, the DNA Polymerase Accessory Subunit from Kaposi's Sarcoma-Associated Herpesvirus

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    Baltz, Jennifer L.; Filman, David J.; Ciustea, Mihai; Silverman, Janice Elaine Y.; Lautenschlager, Catherine L.; Coen, Donald M.; Ricciardi, Robert P.; Hogle, James M.; (UPENN)

    2009-12-01

    Kaposi's sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi's sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 {angstrom}. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predicted to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8's mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.

  7. The Crystal Structure of PF-8, the DNA Polymerase Accessory Subunit from Kaposi's Sarcoma-Associated Herpesvirus▿

    Science.gov (United States)

    Baltz, Jennifer L.; Filman, David J.; Ciustea, Mihai; Silverman, Janice Elaine Y.; Lautenschlager, Catherine L.; Coen, Donald M.; Ricciardi, Robert P.; Hogle, James M.

    2009-01-01

    Kaposi's sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi's sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 Å. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predicted to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8's mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins. PMID:19759157

  8. Kaposi's sarcoma-associated herpesvirus LANA recruits the DNA polymerase clamp loader to mediate efficient replication and virus persistence.

    Science.gov (United States)

    Sun, Qiming; Tsurimoto, Toshiki; Juillard, Franceline; Li, Lin; Li, Shijun; De León Vázquez, Erika; Chen, She; Kaye, Kenneth

    2014-08-12

    Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and persists as a multiple-copy, extrachromosomal, circular episome. To persist, the viral genome must replicate with each cell cycle. The KSHV latency-associated nuclear antigen (LANA) mediates viral DNA replication and persistence, but little is known regarding the underlying mechanisms. We find that LANA recruits replication factor C (RFC), the DNA polymerase clamp [proliferating cell nuclear antigen (PCNA)] loader, to drive DNA replication efficiently. Mutated LANA lacking RFC interaction was deficient for LANA-mediated DNA replication and episome persistence. RFC depletion had a negative impact on LANA's ability to replicate and maintain viral DNA in cells containing artificial KSHV episomes or in infected cells, leading to loss of virus. LANA substantially increased PCNA loading onto DNA in vitro and recruited RFC and PCNA to KSHV DNA in cells. These findings suggest that PCNA loading is a rate-limiting step in DNA replication that is incompatible with viral survival. LANA enhancement of PCNA loading permits efficient virus replication and persistence, revealing a previously unidentified mechanism for KSHV latency.

  9. New insights into the expression and functions of the Kaposi's sarcoma-associated herpesvirus long noncoding PAN RNA.

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    Conrad, Nicholas K

    2016-01-02

    The Kaposi's sarcoma-associated herpesvirus (KSHV) is a clinically relevant pathogen associated with several human diseases that primarily affect immunocompromised individuals. KSHV encodes a noncoding polyadenylated nuclear (PAN) RNA that is essential for viral propagation and viral gene expression. PAN RNA is the most abundant viral transcript produced during lytic replication. The accumulation of PAN RNA depends on high levels of transcription driven by the Rta protein, a KSHV transcription factor necessary and sufficient for latent-to-lytic phase transition. In addition, KSHV uses several posttranscriptional mechanisms to stabilize PAN RNA. A cis-acting element, called the ENE, prevents PAN RNA decay by forming a triple helix with its poly(A) tail. The viral ORF57 and the cellular PABPC1 proteins further contribute to PAN RNA stability during lytic phase. PAN RNA functions are only beginning to be uncovered, but PAN RNA has been proposed to control gene expression by several different mechanisms. PAN RNA associates with the KSHV genome and may regulate gene expression by recruiting chromatin-modifying factors. Moreover, PAN RNA binds the viral latency-associated nuclear antigen (LANA) protein and decreases its repressive activity by sequestering it from the viral genome. Surprisingly, PAN RNA was found to associate with translating ribosomes, so this noncoding RNA may be additionally used to produce viral peptides. In this review, I highlight the mechanisms of PAN RNA accumulation and describe recent insights into potential functions of PAN RNA. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells

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    Cai, Xuezhong; Lu, Shihua; Zhang, Zhihong; Gonzalez, Carlos M.; Damania, Blossom; Cullen, Bryan R.

    2005-01-01

    MicroRNAs (miRNAs) are an endogenously encoded class of small RNAs that have been proposed to function as key posttranscriptional regulators of gene expression in a range of eukaryotic species, including humans. The small size of miRNA precursors makes them potentially ideal for use by viruses as inhibitors of host cell defense pathways. Here, we demonstrate that the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an array of 11 distinct miRNAs, all of which are expressed at readily detectable levels in latently KSHV infected cells. Individual KSHV miRNAs were expressed at up to 2,200 copies per cell. The KSHV miRNAs are expressed from what appears to be a single genetic locus that largely coincides with an ≈4-kb noncoding sequence located between the KSHV v-cyclin and K12/Kaposin genes, both of which are also expressed in latently infected cells. Computer analysis of potential mRNA targets for these viral miRNAs identified a number of interesting candidate genes, including several mRNAs previously shown to be down-regulated in KSHV-infected cells. We hypothesize that these viral miRNAs play a critical role in the establishment and/or maintenance of KSHV latent infection in vivo and, hence, in KSHV-induced oncogenesis. PMID:15800047

  11. Asynchronous Progression through the Lytic Cascade and Variations in Intracellular Viral Loads Revealed by High-Throughput Single-Cell Analysis of Kaposi's Sarcoma-Associated Herpesvirus Infection

    OpenAIRE

    Adang, Laura A.; Parsons, Christopher H.; Kedes, Dean H.

    2006-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus-8) is frequently tumorigenic in immunocompromised patients. The average intracellular viral copy number within infected cells, however, varies markedly by tumor type. Since the KSHV-encoded latency-associated nuclear antigen (LANA) tethers viral episomes to host heterochromatin and displays a punctate pattern by fluorescence microscopy, we investigated whether accurate quantification of individual LANA dots is predictive of in...

  12. Was Kaposi's sarcoma-associated herpesvirus introduced into China via the ancient Silk Road? An evolutionary perspective.

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    Liu, Zhenqiu; Fang, Qiwen; Zuo, Jialu; Minhas, Veenu; Wood, Charles; He, Na; Zhang, Tiejun

    2017-07-07

    Kaposi's sarcoma-associated herpesvirus (KSHV) has become widely dispersed worldwide since it was first reported in 1994, but the seroprevalence of KSHV varies geographically. KSHV is relatively ubiquitous in Mediterranean areas and the Xinjiang Uygur Autonomous Region, China. The origin of KSHV has long been puzzling. In the present study, we collected and analysed 154 KSHV ORF-K1 sequences obtained from samples originating from Xinjiang, Italy, Greece, Iran and southern Siberia using Bayesian evolutionary analysis in BEAST to test the hypothesis that KSHV was introduced into Xinjiang via the ancient Silk Road. According to the phylogenetic analysis, 72 sequences were subtype A and 82 subtype C, with C2 (n = 56) being the predominant subtype. The times to the most recent common ancestors (tMRCAs) of KSHV were 29,872 years (95% highest probability density [HPD], 26,851-32,760 years) for all analysed sequences and 2037 years (95% HPD, 1843-2229 years) for Xinjiang sequences in particular. The tMRCA of Xinjiang KSHV was exactly matched with the time period of the ancient Silk Road approximately two thousand years ago. This route began in Chang'an, the capital of the Han dynasty of China, and crossed Central Asia, ending in the Roman Empire. The evolution rate of KSHV was slow, with 3.44 × 10-6 substitutions per site per year (95% HPD, 2.26 × 10-6 to 4.71 × 10-6), although 11 codons were discovered to be under positive selection pressure. The geographic distances from Italy to Iran and Xinjiang are more than 4000 and 7000 kilometres, respectively, but no explicit relationship between genetic distance and geographic distance was detected.

  13. Regulation of the Abundance of Kaposi's Sarcoma-Associated Herpesvirus ORF50 Protein by Oncoprotein MDM2.

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    Tzu-Hsuan Chang

    2016-10-01

    Full Text Available The switch between latency and the lytic cycle of Kaposi's sarcoma-associated herpesvirus (KSHV is controlled by the expression of virally encoded ORF50 protein. Thus far, the regulatory mechanism underlying the protein stability of ORF50 is unknown. Our earlier studies have demonstrated that a protein abundance regulatory signal (PARS at the ORF50 C-terminal region modulates its protein abundance. The PARS region consists of PARS-I (aa 490-535 and PARS-II (aa 590-650, and mutations in either component result in abundant expression of ORF50. Here, we show that ORF50 protein is polyubiquitinated and its abundance is controlled through the proteasomal degradation pathway. The PARS-I motif mainly functions as a nuclear localization signal in the control of ORF50 abundance, whereas the PARS-II motif is required for the binding of ubiquitin enzymes in the nucleus. We find that human oncoprotein MDM2, an ubiquitin E3 ligase, is capable of interacting with ORF50 and promoting ORF50 degradation in cells. The interaction domains between both proteins are mapped to the PARS region of ORF50 and the N-terminal 220-aa region of MDM2. Additionally, we identify lysine residues at positions 152 and 154 in the N-terminal domain of ORF50 critically involved in MDM2-mediated downregulation of ORF50 levels. Within KSHV-infected cells, the levels of MDM2 were greatly reduced during viral lytic cycle and genetic knockdown of MDM2 in these cells favored the enhancement of ORF50 expression, supporting that MDM2 is a negative regulator of ORF50 expression. Collectively, the study elucidates the regulatory mechanism of ORF50 stability and implicates that MDM2 may have a significant role in the maintenance of viral latency by lowering basal level of ORF50.

  14. Hsp70 Isoforms Are Essential for the Formation of Kaposi's Sarcoma-Associated Herpesvirus Replication and Transcription Compartments.

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    Belinda Baquero-Pérez

    2015-11-01

    Full Text Available Kaposi's sarcoma-associated herpesvirus (KSHV is an oncogenic herpesvirus associated with various AIDS-related malignancies. Like other herpesviruses, multiple processes required for KSHV lytic replication, including viral transcription, viral DNA synthesis and capsid assembly occur in virus-induced intranuclear structures, termed replication and transcription compartments (RTCs. Here we utilised a novel methodology, combining subcellular fractionation and quantitative proteomics, to identify cellular proteins which are recruited to KSHV-induced RTCs and thus play a key role in KSHV lytic replication. We show that several isoforms of the HSP70 chaperone family, Hsc70 and iHsp70, are redistributed from the cytoplasm into the nucleus coinciding with the initial formation of KSHV-induced RTCs. We demonstrate that nuclear chaperone foci are dynamic, initially forming adjacent to newly formed KSHV RTCs, however during later time points the chaperones move within KSHV RTCs and completely co-localise with actively replicating viral DNA. The functional significance of Hsp70 isoforms recruitment into KSHV RTCs was also examined using the specific Hsp70 isoform small molecule inhibitor, VER-155008. Intriguingly, results highlight an essential role of Hsp70 isoforms in the KSHV replication cycle independent of protein stability and maturation. Notably, inhibition of Hsp70 isoforms precluded KSHV RTC formation and RNA polymerase II (RNAPII relocalisation to the viral genome leading to the abolishment of global KSHV transcription and subsequent viral protein synthesis and DNA replication. These new findings have revealed novel mechanisms that regulate KSHV lytic replication and highlight the potential of HSP70 inhibitors as novel antiviral agents.

  15. Efficient infection by a recombinant Kaposi's sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: application for genetic analysis.

    Science.gov (United States)

    Zhou, Fu-Chun; Zhang, Yan-Jin; Deng, Jian-Hong; Wang, Xin-Ping; Pan, Hong-Yi; Hettler, Evelyn; Gao, Shou-Jiang

    2002-06-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma and several other malignancies. The lack of an efficient infection system has impeded the understanding of KSHV-related pathogenesis. A genetic approach was used to isolate infectious KSHV. Recombinant bacteria artificial chromosome (BAC) KSHV containing hygromycin resistance and green fluorescent protein (GFP) markers was generated by homologous recombination in KSHV-infected BCBL-1 cells. Recombinant KSHV genomes from cell clones that were resistant to hygromycin, expressed GFP, and produced infectious virions after induction with tetradecanoyl phorbol acetate (TPA) were rescued in Escherichia coli and reconstituted in 293 cells. Several 293 cell lines resulting from infection with recombinant virions induced from a full-length recombinant KSHV genome, named BAC36, were obtained. BAC36 virions established stable latent infection in 293 cells, harboring 1 to 2 copies of viral genome per cell and expressing viral latent proteins, with approximately 0.5% of cells undergoing spontaneous lytic replication, which is reminiscent of KSHV infection in Kaposi's sarcoma tumors. TPA treatment induced BAC36-infected 293 cell lines into productive lytic replication, expressing lytic proteins and producing virions that efficiently infected normal 293 cells with a approximately 50% primary infection rate. BAC36 virions were also infectious to HeLa and E6E7-immortalized human endothelial cells. Since BAC36 can be efficiently shuttled between bacteria and mammalian cells, it is useful for KSHV genetic analysis. The feasibility of the system was illustrated through the generation of a KSHV mutant with the vIRF gene deleted. This cellular model is useful for the investigation of KSHV infection and pathogenesis.

  16. Risk of Kaposi's sarcoma-associated herpes virus transmission from donor allografts among Italian posttransplant Kaposi's sarcoma patients.

    Science.gov (United States)

    Parravicini, C; Olsen, S J; Capra, M; Poli, F; Sirchia, G; Gao, S J; Berti, E; Nocera, A; Rossi, E; Bestetti, G; Pizzuto, M; Galli, M; Moroni, M; Moore, P S; Corbellino, M

    1997-10-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a newly discovered herpes virus found in all forms of Kaposi's sarcoma (KS) including KS among immunosuppressed transplant patients. It is unknown whether this virus is transmitted by organ transplantation or is reactivated during immunosuppression among those patients infected before transplantation. To investigate the risk of KSHV transmission during organ transplantation, we conducted a case-control study of transplant recipients with and without KS matched to their respective donors. Sera were collected at time of transplantation and tested in a randomized and blinded fashion using four KSHV serologic assays testing for antibodies to both latent and lytic phase antigens. Ten (91%) of 11 organ recipients who developed KS were seropositive prior to transplantation by one or more of the assays compared with two (12%) of 17 control organ recipients (OR = 75, 95% CI = 4.7, 3500). KS cases were more likely to have been born in southern Italy where KS is endemic than the recipient controls or either donor group. Only four (36%) of 11 donors to case patients and three (18%) of 17 donors to control patients were seropositive (P = .38, two-tailed Fisher's exact test). KSHV transmission could not be ruled out for the single KS patient seronegative at transplantation and clear evidence for organ-related transmission was found for another KS patient outside of the case-control study. Antibodies to KSHV are detectable in the sera from most transplant recipients before initiation of immunosuppressive treatment suggesting that KS among immunosuppressed transplant patients is primarily due to virus reactivation. KSHV transmission, however, from an infected allograft can occur, and our study reports the first documented case of person-to-person transmission of KSHV.

  17. Exploitation of the complement system by oncogenic Kaposi's sarcoma-associated herpesvirus for cell survival and persistent infection.

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    Myung-Shin Lee

    2014-09-01

    Full Text Available During evolution, herpesviruses have developed numerous, and often very ingenious, strategies to counteract efficient host immunity. Specifically, Kaposi's sarcoma-associated herpesvirus (KSHV eludes host immunity by undergoing a dormant stage, called latency wherein it expresses a minimal number of viral proteins to evade host immune activation. Here, we show that during latency, KSHV hijacks the complement pathway to promote cell survival. We detected strong deposition of complement membrane attack complex C5b-9 and the complement component C3 activated product C3b on Kaposi's sarcoma spindle tumor cells, and on human endothelial cells latently infected by KSHV, TIME-KSHV and TIVE-LTC, but not on their respective uninfected control cells, TIME and TIVE. We further showed that complement activation in latently KSHV-infected cells was mediated by the alternative complement pathway through down-regulation of cell surface complement regulatory proteins CD55 and CD59. Interestingly, complement activation caused minimal cell death but promoted the survival of latently KSHV-infected cells grown in medium depleted of growth factors. We found that complement activation increased STAT3 tyrosine phosphorylation (Y705 of KSHV-infected cells, which was required for the enhanced cell survival. Furthermore, overexpression of either CD55 or CD59 in latently KSHV-infected cells was sufficient to inhibit complement activation, prevent STAT3 Y705 phosphorylation and abolish the enhanced survival of cells cultured in growth factor-depleted condition. Together, these results demonstrate a novel mechanism by which an oncogenic virus subverts and exploits the host innate immune system to promote viral persistent infection.

  18. Prevalence of Kaposi's sarcoma-associated herpesvirus infection in sex workers and women from the general population in Spain.

    Science.gov (United States)

    de Sanjosé, Sílvia; Marshall, Vickie; Solà, Judit; Palacio, Virgilio; Almirall, Rosa; Goedert, James J; Bosch, F Xavier; Whitby, Denise

    2002-03-01

    Transmission routes of Kaposi's sarcoma-associated herpesvirus (KSHV) in the general population are poorly understood. Whereas sexual transmission appears to be common in homosexual men, the evidence for heterosexual transmission is less convincing. In our study, prevalence of KSHV infection was examined among women in the Spanish general population and among sex workers. Subjects consisted of 100 prostitutes and 100 women randomly sampled from the general population and age-matched to the prostitutes. Women had a personal interview and gynecologic examinations in which a blood sample, cervical cells and oral cells were obtained. Peripheral blood mononuclear cells (PBMC), oral and cervical samples were tested for KSHV DNA by quantitative real-time PCR. Sera were tested for antibodies against human immunodeficiency virus (HIV) by ELISA and against KSHV by latent IFA and K8.1 ELISA. Women who were positive in either serologic assay or PCR were considered infected by KSHV. Human papillomavirus (HPV) DNA in cervical scrapes were evaluated using the Hybrid Capture System. The study population had an average age of 30 years and were HIV-negative. Women from the general population were largely of Spanish nationality, and 61% reported lifetime monogamy. The majority of the prostitutes (76%) were immigrants, primarily from South America. Sex workers were twice as likely to be infected with KSHV than women in the general population (16% vs. 8%, prevalence odds ratio [OR] = 2.2). KSHV was more prevalent among HPV DNA-positive women (OR = 2.5) and among women with an early age at first sexual intercourse (OR = 2.7, p women in the general population. All PBMC samples were negative. These results suggest that in low-risk countries for KSHV, oral shedding and heterosexual contacts are potential pathways for KSHV transmission. Copyright 2001 Wiley-Liss, Inc.

  19. Kaposi's sarcoma-associated herpesvirus ORF57 protein binds and protects a nuclear noncoding RNA from cellular RNA decay pathways.

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    Brooke B Sahin

    2010-03-01

    Full Text Available The control of RNA stability is a key determinant in cellular gene expression. The stability of any transcript is modulated through the activity of cis- or trans-acting regulatory factors as well as cellular quality control systems that ensure the integrity of a transcript. As a result, invading viral pathogens must be able to subvert cellular RNA decay pathways capable of destroying viral transcripts. Here we report that the Kaposi's sarcoma-associated herpesvirus (KSHV ORF57 protein binds to a unique KSHV polyadenylated nuclear RNA, called PAN RNA, and protects it from degradation by cellular factors. ORF57 increases PAN RNA levels and its effects are greatest on unstable alleles of PAN RNA. Kinetic analysis of transcription pulse assays shows that ORF57 protects PAN RNA from a rapid cellular RNA decay process, but ORF57 has little effect on transcription or PAN RNA localization based on chromatin immunoprecipitation and in situ hybridization experiments, respectively. Using a UV cross-linking technique, we further demonstrate that ORF57 binds PAN RNA directly in living cells and we show that binding correlates with function. In addition, we define an ORF57-responsive element (ORE that is necessary for ORF57 binding to PAN RNA and sufficient to confer ORF57-response to a heterologous intronless beta-globin mRNA, but not its spliced counterparts. We conclude that ORF57 binds to viral transcripts in the nucleus and protects them from a cellular RNA decay pathway. We propose that KSHV ORF57 protein functions to enhance the nuclear stability of intronless viral transcripts by protecting them from a cellular RNA quality control pathway.

  20. Epstein-Barr virus (EBV Rta-mediated EBV and Kaposi's sarcoma-associated herpesvirus lytic reactivations in 293 cells.

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    Yen-Ju Chen

    Full Text Available Epstein-Barr virus (EBV Rta belongs to a lytic switch gene family that is evolutionarily conserved in all gamma-herpesviruses. Emerging evidence indicates that cell cycle arrest is a common means by which herpesviral immediate-early protein hijacks the host cell to advance the virus's lytic cycle progression. To examine the role of Rta in cell cycle regulation, we recently established a doxycycline (Dox-inducible Rta system in 293 cells. In this cell background, inducible Rta modulated the levels of signature G1 arrest proteins, followed by induction of the cellular senescence marker, SA-β-Gal. To delineate the relationship between Rta-induced cell growth arrest and EBV reactivation, recombinant viral genomes were transferred into Rta-inducible 293 cells. Somewhat unexpectedly, we found that Dox-inducible Rta reactivated both EBV and Kaposi's sarcoma-associated herpesvirus (KSHV, to similar efficacy. As a consequence, the Rta-mediated EBV and KSHV lytic replication systems, designated as EREV8 and ERKV, respectively, were homogenous, robust, and concurrent with cell death likely due to permissive lytic replication. In addition, the expression kinetics of EBV lytic genes in Dox-treated EREV8 cells was similar to that of their KSHV counterparts in Dox-induced ERKV cells, suggesting that a common pathway is used to disrupt viral latency in both cell systems. When the time course was compared, cell cycle arrest was achieved between 6 and 48 h, EBV or KSHV reactivation was initiated abruptly at 48 h, and the cellular senescence marker was not detected until 120 h after Dox treatment. These results lead us to hypothesize that in 293 cells, Rta-induced G1 cell cycle arrest could provide (1 an ideal environment for virus reactivation if EBV or KSHV coexists and (2 a preparatory milieu for cell senescence if no viral genome is available. The latter is hypothetical in a transient-lytic situation.

  1. A hydrophobic domain within the small capsid protein of Kaposi's sarcoma-associated herpesvirus is required for assembly.

    Science.gov (United States)

    Capuano, Christopher M; Grzesik, Peter; Kreitler, Dale; Pryce, Erin N; Desai, Keshal V; Coombs, Gavin; McCaffery, J Michael; Desai, Prashant J

    2014-08-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) capsids can be produced in insect cells using recombinant baculoviruses for protein expression. All six capsid proteins are required for this process to occur and, unlike for alphaherpesviruses, the small capsid protein (SCP) ORF65 is essential for this process. This protein decorates the capsid shell by virtue of its interaction with the capsomeres. In this study, we have explored the SCP interaction with the major capsid protein (MCP) using GFP fusions. The assembly site within the nucleus of infected cells was visualized by light microscopy using fluorescence produced by the SCP-GFP polypeptide, and the relocalization of the SCP to these sites was evident only when the MCP and the scaffold protein were also present - indicative of an interaction between these proteins that ensures delivery of the SCP to assembly sites. Biochemical assays demonstrated a physical interaction between the SCP and MCP, and also between this complex and the scaffold protein. Self-assembly of capsids with the SCP-GFP polypeptide was evident. Potentially, this result can be used to engineer fluorescent KSHV particles. A similar SCP-His6 polypeptide was used to purify capsids from infected cell lysates using immobilized affinity chromatography and to directly label this protein in capsids using chemically derivatized gold particles. Additional studies with SCP-GFP polypeptide truncation mutants identified a domain residing between aa 50 and 60 of ORF65 that was required for the relocalization of SCP-GFP to nuclear assembly sites. Substitution of residues in this region and specifically at residue 54 with a polar amino acid (lysine) disrupted or abolished this localization as well as capsid assembly, whereas substitution with non-polar residues did not affect the interaction. Thus, this study identified a small conserved hydrophobic domain that is important for the SCP-MCP interaction. © 2014 The Authors.

  2. A hydrophobic domain within the small capsid protein of Kaposi’s sarcoma-associated herpesvirus is required for assembly

    Science.gov (United States)

    Capuano, Christopher M.; Grzesik, Peter; Kreitler, Dale; Pryce, Erin N.; Desai, Keshal V.; Coombs, Gavin; McCaffery, J. Michael

    2014-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) capsids can be produced in insect cells using recombinant baculoviruses for protein expression. All six capsid proteins are required for this process to occur and, unlike for alphaherpesviruses, the small capsid protein (SCP) ORF65 is essential for this process. This protein decorates the capsid shell by virtue of its interaction with the capsomeres. In this study, we have explored the SCP interaction with the major capsid protein (MCP) using GFP fusions. The assembly site within the nucleus of infected cells was visualized by light microscopy using fluorescence produced by the SCP–GFP polypeptide, and the relocalization of the SCP to these sites was evident only when the MCP and the scaffold protein were also present – indicative of an interaction between these proteins that ensures delivery of the SCP to assembly sites. Biochemical assays demonstrated a physical interaction between the SCP and MCP, and also between this complex and the scaffold protein. Self-assembly of capsids with the SCP–GFP polypeptide was evident. Potentially, this result can be used to engineer fluorescent KSHV particles. A similar SCP–His6 polypeptide was used to purify capsids from infected cell lysates using immobilized affinity chromatography and to directly label this protein in capsids using chemically derivatized gold particles. Additional studies with SCP–GFP polypeptide truncation mutants identified a domain residing between aa 50 and 60 of ORF65 that was required for the relocalization of SCP–GFP to nuclear assembly sites. Substitution of residues in this region and specifically at residue 54 with a polar amino acid (lysine) disrupted or abolished this localization as well as capsid assembly, whereas substitution with non-polar residues did not affect the interaction. Thus, this study identified a small conserved hydrophobic domain that is important for the SCP–MCP interaction. PMID:24824860

  3. Kaposi's Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N6-Adenosine Methylation To Promote Lytic Replication

    Science.gov (United States)

    Chen, E. Ricky; Nilsen, Timothy W.

    2017-01-01

    ABSTRACT N6-adenosine methylation (m6A) is the most common posttranscriptional RNA modification in mammalian cells. We found that most transcripts encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) genome undergo m6A modification. The levels of m6A-modified mRNAs increased substantially upon stimulation for lytic replication. The blockage of m6A inhibited splicing of the pre-mRNA encoding the replication transcription activator (RTA), a key KSHV lytic switch protein, and halted viral lytic replication. We identified several m6A sites in RTA pre-mRNA crucial for splicing through interactions with YTH domain containing 1 (YTHDC1), an m6A nuclear reader protein, in conjunction with serine/arginine-rich splicing factor 3 (SRSF3) and SRSF10. Interestingly, RTA induced m6A and enhanced its own pre-mRNA splicing. Our results not only demonstrate an essential role of m6A in regulating RTA pre-mRNA splicing but also suggest that KSHV has evolved a mechanism to manipulate the host m6A machinery to its advantage in promoting lytic replication. IMPORTANCE KSHV productive lytic replication plays a pivotal role in the initiation and progression of Kaposi's sarcoma tumors. Previous studies suggested that the KSHV switch from latency to lytic replication is primarily controlled at the chromatin level through histone and DNA modifications. The present work reports for the first time that KSHV genome-encoded mRNAs undergo m6A modification, which represents a new mechanism at the posttranscriptional level in the control of viral replication. PMID:28592530

  4. Kaposi's sarcoma-associated herpesvirus ORF18 and ORF30 are essential for late gene expression during lytic replication.

    Science.gov (United States)

    Gong, Danyang; Wu, Nicholas C; Xie, Yafang; Feng, Jun; Tong, Leming; Brulois, Kevin F; Luan, Harding; Du, Yushen; Jung, Jae U; Wang, Cun-yu; Kang, Mo Kwan; Park, No-Hee; Sun, Ren; Wu, Ting-Ting

    2014-10-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several human malignances. As saliva is likely the major vehicle for KSHV transmission, we studied in vitro KSHV infection of oral epithelial cells. Through infection of two types of oral epithelial cells, normal human oral keratinocytes (NHOKs) and papilloma-immortalized human oral keratinocyte (HOK16B) cells, we found that KSHV can undergo robust lytic replication in oral epithelial cells. By employing de novo lytic infection of HOK16B cells, we studied the functions of two previously uncharacterized genes, ORF18 and ORF30, during the KSHV lytic cycle. For this purpose, an ORF18-deficient virus and an ORF30-deficient virus were generated using a mutagenesis strategy based on bacterial artificial chromosome (BAC) technology. We found that neither ORF18 nor ORF30 is required for immediately early or early gene expression or viral DNA replication, but each is essential for late gene expression during both de novo lytic replication and reactivation. This critical role of ORF18 and ORF30 in late gene expression was also observed during KSHV reactivation. In addition, global analysis of viral transcripts by RNA sequencing indicated that ORF18 and ORF30 control the same set of viral genes. Therefore, we suggest that these two viral ORFs are involved in the same mechanism or pathway that coregulates the viral late genes as a group. While KSHV can infect multiple cell types in vitro, only a few can support a full lytic replication cycle with progeny virions produced. Consequently, KSHV lytic replication is mostly studied through reactivation, which requires chemicals to induce the lytic cycle or overexpression of the viral transcriptional activator, RTA. In this study, we present a robust de novo lytic infection system based on oral epithelial cells. Using this system, we demonstrate the role of two viral ORFs, ORF18 and ORF30, in regulating viral gene expression during KSHV lytic replication. As the major

  5. A microRNA encoded by Kaposi sarcoma-associated herpesvirus promotes B-cell expansion in vivo.

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    Christine Dahlke

    Full Text Available The human gammaherpesvirus Kaposi sarcoma-associated herpesvirus is strongly linked to neoplasms of endothelial and B-cell origin. The majority of tumor cells in these malignancies are latently infected, and latency genes are consequently thought to play a critical role in virus-induced tumorigenesis. One such factor is kshv-miR-K12-11, a viral microRNA that is constitutively expressed in cell lines derived from KSHV-associated tumors, and that shares perfect homology of its seed sequence with the cellular miR-155. Since miR-155 is overexpressed in a number of human tumors, it is conceivable that mimicry of miR-155 by miR-K12-11 may contribute to cellular transformation in KSHV-associated disease. Here, we have performed a side-by-side study of phenotypic alterations associated with constitutive expression of either human miR-155 or viral miR-K12-11 in bone marrow-derived hematopoietic stem cells. We demonstrate that retroviral-mediated gene transfer and hematopoietic progenitor cell transplantation into C57BL/6 mice leads to increased B-cell fractions in lymphoid organs, as well as to enhanced germinal center formation in both microRNA-expressing mouse cohorts. We furthermore identify Jarid2, a component of Polycomb repressive complex 2, as a novel validated target of miR-K12-11, and confirm its downregulation in miR-K12-11 as well as miR-155 expressing bone marrow cells. Our findings confirm and extend previous observations made in other mouse models, and underscore the notion that miR-K12-11 may have arisen to mimic miR-155 functions in KSHV-infected B-cells. The expression of miR-K12-11 may represent one mechanism by which KSHV presumably aims to reprogram naïve B-cells towards supporting long-term latency, which at the same time is likely to pre-dispose infected lymphocytes to malignant transformation.

  6. ESCRT-0 Component Hrs Promotes Macropinocytosis of Kaposi's Sarcoma-Associated Herpesvirus in Human Dermal Microvascular Endothelial Cells.

    Science.gov (United States)

    Veettil, Mohanan Valiya; Kumar, Binod; Ansari, Mairaj Ahmed; Dutta, Dipanjan; Iqbal, Jawed; Gjyshi, Olsi; Bottero, Virginie; Chandran, Bala

    2016-04-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) enters human dermal microvascular endothelial cells (HMVEC-d), its naturalin vivotarget cells, by lipid raft-dependent macropinocytosis. The internalized viral envelope fuses with the macropinocytic membrane, and released capsid is transported to the nuclear vicinity, resulting in the nuclear entry of viral DNA. The endosomal sorting complexes required for transport (ESCRT) proteins, which include ESCRT-0, -I, -II, and -III, play a central role in endosomal trafficking and sorting of internalized and ubiquitinated receptors. Here, we examined the role of ESCRT-0 component Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in KSHV entry into HMVEC-d by macropinocytosis. Knockdown of Hrs by short hairpin RNA (shRNA) transduction resulted in significant decreases in KSHV entry and viral gene expression. Immunofluorescence analysis (IFA) and plasma membrane isolation and proximity ligation assay (PLA) demonstrated the translocation of Hrs from the cytosol to the plasma membrane of infected cells and association with α-actinin-4. In addition, infection induced the plasma membrane translocation and activation of the serine/threonine kinase ROCK1, a downstream target of the RhoA GTPase. Hrs knockdown reduced these associations, suggesting that the recruitment of ROCK1 is an Hrs-mediated event. Interaction between Hrs and ROCK1 is essential for the ROCK1-induced phosphorylation of NHE1 (Na(+)/H(+)exchanger 1), which is involved in the regulation of intracellular pH. Thus, our studies demonstrate the plasma membrane association of ESCRT protein Hrs during macropinocytosis and suggest that KSHV entry requires both Hrs- and ROCK1-dependent mechanisms and that ROCK1-mediated phosphorylation of NHE1 and pH change is an essential event required for the macropinocytosis of KSHV. Macropinocytosis is the major entry pathway of KSHV in human dermal microvascular endothelial cells, the natural target cells of KSHV

  7. Glycolysis, Glutaminolysis, and Fatty Acid Synthesis Are Required for Distinct Stages of Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication.

    Science.gov (United States)

    Sanchez, Erica L; Pulliam, Thomas H; Dimaio, Terri A; Thalhofer, Angel B; Delgado, Tracie; Lagunoff, Michael

    2017-05-15

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS). KSHV infection induces and requires multiple metabolic pathways, including the glycolysis, glutaminolysis, and fatty acid synthesis (FAS) pathways, for the survival of latently infected endothelial cells. To determine the metabolic requirements for productive KSHV infection, we induced lytic replication in the presence of inhibitors of different metabolic pathways. We found that glycolysis, glutaminolysis, and FAS are all required for maximal KSHV virus production and that these pathways appear to participate in virus production at different stages of the viral life cycle. Glycolysis and glutaminolysis, but not FAS, inhibit viral genome replication and, interestingly, are required for different early steps of lytic gene expression. Glycolysis is necessary for early gene transcription, while glutaminolysis is necessary for early gene translation but not transcription. Inhibition of FAS resulted in decreased production of extracellular virions but did not reduce intracellular genome levels or block intracellular virion production. However, in the presence of FAS inhibitors, the intracellular virions are noninfectious, indicating that FAS is required for virion assembly or maturation. KS tumors support both latent and lytic KSHV replication. Previous work has shown that multiple cellular metabolic pathways are required for latency, and we now show that these metabolic pathways are required for efficient lytic replication, providing novel therapeutic avenues for KS tumors. IMPORTANCE KSHV is the etiologic agent of Kaposi's sarcoma, the most common tumor of AIDS patients. KS spindle cells, the main tumor cells, all contain KSHV, mostly in the latent state, during which there is limited viral gene expression. However, a percentage of spindle cells support lytic replication and production of virus and these cells are thought to contribute to overall tumor formation. Our

  8. PAN’s Labyrinth: Molecular Biology of Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) PAN RNA, a Multifunctional Long Noncoding RNA

    Science.gov (United States)

    Rossetto, Cyprian C.; Pari, Gregory S.

    2014-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesivrus, the causative agent of Kaposi’s sarcoma and body cavity lymphomas. During infection KSHV produces a highly abundant long non-coding polyadenylated RNA that is retained in the nucleus known as PAN RNA. Long noncoding RNAs (lncRNA) are key regulators of gene expression and are known to interact with specific chromatin modification complexes, working in cis and trans to regulate gene expression. Data strongly supports a model where PAN RNA is a multifunctional regulatory transcript that controls KSHV gene expression by mediating the modification of chromatin by targeting the KSHV repressed genome. PMID:25375885

  9. DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi's sarcoma-associated herpesvirus latent replication

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Seho [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of); Lim, Chunghun [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of); Lee, Jae Young [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of); Song, Yoon-Jae [Department of Life Science, Kyungwon University, Seongnam-Si, Kyeonggi-Do 461-701 (Korea, Republic of); Park, Junsoo [Division of Biological Science and Technology, Yonsei University, Wonju 220-100 (Korea, Republic of); Choe, Joonho [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of); Seo, Taegun, E-mail: tseo@dongguk.edu [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of)

    2010-04-16

    During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.

  10. Kaposi sarcoma-associated herpes virus targets the lymphotactin receptor with both a broad spectrum antagonist vCCL2 and a highly selective and potent agonist vCCL3

    DEFF Research Database (Denmark)

    Lüttichau, Hans R; Johnsen, Anders H; Jurlander, Jesper

    2007-01-01

    Large DNA viruses such as herpesvirus and poxvirus encode proteins that target and exploit the chemokine system of their host. These proteins have the potential to block or change the orchestrated recruitment of leukocytes to sites of viral infection. The genome of Kaposi sarcoma-associated herpes...

  11. The Gammaherpesviruses Kaposi's Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus 68 Modulate the Toll-Like Receptor-Induced Proinflammatory Cytokine Response

    Science.gov (United States)

    Bussey, Kendra A.; Reimer, Elisa; Todt, Helene; Denker, Brigitte; Gallo, Antonio; Konrad, Andreas; Ottinger, Matthias; Adler, Heiko; Stürzl, Michael; Brune, Wolfram

    2014-01-01

    ABSTRACT The human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease, establishes lifelong latency upon infection. Murine gammaherpesvirus 68 (MHV68) is a well-established model for KSHV. Toll-like receptors (TLRs) play a crucial role for the innate immune response to pathogens. Although KSHV and MHV68 are detected by TLRs, studies suggest they modulate TLR4 and TLR9 signaling, respectively. In this study, we show that in bone marrow-derived macrophages (BMDMs), MHV68 did not induce a detectable proinflammatory cytokine response. Furthermore, MHV68 abrogated the response to TLR2, -4, -7, and -9 agonists in BMDMs. Similarly to observations with MHV68, infection with KSHV efficiently inhibited TLR2 signaling in THP-1 monocytes. Using a KSHV open reading frame (ORF) library, we found that K4.2, ORF21, ORF31, and the replication and transcription activator protein (RTA)/ORF50 inhibited TLR2-dependent nuclear factor kappa B (NF-κB) activation in HEK293 TLR2-yellow fluorescent protein (YFP)- and Flag-TLR2-transfected HEK293T cells. Of the identified ORFs, RTA/ORF50 strongly downregulated TLR2 and TLR4 signaling by reducing TLR2 and TLR4 protein expression. Confocal microscopy revealed that TLR2 and TLR4 were no longer localized to the plasma membrane in cells expressing RTA/ORF50. In this study, we have shown that the gammaherpesviruses MHV68 and KSHV efficiently downmodulate TLR signaling in macrophages and have identified a novel function of RTA/ORF50 in modulation of the innate immune response. IMPORTANCE The Toll-like receptors (TLRs) are an important class of pattern recognition receptors of the innate immune system. They induce a potent proinflammatory cytokine response upon detection of a variety of pathogens. In this study, we found that the gammaherpesviruses murine gammaherpesvirus 68 (MHV68) and Kaposi's sarcoma-associated herpesvirus (KSHV

  12. Chromatin Immunoprecipitation and Microarray Analysis Suggest Functional Cooperation between Kaposi's Sarcoma-Associated Herpesvirus ORF57 and K-bZIP

    Science.gov (United States)

    Hunter, Olga V.; Sei, Emi; Richardson, R. Blake

    2013-01-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 57 (ORF57)-encoded protein (Mta) is a multifunctional regulator of viral gene expression. ORF57 is essential for viral replication, so elucidation of its molecular mechanisms is important for understanding KSHV infection. ORF57 has been implicated in nearly every aspect of viral gene expression, including transcription, RNA stability, splicing, export, and translation. Here we demonstrate that ORF57 interacts with the KSHV K-bZIP protein in vitro and in cell extracts from lytically reactivated infected cells. To further test the biological relevance of the interaction, we performed a chromatin immunoprecipitation and microarray (ChIP-chip) analysis using anti-ORF57 antibodies and a KSHV tiling array. The results revealed four specific areas of enrichment, including the ORF4 and K8 (K-bZIP) promoters, as well as oriLyt, all of which interact with K-bZIP. In addition, ORF57 associated with DNA corresponding to the PAN RNA transcribed region, a known posttranscriptional target of ORF57. All of the peaks were RNase insensitive, demonstrating that ORF57 association with the viral genome is unlikely to be mediated exclusively by an RNA tether. Our data demonstrate that ORF57 associates with the viral genome by using at least two modes of recruitment, and they suggest that ORF57 and K-bZIP coregulate viral gene expression during lytic infection. PMID:23365430

  13. NEDDylation is essential for Kaposi's sarcoma-associated herpesvirus latency and lytic reactivation and represents a novel anti-KSHV target.

    Directory of Open Access Journals (Sweden)

    David J Hughes

    2015-03-01

    Full Text Available Kaposi's sarcoma-associated herpesvirus (KSHV is the causative agent of Kaposi's sarcoma (KS and primary effusion lymphoma (PEL, which are aggressive malignancies associated with immunocompromised patients. For many non-viral malignancies, therapeutically targeting the ubiquitin proteasome system (UPS has been successful. Likewise, laboratory studies have demonstrated that inhibition of the UPS might provide a promising avenue for the treatment of KSHV-associated diseases. The largest class of E3 ubiquitin ligases are the cullin-RING ligases (CRLs that are activated by an additional ubiquitin-like protein, NEDD8. We show that pharmacological inhibition of NEDDylation (using the small molecule inhibitor MLN4924 is cytotoxic to PEL cells by inhibiting NF-κB. We also show that CRL4B is a novel regulator of latency as its inhibition reactivated lytic gene expression. Furthermore, we uncovered a requirement for NEDDylation during the reactivation of the KSHV lytic cycle. Intriguingly, inhibition prevented viral DNA replication but not lytic cycle-associated gene expression, highlighting a novel mechanism that uncouples these two features of KSHV biology. Mechanistically, we show that MLN4924 treatment precluded the recruitment of the viral pre-replication complex to the origin of lytic DNA replication (OriLyt. These new findings have revealed novel mechanisms that regulate KSHV latency and reactivation. Moreover, they demonstrate that inhibition of NEDDylation represents a novel approach for the treatment of KSHV-associated malignancies.

  14. KSHV 2.0: a comprehensive annotation of the Kaposi's sarcoma-associated herpesvirus genome using next-generation sequencing reveals novel genomic and functional features.

    Directory of Open Access Journals (Sweden)

    Carolina Arias

    2014-01-01

    Full Text Available Productive herpesvirus infection requires a profound, time-controlled remodeling of the viral transcriptome and proteome. To gain insights into the genomic architecture and gene expression control in Kaposi's sarcoma-associated herpesvirus (KSHV, we performed a systematic genome-wide survey of viral transcriptional and translational activity throughout the lytic cycle. Using mRNA-sequencing and ribosome profiling, we found that transcripts encoding lytic genes are promptly bound by ribosomes upon lytic reactivation, suggesting their regulation is mainly transcriptional. Our approach also uncovered new genomic features such as ribosome occupancy of viral non-coding RNAs, numerous upstream and small open reading frames (ORFs, and unusual strategies to expand the virus coding repertoire that include alternative splicing, dynamic viral mRNA editing, and the use of alternative translation initiation codons. Furthermore, we provide a refined and expanded annotation of transcription start sites, polyadenylation sites, splice junctions, and initiation/termination codons of known and new viral features in the KSHV genomic space which we have termed KSHV 2.0. Our results represent a comprehensive genome-scale image of gene regulation during lytic KSHV infection that substantially expands our understanding of the genomic architecture and coding capacity of the virus.

  15. Asynchronous progression through the lytic cascade and variations in intracellular viral loads revealed by high-throughput single-cell analysis of Kaposi's sarcoma-associated herpesvirus infection.

    Science.gov (United States)

    Adang, Laura A; Parsons, Christopher H; Kedes, Dean H

    2006-10-01

    Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus-8) is frequently tumorigenic in immunocompromised patients. The average intracellular viral copy number within infected cells, however, varies markedly by tumor type. Since the KSHV-encoded latency-associated nuclear antigen (LANA) tethers viral episomes to host heterochromatin and displays a punctate pattern by fluorescence microscopy, we investigated whether accurate quantification of individual LANA dots is predictive of intracellular viral genome load. Using a novel technology that integrates single-cell imaging with flow cytometry, we found that both the number and the summed immunofluorescence of individual LANA dots are directly proportional to the amount of intracellular viral DNA. Moreover, combining viral (immediate early lytic replication and transcription activator [RTA] and late lytic K8.1) and cellular (syndecan-1) staining with image-based flow cytometry, we were also able to rapidly and simultaneously distinguish among cells supporting latent, immediate early lytic, early lytic, late lytic, and a potential fourth "delayed late" category of lytic replication. Applying image-based flow cytometry to KSHV culture models, we found that de novo infection results in highly varied levels of intracellular viral load and that lytic induction of latently infected cells likewise leads to a heterogeneous population at various stages of reactivation. These findings additionally underscore the potential advantages of studying KSHV biology with high-throughput analysis of individual cells.

  16. KSHV 2.0: a comprehensive annotation of the Kaposi's sarcoma-associated herpesvirus genome using next-generation sequencing reveals novel genomic and functional features.

    Science.gov (United States)

    Arias, Carolina; Weisburd, Ben; Stern-Ginossar, Noam; Mercier, Alexandre; Madrid, Alexis S; Bellare, Priya; Holdorf, Meghan; Weissman, Jonathan S; Ganem, Don

    2014-01-01

    Productive herpesvirus infection requires a profound, time-controlled remodeling of the viral transcriptome and proteome. To gain insights into the genomic architecture and gene expression control in Kaposi's sarcoma-associated herpesvirus (KSHV), we performed a systematic genome-wide survey of viral transcriptional and translational activity throughout the lytic cycle. Using mRNA-sequencing and ribosome profiling, we found that transcripts encoding lytic genes are promptly bound by ribosomes upon lytic reactivation, suggesting their regulation is mainly transcriptional. Our approach also uncovered new genomic features such as ribosome occupancy of viral non-coding RNAs, numerous upstream and small open reading frames (ORFs), and unusual strategies to expand the virus coding repertoire that include alternative splicing, dynamic viral mRNA editing, and the use of alternative translation initiation codons. Furthermore, we provide a refined and expanded annotation of transcription start sites, polyadenylation sites, splice junctions, and initiation/termination codons of known and new viral features in the KSHV genomic space which we have termed KSHV 2.0. Our results represent a comprehensive genome-scale image of gene regulation during lytic KSHV infection that substantially expands our understanding of the genomic architecture and coding capacity of the virus.

  17. Kaposi's-sarcoma-associated-herpesvirus-activated dendritic cells promote HIV-1 trans-infection and suppress CD4{sup +} T cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Wan; Qin, Yan; Bai, Lei [Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, the Chinese Academy of Sciences, Shanghai (China); Graduate School of the Chinese Academy of Sciences, Beijing (China); Lan, Ke [Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, the Chinese Academy of Sciences, Shanghai (China); Wang, Jian-Hua, E-mail: Jh_wang@sibs.ac.cn [Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, the Chinese Academy of Sciences, Shanghai (China)

    2013-06-05

    Infection of Kaposi's sarcoma-associated herpesvirus (KSHV) is commonly occurred in AIDS patients. KSHV and HIV-1 act cooperatively in regulating infection with each other and in human carcinogenesis. Dendritic cells (DCs), as the pivotal cells in host immunity, may be modulated by both viruses, for immunoevasion and dissemination, therefore, the interaction between DCs and each virus has been a prior focus for pathogenesis elucidation. Here, we assessed the potential effect of KSHV on DC–HIV-1 interaction. We found that KSHV stimulation could promote maturation of monocyte-derived DCs (MDDCs) and impaired the ability of MDDCs to drive proliferation of resting CD4{sup +} T cells, demonstrating the immunosuppression induced by KSHV. More importantly, KSHV-stimulated MDDCs could capture more HIV-1 and efficiently transferred these infectious viruses to Hut/CCR5 T cell line. Our results reveal the novel modulation of DC-mediated HIV-1 dissemination by KSHV, and highlight the importance of studying DC–HIV-1 interaction to elucidate HIV/AIDS pathogenesis. - Highlights: ► KSHV impaired the ability of MDDCs to drive proliferation of resting CD4{sup +} T cells. ► KSHV stimulation matured MDDCs and enhanced HIV-1 endocytosis. ► KSHV stimulated MDDCs increased ICAM-1 expression and tighten contact with T cells. ► KSHV-stimulated MDDCs promoted HIV-1 trans-infection of CD4{sup +} T cells.

  18. Seroprevalence and risk factors of Kaposi's sarcoma-associated herpesvirus infection among the general Uygur population from south and north region of Xinjiang, China

    Directory of Open Access Journals (Sweden)

    Wang Hui

    2011-12-01

    Full Text Available Abstract Background Kaposi sarcoma (KS is a complex multifocal neoplasm and is the major cause of death for about 50% of acquired immunodeficiency syndrome (AIDS patients. Kaposi's sarcoma-associated herpesvirus (KSHV is an oncogenic virus with a causal role in the development of all types of KS. KS is prevalent among the Uygur people in Xinjiang, especially in south area. Here we carried out a cross-sectional study among 1534 general Uygur individuals from south and north region of Xinjiang to assess the seroprevalence of KSHV and to identify the potential correlation between KSHV seroprevalence and KS incidence. Results Seroprevalence of KSHV in South and North Xinjiang was 23.1% and 25.9%, respectively. Older age was independently associated with higher KSHV seroprevalence. In subjects from South Xinjiang, lower educational level and reported drinking were each independently associated with higher KSHV seroprevalence. Furthermore, the antibody titer was significantly lower in both south and north KSHV seropositive individuals compared with KS patients, as analyzed by gradient dilution (P Conclusion KSHV is highly prevalent in the general Uygur population in both South and North Xinjiang. Interestingly, the infection rate of KSHV in these two geographical areas did not correlate well with KS incidence. Perhaps unknown factors exist that promote the progression of KSHV infection to KS development in the local minority groups.

  19. Kaposi’s Sarcoma-Associated Herpesvirus K3 and K5 Proteins Down Regulate Both DC-SIGN and DC-SIGNR

    Science.gov (United States)

    Karki, Roshan; Tartell, Michael A.; Means, Robert E.

    2013-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of multicentric Castleman’s disease, primary effusion lymphoma and Kaposi’s sarcoma. In this study, we show that like the C-type lectin DC-SIGN, the closely related DC-SIGNR can also enhance KSHV infection. Following infection, they are both targeted for down modulation and our data indicate that the KSHV MARCH-family ubiquitin ligase K5 is mediating this regulation and subsequent targeting for degradation of DC-SIGN and DC-SIGNR in the context of the virus. The closely related viral K3 protein, is also able to target these lectins in exogenous expressions studies, but only weakly during viral infection. In addition to requiring a functional RING-CH domain, several protein trafficking motifs in the C-terminal region of both K3 and K5 are important in regulation of DC-SIGN and DC-SIGNR. Further exploration of this modulation revealed that DC-SIGN is endocytosed from the cell surface in THP-1 monocytes, but degraded from an internal location with minimal endocytosis in HEK-293 cells. Pull-down data indicate that both K3 and K5 preferentially associate with immature forms of the lectins, mediating their ubiquitylation and degradation. Together, these data emphasize the molecular complexities of K3 and K5, while expanding the repertoire of targets of these two viral proteins. PMID:23460925

  20. Generation of high-titre virus stocks using BrK.219, a B-cell line infected stably with recombinant Kaposi's sarcoma-associated herpesvirus.

    Science.gov (United States)

    Kati, Semra; Hage, Elias; Mynarek, Martin; Ganzenmueller, Tina; Indenbirken, Daniela; Grundhoff, Adam; Schulz, Thomas F

    2015-06-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a gamma-2-lymphotropic human oncogenic herpesvirus associated with Kaposi's sarcoma (KS) and two B-cell lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). KSHV establishes latency soon after infection in vivo and in vitro. Consequently, it is technically difficult to generate high-titre virus stocks required for infection experiments in tissue culture. Currently used methods of KSHV stock production involve induction of the lytic/productive cycle in PEL cell lines or in adherent cell lines harbouring recombinant KSHV genomes. In this study, the BJAB-derived B-cell line BrK.219, which is infected latently with a recombinant KSHV (rKSHV.219), is used to produce high-titre virus stocks. BrK.219 cells enter the lytic KSHV replication cycle upon cross-linking of B-cell receptors (BCRs) with anti-IgM antibodies without the need for additional, potentially toxic chemical inducers. High cell concentrations can be cultured and induced easily in spinner flasks, saving time and resources. The established protocol allows the generation of KSHV virus stocks with titres of up to 10(6) IU/ml in unconcentrated culture supernatants, representing a 10(3)-10(4)-fold improvement compared to conventional methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Herpes simplex virus type 2 triggers reactivation of Kaposi's sarcoma-associated herpesvirus from latency and collaborates with HIV-1 Tat.

    Science.gov (United States)

    Tang, Qiao; Qin, Di; Lv, Zhigang; Zhu, Xiaolei; Ma, Xinting; Yan, Qin; Zeng, Yi; Guo, Yuanyuan; Feng, Ninghan; Lu, Chun

    2012-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) infection was necessary but not sufficient for Kaposi's sarcoma (KS) development without other cofactors. Previously, we identified that both human immunodeficiency type 1 (HIV-1) Tat and herpes simplex virus 1 (HSV-1) were important cofactors reactivating KSHV from latency. Here, we further investigated the potential of herpes simplex virus 2 (HSV-2) to influence KSHV replication and examined the role of Tat in this procedure. We demonstrated that HSV-2 was a potentially important factor in the pathogenesis of KS, as determined by production of lytic phase mRNA transcripts, viral proteins and infectious viral particles in BCBL-1 cells. These results were further confirmed by an RNA interference experiment using small interfering RNA targeting KSHV Rta and a luciferase reporter assay testing Rta promoter-driven luciferase activity. Mechanistic studies showed that HSV-2 infection activated nuclear factor-kappa B (NF-κB) signaling pathway. Inhibition of NF-κB pathway enhanced HSV-2-mediated KSHV activation, whereas activation of NF-κB pathway suppressed KSHV replication in HSV-2-infected BCBL-1 cells. Additionally, ectopic expression of Tat enhanced HSV-2-induced KSHV replication. These novel findings suggest a role of HSV-2 in the pathogenesis of KS and provide the first laboratory evidence that Tat may participate HSV-2-mediated KSHV activation, implying the complicated pathogenesis of acquired immunodeficiency syndrome (AIDS)-related KS (AIDS-KS) patients.

  2. Herpes simplex virus type 2 triggers reactivation of Kaposi's sarcoma-associated herpesvirus from latency and collaborates with HIV-1 Tat.

    Directory of Open Access Journals (Sweden)

    Qiao Tang

    Full Text Available Kaposi's sarcoma-associated herpesvirus (KSHV infection was necessary but not sufficient for Kaposi's sarcoma (KS development without other cofactors. Previously, we identified that both human immunodeficiency type 1 (HIV-1 Tat and herpes simplex virus 1 (HSV-1 were important cofactors reactivating KSHV from latency. Here, we further investigated the potential of herpes simplex virus 2 (HSV-2 to influence KSHV replication and examined the role of Tat in this procedure. We demonstrated that HSV-2 was a potentially important factor in the pathogenesis of KS, as determined by production of lytic phase mRNA transcripts, viral proteins and infectious viral particles in BCBL-1 cells. These results were further confirmed by an RNA interference experiment using small interfering RNA targeting KSHV Rta and a luciferase reporter assay testing Rta promoter-driven luciferase activity. Mechanistic studies showed that HSV-2 infection activated nuclear factor-kappa B (NF-κB signaling pathway. Inhibition of NF-κB pathway enhanced HSV-2-mediated KSHV activation, whereas activation of NF-κB pathway suppressed KSHV replication in HSV-2-infected BCBL-1 cells. Additionally, ectopic expression of Tat enhanced HSV-2-induced KSHV replication. These novel findings suggest a role of HSV-2 in the pathogenesis of KS and provide the first laboratory evidence that Tat may participate HSV-2-mediated KSHV activation, implying the complicated pathogenesis of acquired immunodeficiency syndrome (AIDS-related KS (AIDS-KS patients.

  3. Phosphorylation of Kaposi's Sarcoma-Associated Herpesvirus Processivity Factor ORF59 by a Viral Kinase Modulates Its Ability To Associate with RTA and oriLyt

    Science.gov (United States)

    McDowell, Maria E.; Purushothaman, Pravinkumar; Rossetto, Cyprian C.; Pari, Gregory S.

    2013-01-01

    ORF59 of Kaposi's sarcoma-associated herpesvirus (KSHV) plays an essential role in viral lytic replication by providing DNA processivity activity to the viral DNA polymerase (ORF9). ORF59 forms a homodimer in the cytoplasm and binds and translocates ORF9 into the nucleus, where it secures ORF9 to the origin of lytic DNA replication (oriLyt) in order to synthesize long DNA fragments during replication. ORF59 binds to oriLyt through an immediate early protein, replication and transcription activator (RTA). Here, we show that viral kinase (ORF36) phosphorylates serines between amino acids 376 and 379 of ORF59 and replacement of the Ser378 residue with alanine significantly impairs phosphorylation. Although mutating these serine residues had no effect on binding between ORF59 and ORF9, viral polymerase, or ORF36, the viral kinase, it significantly reduced the ability of ORF59 to bind to RTA. The results for the mutant in which Ser376 to Ser379 were replaced by alanine showed that both Ser378 and Ser379 contribute to binding to RTA. Additionally, the Ser376, Ser378, and Ser379 residues were found to be critical for binding of ORF59 to oriLyt and its processivity function. Ablation of these phosphorylation sites reduced the production of virion particles, suggesting that phosphorylation is critical for ORF59 activity and viral DNA synthesis. PMID:23678174

  4. Human Mesenchymal Stem Cells of Diverse Origins Support Persistent Infection with Kaposi’s Sarcoma-Associated Herpesvirus and Manifest Distinct Angiogenic, Invasive, and Transforming Phenotypes

    Directory of Open Access Journals (Sweden)

    Myung-Shin Lee

    2016-01-01

    Full Text Available Kaposi’s sarcoma (KS, a highly angiogenic and invasive tumor often involving different organ sites, including the oral cavity, is caused by infection with Kaposi’s sarcoma-associated herpesvirus (KSHV. Diverse cell markers have been identified on KS tumor cells, but their origin remains an enigma. We previously showed that KSHV could efficiently infect, transform, and reprogram rat primary mesenchymal stem cells (MSCs into KS-like tumor cells. In this study, we showed that human primary MSCs derived from diverse organs, including bone marrow (MSCbm, adipose tissue (MSCa, dental pulp, gingiva tissue (GMSC, and exfoliated deciduous teeth, were permissive to KSHV infection. We successfully established long-term cultures of KSHV-infected MSCa, MSCbm, and GMSC (LTC-KMSCs. While LTC-KMSCs had lower proliferation rates than the uninfected cells, they expressed mixtures of KS markers and displayed differential angiogenic, invasive, and transforming phenotypes. Genetic analysis identified KSHV-derived microRNAs that mediated KSHV-induced angiogenic activity by activating the AKT pathway. These results indicated that human MSCs could be the KSHV target cells in vivo and established valid models for delineating the mechanism of KSHV infection, replication, and malignant transformation in biologically relevant cell types.

  5. Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs Target GADD45B To Protect Infected Cells from Cell Cycle Arrest and Apoptosis.

    Science.gov (United States)

    Liu, Xiaoyan; Happel, Christine; Ziegelbauer, Joseph M

    2017-02-01

    Kaposi's sarcoma is one of the most common malignancies in HIV-infected individuals. The responsible agent, Kaposi's sarcoma-associated herpesvirus (KSHV; HHV8), expresses multiple microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. After infection in primary endothelial cells with KSHV, growth arrest DNA damage-inducible gene 45 beta (GADD45B) is one of the most repressed genes using genomic expression profiling. GADD45B was also repressed in mRNA expression profiling experiments when KSHV miRNAs were introduced to uninfected cells. We hypothesized that KSHV miRNAs target human GADD45B to protect cells from consequences of DNA damage, which can be triggered by viral infection. Expression of GADD45B protein is induced by the p53 activator, Nutlin-3, and KSHV miRNA-K9 inhibits this induction. In addition, Nutlin-3 increased apoptosis and cell cycle arrest based on flow cytometry assays. KSHV miR-K9 protected primary endothelial cells from apoptosis and cell cycle arrest following Nutlin-3 treatment. Similar protective phenotypes were seen for targeting GADD45B with short interfering RNAs (siRNAs), as with miR-K9. KSHV miR-K9 also decreased the protein levels of cleaved caspase-3, cleaved caspase-7, and cleaved poly(ADP-ribose) polymerase (PARP). In B lymphocytes latently infected with KSHV, specific inhibitors of KSHV miR-K9 led to increased GADD45B expression and apoptosis, indicating that miR-K9 is important for reducing apoptosis in infected cells. Furthermore, ectopic expression of GADD45B in KSHV-infected cells promoted apoptosis. Together, these results identify a new miRNA target and demonstrate that KSHV miRNAs are important for protecting infected cells from DNA damage responses. Kaposi's sarcoma-associated herpesvirus is a leading cause of cancers in individuals with AIDS. Promoting survival of infected cells is essential for maintaining viral infections. A virus needs to combat various cellular defense

  6. p130Cas scaffolds the signalosome to direct adaptor-effector cross talk during Kaposi's sarcoma-associated herpesvirus trafficking in human microvascular dermal endothelial cells.

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    Bandyopadhyay, Chirosree; Veettil, Mohanan Valiya; Dutta, Sujoy; Chandran, Bala

    2014-12-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface receptors, such as heparan sulfate, integrins (α3β1, αVβ3, and αVβ5), and EphrinA2 (EphA2), and activates focal adhesion kinase (FAK), Src, phosphoinositol 3-kinase (PI3-K), c-Cbl, and RhoA GTPase signal molecules early during lipid raft (LR)-dependent productive macropinocytic entry into human dermal microvascular endothelial cells. Our recent studies have identified CIB1 as a signal amplifier facilitating EphA2 phosphorylation and subsequent cytoskeletal cross talk during KSHV macropinocytosis. Although CIB1 lacks an enzymatic activity and traditional adaptor domain or known interacting sequence, it associated with the KSHV entry signal complex and the CIB1-KSHV association was sustained over 30 min postinfection. To identify factors scaffolding the EphA2-CIB1 signal axis, the role of major cellular scaffold protein p130Cas (Crk-associated substrate of Src) was investigated. Inhibitor and small interfering RNA (siRNA) studies demonstrated that KSHV induced p130Cas in an EphA2-, CIB1-, and Src-dependent manner. p130Cas and Crk were associated with KSHV, LRs, EphA2, and CIB1 early during infection. Live-cell microscopy and biochemical studies demonstrated that p130Cas knockdown did not affect KSHV entry but significantly reduced productive nuclear trafficking of viral DNA and routed KSHV to lysosomal degradation. p130Cas aided in scaffolding adaptor Crk to downstream guanine nucleotide exchange factor phospho-C3G possibly to coordinate GTPase signaling during KSHV trafficking. Collectively, these studies demonstrate that p130Cas acts as a bridging molecule between the KSHV-induced entry signal complex and the downstream trafficking signalosome in endothelial cells and suggest that simultaneous targeting of KSHV entry receptors with p130Cas would be an attractive potential avenue for therapeutic intervention in KSHV infection. Eukaryotic cell adaptor molecules, without any intrinsic

  7. Piracy of prostaglandin E2/EP receptor-mediated signaling by Kaposi's sarcoma-associated herpes virus (HHV-8) for latency gene expression: strategy of a successful pathogen.

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    George Paul, Arun; Sharma-Walia, Neelam; Kerur, Nagaraj; White, Carl; Chandran, Bala

    2010-05-01

    Kaposi's sarcoma-associated herpes virus (KSHV) is implicated in the pathogenesis of KS, a chronic inflammation-associated malignancy. Cyclooxygenase-2 (COX-2) and its metabolite prostaglandin E2 (PGE2), two pivotal proinflammatory/oncogeneic molecules, are proposed to play roles in the expression of major KSHV latency-associated nuclear antigen-1 (LANA-1). Microsomal PGE2 synthase, PGE2, and its receptors (EP1, EP2, EP3, and EP4) were detected in KS lesions with the distinct staining of EP2/EP4 in KS lesions. In latently infected endothelial TIVE-LTC cells, EP receptor antagonists downregulated LANA-1 expression as well as Ca(2+), p-Src, p-PI3K, p-PKCzeta/lambda, and p-NF-kappaB, which are also some of the signal molecules proposed to be important in KS pathogenesis. Exogenous PGE2 and EP receptor agonists induced the LANA-1 promoter in 293 cells, and YY1, Sp1, Oct-1, Oct-6, C/EBP, and c-Jun transcription factors seem to be involved in this induction. PGE2/EP receptor-induced LANA-1 promoter activity was downregulated significantly by the inhibition of Ca(2+), p-Src, p-PI3K, p-PKCzeta/lambda, and p-NF-kappaB. These findings implicate the inflammatory PGE2/EP receptors and the associated signal molecules in herpes virus latency and uncover a novel paradigm that shows the evolution of KSHV genome plasticity to use inflammatory response for its survival advantage of maintaining latent gene expression. These data also suggest that potential use of anti-COX-2 and anti-EP receptor therapy may not only ameliorate the chronic inflammation associated with KS but could also lead to elimination of the KSHV latent infection and the associated KS lesions. (c)2010 AACR.

  8. CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV lytic replication by modulating viral gene transcription.

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    Da-Jiang Li

    2014-01-01

    Full Text Available Kaposi's sarcoma-associated herpesvirus (KSHV is a human herpesvirus that causes Kaposi's sarcoma and is associated with the development of lymphoproliferative diseases. KSHV reactivation from latency and virion production is dependent on efficient transcription of over eighty lytic cycle genes and viral DNA replication. CTCF and cohesin, cellular proteins that cooperatively regulate gene expression and mediate long-range DNA interactions, have been shown to bind at specific sites in herpesvirus genomes. CTCF and cohesin regulate KSHV gene expression during latency and may also control lytic reactivation, although their role in lytic gene expression remains incompletely characterized. Here, we analyze the dynamic changes in CTCF and cohesin binding that occur during the process of KSHV viral reactivation and virion production by high resolution chromatin immunoprecipitation and deep sequencing (ChIP-Seq and show that both proteins dissociate from viral genomes in kinetically and spatially distinct patterns. By utilizing siRNAs to specifically deplete CTCF and Rad21, a cohesin component, we demonstrate that both proteins are potent restriction factors for KSHV replication, with cohesin knockdown leading to hundred-fold increases in viral yield. High-throughput RNA sequencing was used to characterize the transcriptional effects of CTCF and cohesin depletion, and demonstrated that both proteins have complex and global effects on KSHV lytic transcription. Specifically, both proteins act as positive factors for viral transcription initially but subsequently inhibit KSHV lytic transcription, such that their net effect is to limit KSHV RNA accumulation. Cohesin is a more potent inhibitor of KSHV transcription than CTCF but both proteins are also required for efficient transcription of a subset of KSHV genes. These data reveal novel effects of CTCF and cohesin on transcription from a relatively small genome that resemble their effects on the cellular

  9. Ex-vivo recognition of late-lytic CD8 epitopes specific for Kaposi's sarcoma-associated herpesvirus (KSHV) by HIV/KSHV-coinfected individuals.

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    Robey, Rebecca C; Mletzko, Salvinia; Bower, Mark; Meys, Rhonda; Boffito, Marta; Nelson, Mark; Bunker, Christopher B; Gotch, Frances M

    2011-06-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), the most common cancer in individuals with untreated HIV/AIDS. Host control of KSHV infection and KS oncogenesis by CD8 T cells remains underexplored. Although KSHV CD8 epitopes have been identified, the responses they elicit are weak and little is known about their relative importance. We sought to make a direct comparison of the recognition of a selection of the best-described known epitopes by a cohort of KSHV-seropositive, HIV-co-infected individuals, in order to assess the relative dominance of these epitopes. We further sought to identify novel epitopes from within a candidate immunogenic protein encoded by KSHV ORF28. MHC binding and denaturation assays identified putative novel A*0201-restricted epitopes from within the late-lytic glycoprotein ORF28. Recognition of these candidate epitopes was tested in a cohort of KSHV-seropositive, HIV-1-seropositive, A*0201-positive individuals by ex vivo ELISPOT, and compared with recognition of nine previously described epitopes. One novel late-lytic epitope from ORF28 was recognized by 7.1% of individuals, and was used for further investigation of KSHV-specific T cells using multimer technology. One known late-lytic epitope from the glycoprotein-encoding K8.1 was recognized by 71.4% of individuals, and represented an immunodominant KSHV epitope, but was too hydrophobic for multimer synthesis. This study identifies two KSHV CD8 epitopes derived from late-lytic antigens that are recognized by KSHV-seropositive, HIV co-infected individuals, and will be useful in future immunological studies into the CD8 response against KSHV in similar patient cohorts.

  10. Comparative Study of Kaposi's Sarcoma-Associated Herpesvirus Serological Assays Using Clinically and Serologically Defined Reference Standards and Latent Class Analysis▿

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    Nascimento, Maria Claudia; de Souza, Vanda Akico; Sumita, Laura Masami; Freire, Wilton; Munoz, Fernando; Kim, Joseph; Pannuti, Claudio S.; Mayaud, Philippe

    2007-01-01

    Accurate determination of infection with Kaposi's sarcoma-associated herpesvirus (KSHV) has been hindered by the lack of a “gold standard” for comparison of serological assays used to estimate KSHV prevalence in serosurveys conducted in different settings. We have evaluated the performance of five in-house (developed at University College London [UCL], United Kingdom, and at the virology laboratory of the Instituto de Medicine Tropical [IMT] in Sao Paulo, Brazil) and two commercial (ABI and DIAVIR) serological assays to detect antibodies to latency-associated nuclear antigen (LANA) and to lytic KSHV antigens. We used a variety of serum samples assembled to represent populations likely to be at high, intermediate, and low risk of KSHV infection in Brazil. Composite reference standard panels were prepared based on clinical and serological parameters, against which assay performances were assessed using conventional Bayesian statistics and latent class analysis (LCA). Against the clinical reference standard, in-house immunofluorescence assays to detect anti-LANA antibodies (IFA-LANA) produced at UCL and IMT had similar performances, with sensitivities of 61% (95% confidence interval [CI], 48% to 74%) and 72% (95% CI, 58% to 83%) and specificities of 99% (95% CI, 94% to 100%) and 100% (95% CI, 96% to 100%), respectively, and only the IMT IFA-LANA was included in LCA, together with the IMT IFA-lytic and four enzyme-linked immunosorbent assays (ELISAs). The LCA indicated that the IMT whole-virus ELISA performed best (sensitivity, 87% [95% CI, 81% to 91%]; and specificity, 100% [95% CI, 98% to 100%]), confirming the results obtained with the conventional statistical approach. Commercially available ELISA-based tests yielded the lowest specificities using a spectrum of serum samples. The evaluation of KSHV serological assays is warranted before planning serosurveys in various settings. PMID:17182752

  11. Kaposi's sarcoma-associated herpesvirus noncoding polyadenylated nuclear RNA interacts with virus- and host cell-encoded proteins and suppresses expression of genes involved in immune modulation.

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    Rossetto, Cyprian C; Pari, Gregory S

    2011-12-01

    During lytic infection, Kaposi's sarcoma-associated herpesvirus (KSHV) expresses a polyadenylated nuclear RNA (PAN RNA). This noncoding RNA (ncRNA) is localized to the nucleus and is the most abundant viral RNA during lytic infection; however, to date, the role of PAN RNA in the virus life cycle is unknown. Many examples exist where ncRNAs have a defined key regulatory function controlling gene expression by various mechanisms. Our goal for this study was to identify putative binding partners for PAN RNA in an effort to elucidate a possible function for the transcript in KSHV infection. We employed an in vitro affinity protocol where PAN RNA was used as bait for factors present in BCBL-1 cell nuclear extract to show that PAN RNA interacts with several virus- and host cell-encoded factors, including histones H1 and H2A, mitochondrial and cellular single-stranded binding proteins (SSBPs), and interferon regulatory factor 4 (IRF4). RNA chromatin immunoprecipitation (ChIP) assays confirmed that PAN RNA interacted with these factors in the infected cell environment. A luciferase reporter assay showed that PAN RNA expression interfered with the ability of IRF4/PU.1 to activate the interleukin-4 (IL-4) promoter, strongly suggesting a role for PAN RNA in immune modulation. Since the proteomic screen and functional data suggested a role in immune responses, we investigated if constitutive PAN RNA expression could affect other genes involved in immune responses. PAN RNA expression decreased expression of gamma interferon, interleukin-18, alpha interferon 16, and RNase L. These data strongly suggest that PAN RNA interacts with viral and cellular proteins and can function as an immune modulator.

  12. Interaction of Kaposi's sarcoma-associated herpesvirus ORF59 with oriLyt is dependent on binding with K-Rta.

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    Rossetto, Cyprian C; Susilarini, Ni Ketut; Pari, Gregory S

    2011-04-01

    Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) displays two distinct life stages, latency and lytic reactivation. Progression through the lytic cycle and replication of the viral genome constitute an essential step toward the production of infectious virus and human disease. KSHV K-RTA has been shown to be the major transactivator required for the initiation of lytic reactivation. In the transient-cotransfection replication assay, K-Rta is the only noncore protein required for DNA synthesis. K-Rta was shown to interact with both C/EBPα binding motifs and the R response elements (RRE) within oriLyt. It is postulated that K-Rta acts in part to facilitate the recruitment of replication factors to oriLyt. In order to define the role of K-Rta in the initiation of lytic DNA synthesis, we show an interaction with ORF59, the DNA polymerase processivity factor (PF), one of the eight virally encoded proteins necessary for origin-dependent DNA replication. Using the chromatin immunoprecipitation (ChIP) assay, both K-Rta and ORF59 interact with the RRE and C/EBPα binding motifs within oriLyt in cells harboring the KSHV bacterial artificial chromosome (BAC). A transient-transfection ChIP assay demonstrated that the interaction of ORF59 with oriLyt is dependent on binding with K-Rta and that ORF59 fails to bind to oriLyt in the absence of K-Rta. Also, using the cotransfection replication assay, overexpression of the interaction domain of K-Rta with ORF59 has a dominant negative effect on oriLyt amplification, suggesting that the interaction of K-Rta with ORF59 is essential for DNA synthesis and supporting the hypothesis that K-Rta facilitates the formation of a replication complex at oriLyt.

  13. Ser-634 and Ser-636 of Kaposi's Sarcoma-Associated Herpesvirus RTA are Involved in Transactivation and are Potential Cdk9 Phosphorylation Sites.

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    Tsai, Wan-Hua; Wang, Pei-Wen; Lin, Shu-Yu; Wu, I-Lin; Ko, Ying-Chieh; Chen, Yu-Lian; Li, Mengtao; Lin, Su-Fang

    2012-01-01

    The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV), K-RTA, is a lytic switch protein that moderates the reactivation process of KSHV latency. By mass spectrometric analysis of affinity purified K-RTA, we showed that Thr-513 or Thr-514 was the primary in vivo phosphorylation site. Thr-513 and Thr-514 are proximal to the nuclear localization signal ((527)KKRK(530)) and were previously hypothesized to be target sites of Ser/Thr kinase hKFC. However, substitutions of Thr with Ala at 513 and 514 had no effect on K-RTA subcellular localization or transactivation activity. By contrast, replacement of Ser with Ala at Ser-634 and Ser-636 located in a Ser/Pro-rich region of K-RTA, designated as S634A/S636A, produced a polypeptide with ∼10 kDa shorter in molecular weight and reduced transactivation in a luciferase reporter assay relative to the wild type. In contrast to prediction, the decrease in molecular weight was not due to lack of phosphorylation because the overall Ser and Thr phosphorylation state in K-RTA and S634A/S636A were similar, excluding that Ser-634 or Ser-636 motif served as docking sites for consecutive phosphorylation. Interestingly, S634A/S636A lost ∼30% immuno-reactivity to MPM2, an antibody specific to pSer/pThr-Pro motif, indicating that (634)SPSP(637) motif was in vivo phosphorylated. By in vitro kinase assay, we showed that K-RTA is a substrate of CDK9, a Pro-directed Ser/Thr kinase central to transcriptional regulation. Importantly, the capability of K-RTA in associating with endogenous CDK9 was reduced in S634A/S636A, which suggested that Ser-634 and Ser-636 may be involved in CDK9 recruitment. In agreement, S634A/S636A mutant exhibited ∼25% reduction in KSHV lytic cycle reactivation relative to that by the wild type K-RTA. Taken together, our data propose that Ser-634 and Ser-636 of K-RTA are phosphorylated by host transcriptional kinase CDK9 and such a process contributes to a full

  14. Ser-634 and Ser-636 of Kaposi’s sarcoma-associated herpesvirus RTA are involved in transactivation and are potential CDK9 phosphorylation sites

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    Wan-Hua eTsai

    2012-02-01

    Full Text Available The replication and transcription activator (RTA of Kaposi’s sarcoma-associated herpesvirus (KSHV, K-RTA, is a lytic switch protein that moderates the reactivation process of KSHV latency. By mass spectrometric analysis of affinity-purified K-RTA, we showed that Thr-513 or Thr-514 was the primary in vivo phosphorylation site. Thr-513 and Thr-514 are proximal to the nuclear localization signal (527KKRK530 and were previously hypothesized to be target sites of Ser/Thr kinase hKFC. However, substitutions of Thr with Ala at 513 and 514 had no effect on K-RTA subcellular localization or transactivation activity. By contrast, replacement of Ser with Ala at Ser-634 and Ser-636 located in a Ser/Pro-rich region of K-RTA, designated as S634A/S636A, produced a polypeptide with ∼10 kDa shorter in molecular weight and reduced transactivation in a luciferase reporter assay relative to the wild type. In contrast to prediction, the decrease in molecular weight was not due to lack of phosphorylation because the overall Ser and Thr phosphorylation state in K-RTA and S634A/S636A were similar, excluding that Ser-634 or Ser-636 motif served as docking sites for consecutive phosphorylation. Interestingly, S634A/S636A lost ~30% immuno-reactivity to MPM2, an antibody specific to pSer/pThr-Pro motif, indicating that 634SPSP637 motif was in vivo phosphorylated. By in vitro kinase assay, we showed that K-RTA is a substrate of CDK9, a Pro-directed Ser/Thr kinase central to transcriptional regulation. Importantly, the capability of K-RTA in associating with endogenous CDK9 was reduced in S634A/S636A, which suggested that Ser-634 and Ser-636 may be involved in CDK9 recruitment. In agreement, S634A/S636A mutant exhibited ~30% reduction in KSHV lytic cycle reactivation relative to that by the wild type K-RTA. Taken together, our data propose that Ser-634 and Ser-636 of K-RTA are phosphorylated by host transcriptional kinase CDK9 and such a process contributes to a full

  15. The CD8 and CD4 T-cell response against Kaposi's sarcoma-associated herpesvirus is skewed towards early and late lytic antigens.

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    Rebecca C Robey

    Full Text Available Kaposi's sarcoma-associated herpesvirus (KSHV is causally related to Kaposi's sarcoma (KS, the most common malignancy in untreated individuals with HIV/AIDS. The adaptive T-cell immune response against KSHV has not been fully characterized. To achieve a better understanding of the antigenic repertoire of the CD8 and CD4 T-cell responses against KSHV, we constructed a library of lentiviral expression vectors each coding for one of 31 individual KSHV open reading frames (ORFs. We used these to transduce monocyte-derived dendritic cells (moDCs isolated from 14 KSHV-seropositive (12 HIV-positive and 7 KSHV-seronegative (4 HIV-positive individuals. moDCs were transduced with up to 3 KSHV ORFs simultaneously (ORFs grouped according to their expression during the viral life cycle. Transduced moDCs naturally process the KSHV genes and present the resulting antigens in the context of MHC class I and II. Transduced moDCs were cultured with purified autologous T cells and the CD8 and CD4 T-cell proliferative responses to each KSHV ORF (or group was assessed using a CFSE dye-based assay. Two pools of early lytic KSHV genes ([ORF8/ORF49/ORF61] and [ORF59/ORF65/K4.1] were frequently-recognized targets of both CD8 and CD4 T cells from KSHV seropositive individuals. One pool of late lytic KSHV genes ([ORF28/ORF36/ORF37] was a frequently-recognized CD8 target and another pool of late genes ([ORF33/K1/K8.1] was a frequently-recognized CD4 target. We report that both the CD8 and CD4 T-cell responses against KSHV are skewed towards genes expressed in the early and late phases of the viral lytic cycle, and identify some previously unknown targets of these responses. This knowledge will be important to future immunological investigations into KSHV and may eventually lead to the development of better immunotherapies for KSHV-related diseases.

  16. HITS-CLIP analysis uncovers a link between the Kaposi's sarcoma-associated herpesvirus ORF57 protein and host pre-mRNA metabolism.

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    Emi Sei

    2015-02-01

    Full Text Available The Kaposi's sarcoma associated herpesvirus (KSHV is an oncogenic virus that causes Kaposi's sarcoma, primary effusion lymphoma (PEL, and some forms of multicentric Castleman's disease. The KSHV ORF57 protein is a conserved posttranscriptional regulator of gene expression that is essential for virus replication. ORF57 is multifunctional, but most of its activities are directly linked to its ability to bind RNA. We globally identified virus and host RNAs bound by ORF57 during lytic reactivation in PEL cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP. As expected, ORF57-bound RNA fragments mapped throughout the KSHV genome, including the known ORF57 ligand PAN RNA. In agreement with previously published ChIP results, we observed that ORF57 bound RNAs near the oriLyt regions of the genome. Examination of the host RNA fragments revealed that a subset of the ORF57-bound RNAs was derived from transcript 5' ends. The position of these 5'-bound fragments correlated closely with the 5'-most exon-intron junction of the pre-mRNA. We selected four candidates (BTG1, EGR1, ZFP36, and TNFSF9 and analyzed their pre-mRNA and mRNA levels during lytic phase. Analysis of both steady-state and newly made RNAs revealed that these candidate ORF57-bound pre-mRNAs persisted for longer periods of time throughout infection than control RNAs, consistent with a role for ORF57 in pre-mRNA metabolism. In addition, exogenous expression of ORF57 was sufficient to increase the pre-mRNA levels and, in one case, the mRNA levels of the putative ORF57 targets. These results demonstrate that ORF57 interacts with specific host pre-mRNAs during lytic reactivation and alters their processing, likely by stabilizing pre-mRNAs. These data suggest that ORF57 is involved in modulating host gene expression in addition to KSHV gene expression during lytic reactivation.

  17. Human Immunodeficiency Virus Type 1 Tat Accelerates Kaposi Sarcoma-Associated Herpesvirus Kaposin A-Mediated Tumorigenesis of Transformed Fibroblasts In Vitro as well as in Nude and Immunocompetent Mice

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    Xiuying Chen

    2009-12-01

    Full Text Available Kaposi sarcoma-associated herpesvirus (KSHV is necessary but not sufficient to cause Kaposi sarcoma (KS. Coinfection with human immunodeficiency virus type 1 (HIV-1, in the absence of antiretroviral suppressive therapy, drastically increases the risk of KS. Previously, we identified that HIV-1 transactivative transcription protein (Tat was an important cofactor that activated lytic cycle replication of KSHV. Here, we further investigated the potential of Tat to influence tumorigenesis induced by KSHV Kaposin A, a product of KSHV that was encoded by the open reading frame K12 (a KSHV-transforming gene. By using colony formation in soft agar, 3H-TdR incorporation, cell cycle, and microarray gene expression analyses, we demonstrated that Tat enhanced proliferation as well as mitogen-activated protein kinase, signal transducer and activator of transcription 3, and phosphatidylinositol 3-kinase/protein kinase B signaling induced by Kaposin A in NIH3T3 cells. Animal experiments further demonstrated that Tat accelerated tumorigenesis by Kaposin A in athymic nu/nu mice. Cells obtained from primary tumors of nude mice succeeded inducing tumors in immunocompetent mice. These data suggest that Tat can accelerate tumorigenesis induced by Kaposin A. Our data present the first line of evidence that Tat may participate in KS pathogenesis by collaborating with Kaposin A in acquired immunodeficiency syndrome (AIDS-related KS (AIDS-KS patients. Our data also suggest that the model for Kaposin and Tat-mediated oncogenesis will contribute to our understanding of the pathogenesis of AIDS-KS at the molecular level and may even be important in exploring a novel therapeutic method for AIDS-KS.

  18. Activation of PI3K/AKT and ERK MAPK signal pathways is required for the induction of lytic cycle replication of Kaposi's Sarcoma-associated herpesvirus by herpes simplex virus type 1

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    Lv Zhigang

    2011-10-01

    Full Text Available Abstract Background Kaposi's sarcoma-associated herpesvirus (KSHV is causally linked to several acquired immunodeficiency syndrome-related malignancies, including Kaposi's sarcoma (KS, primary effusion lymphoma (PEL and a subset of multicentric Castleman's disease. Regulation of viral lytic replication is critical to the initiation and progression of KS. Recently, we reported that herpes simplex virus type 1 (HSV-1 was an important cofactor that activated lytic cycle replication of KSHV. Here, we further investigated the possible signal pathways involved in HSV-1-induced reactivation of KSHV. Results By transfecting a series of dominant negative mutants and protein expressing constructs and using pharmacologic inhibitors, we found that either Janus kinase 1 (JAK1/signal transducer and activator of transcription 3 (STAT3 or JAK1/STAT6 signaling failed to regulate HSV-1-induced KSHV replication. However, HSV-1 infection of BCBL-1 cells activated phosphatidylinositol 3-kinase (PI3K/protein kinase B (PKB, also called AKT pathway and inactivated phosphatase and tensin homologue deleted on chromosome ten (PTEN and glycogen synthase kinase-3β (GSK-3β. PTEN/PI3K/AKT/GSK-3β pathway was found to be involved in HSV-1-induced KSHV reactivation. Additionally, extracellular signal-regulated protein kinase (ERK mitogen-activated protein kinase (MAPK pathway also partially contributed to HSV-1-induced KSHV replication. Conclusions HSV-1 infection stimulated PI3K/AKT and ERK MAPK signaling pathways that in turn contributed to KSHV reactivation, which provided further insights into the molecular mechanism controlling KSHV lytic replication, particularly in the context of HSV-1 and KSHV co-infection.

  19. HLA polymorphisms and detection of kaposi sarcoma-associated herpesvirus DNA in saliva and peripheral blood among children and their mothers in the uganda sickle cell anemia KSHV Study

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    Figueiredo Constança

    2010-11-01

    Full Text Available Abstract Kaposi sarcoma-associated herpesvirus (KSHV, also called Human herpesvirus 8 or HHV8 is a γ-2 herpesvirus that causes Kaposi sarcoma. KSHV seroprevalence rates vary geographically with variable rates recorded in different sub Sahara African countries, suggesting that effects of genetic and/or environmental factors may influence the risk of infection. One study conducted in South Africa, where KSHV seroprevalence is relatively low, found that carriage of human leukocyte antigen (HLA alleles HLA-A*6801, HLA-A*30, HLA-A*4301, and HLA-DRB1*04 was associated with increased shedding of KSHV DNA in saliva. Confirmation of those results would strengthen the hypothesis that genetic factors may influence KSHV distribution by modulating KSHV shedding in saliva. To explore these associations in another setting, we used high resolution HLA-A, B, and DRB1 typing on residual samples from the Uganda Sickle Cell Anemia KSHV study, conducted in a high KSHV seroprevalence region, to investigate associations between HLA and KSHV shedding in saliva or peripheral blood among 233 children and their mothers. HLA-A and HLA-DRB1 alleles were not associated with KSHV shedding in our study, but our study was small and was not adequately powered to exclude small associations. In exploratory analyses, we found marginal association of KSHV DNA shedding in saliva but not in peripheral blood among children carrying HLA- B*4415 and marginal association of KSHV DNA shedding in peripheral blood but not in saliva among children carrying HLA- B*0801 alleles. The contribution of individual HLA polymorphisms to KSHV shedding is important but it may vary in different populations. Larger population-based studies are needed to estimate the magnitude and direction of association of HLA with KSHV shedding and viral control.

  20. Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen and Angiogenin Interact with Common Host Proteins, Including Annexin A2, Which Is Essential for Survival of Latently Infected Cells

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    Paudel, Nitika; Sadagopan, Sathish; Balasubramanian, Sandhya

    2012-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) infection and latency-associated nuclear antigen (LANA-1) upregulate the multifunctional protein angiogenin (ANG). Our studies demonstrate that silencing ANG or inhibiting its nuclear translocation downregulates KSHV LANA-1 expression and ANG is necessary for KSHV latency, anti-apoptosis and angiogenesis (Sadagopan et al., J. Virol. 83:3342–3364, 2009; Sadagopan et al., J Virol. 85:2666–2685, 2011). Here we show that LANA-1 interacts with ANG and colocalizes in latently infected endothelial telomerase-immortalized human umbilical vein endothelial (TIVE-LTC) cells. Mass spectrometric analyses of TIVE-LTC proteins immunoprecipitated by anti-LANA-1 and ANG antibodies identified 28 common cellular proteins such as ribosomal proteins, structural proteins, tRNA synthetases, metabolic pathway enzymes, chaperons, transcription factors, antioxidants, and ubiquitin proteosome proteins. LANA-1 and ANG interaction with one of the proteins, annexin A2, was validated. Annexin A2 has been shown to play roles in cell proliferation, apoptosis, plasmin generation, exocytosis, endocytosis, and cytoskeleton reorganization. It is also known to associate with glycolytic enzyme 3-phosphoglyceratekinase in the primer recognition protein (PRP) complex that interacts with DNA polymerase α in the lagging strand of DNA during replication. A higher level of annexin A2 is expressed in KSHV+ but not in Epstein-Barr virus (EBV)+ B-lymphoma cell lines. Annexin A2 colocalized with several LANA-1 punctate spots in KSHV+ body cavity B-cell lymphoma (BCBL-1) cells. In triple-staining analyses, we observed annexin A2-ANG-LANA-1, annexin A2-ANG, and ANG-LANA-1 colocalizations. Annexin A2 appeared as punctate nuclear dots in LANA-1-positive TIVE-LTC cells. In LANA-1-negative TIVE-LTC cells, annexin A2 was detected predominately in the cytoplasm, with some nuclear spots, and colocalization with ANG was observed mostly in the cytoplasm. Annexin A2

  1. Similar activation of signal transduction pathways by the herpesvirus-encoded chemokine receptors US28 and ORF74

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    McLean, Katherine A; Holst, Peter J; Martini, Lene

    2004-01-01

    The virally encoded chemokine receptors US28 from human cytomegalovirus and ORF74 from human herpesvirus 8 are both constitutively active. We show that both receptors constitutively activate the transcription factors nuclear factor of activated T cells (NFAT) and cAMP response element binding...... viral gene expression similarly. As ORF74 is a known inducer of neoplasia, these findings may have important implications for cytomegalovirus-associated pathogenicity....

  2. Gammaherpesviral Tegument Proteins, PML-Nuclear Bodies and the Ubiquitin-Proteasome System

    Directory of Open Access Journals (Sweden)

    Florian Full

    2017-10-01

    Full Text Available Gammaherpesviruses like Epstein-Barr virus (EBV and Kaposi’s sarcoma-associated herpesvirus (KSHV subvert the ubiquitin proteasome system for their own benefit in order to facilitate viral gene expression and replication. In particular, viral tegument proteins that share sequence homology to the formylglycineamide ribonucleotide amidotransferase (FGARAT, or PFAS, an enzyme in the cellular purine biosynthesis, are important for disrupting the intrinsic antiviral response associated with Promyelocytic Leukemia (PML protein-associated nuclear bodies (PML-NBs by proteasome-dependent and independent mechanisms. In addition, all herpesviruses encode for a potent ubiquitin protease that can efficiently remove ubiquitin chains from proteins and thereby interfere with several different cellular pathways. In this review, we discuss mechanisms and functional consequences of virus-induced ubiquitination and deubiquitination for early events in gammaherpesviral infection.

  3. Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells.

    Science.gov (United States)

    Hayakawa, Kazuo; Ikeya, Makoto; Fukuta, Makoto; Woltjen, Knut; Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo; Otsuka, Takanobu; Toguchida, Junya

    2013-03-22

    Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT-SSX. Although precise function of SYT-SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT-SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT-SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT-SSX2 gene. SYT-SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT-SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT-SSX, respectively. Association of these genes with SYT-SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly different in response to the induction of SYT-SSX, and more than half of SYT-SSX target genes in hPSCs were not induced in hMSCs. These results suggest the importance of cellular context for correct understanding of SYT-SSX function, and demonstrated how our new system will help to overcome this issue. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Hayakawa, Kazuo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Ikeya, Makoto [Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Fukuta, Makoto [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Woltjen, Knut [Department of Reprogramming Sciences, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Otsuka, Takanobu [Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Toguchida, Junya, E-mail: togjun@frontier.kyoto-u.ac.jp [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto (Japan)

    2013-03-22

    Highlights: ► We tried to identify targets of synovial sarcoma (SS)-associated SYT–SSX fusion gene. ► We established pluripotent stem cell (PSC) lines with inducible SYT–SSX gene. ► SYT–SSX responsive genes were identified by the induction of SYT–SSX in PSC. ► SS-related genes were selected from database by in silico analyses. ► 51 genes were finally identified among SS-related genes as targets of SYT–SSX in PSC. -- Abstract: Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT–SSX. Although precise function of SYT–SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT–SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT–SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT–SSX2 gene. SYT–SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24 h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24 h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT–SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT–SSX, respectively. Association of these genes with SYT–SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly different in response to the induction of SYT–SSX, and more than half of SYT–SSX target genes in hPSCs were not induced in hMSCs. These results suggest the importance of cellular context for correct understanding of SYT–SSX function, and demonstrated how our new system will help to overcome this issue.

  5. Surgical management of retroperitoneal sarcomas associated with external and intraoperative electron beam radiotherapy.

    Science.gov (United States)

    Bobin, J Y; Al-Lawati, T; Granero, L E; Adham, M; Romestaing, P; Chapet, O; Issac, S; Gerard, J P

    2003-10-01

    To report outcomes of adults with retroperitoneal sarcoma (RS) treated by surgery, external beam radiotherapy (EBRT) and intraoperative electron beam radiotherapy (IORT). From July 1988 to February 2001; 24 patients with primary and recurrent RS were diagnosed and treated. The median dose and energy of IORT delivered was 15 Gy/9meV. EBRT dose varies between 45-50 Gy. There were five primary and 19 recurrent tumours. One primary and five recurrent tumours underwent R0 resection. There were 12 liposarcomas and 19 grade I tumours; 13 patients developed local recurrence and three developed distant metastases.Twenty-two patients received IORT associated with EBRT: 11 developed recurrences. Six patients developed Neurotoxicity (4 grade II and 2 grade III). Disease free survival and overall survival at 5 years was 28 and 56% respectively. EBRT with IORT treatment is a promising technique for local control. Lower recurrence rates are associated with radical (R0) surgical procedures.

  6. Sarcomas associated with hereditary nonpolyposis colorectal cancer: broad anatomical and morphological spectrum

    DEFF Research Database (Denmark)

    Nilbert, Mef; Therkildsen, Christina; Nissen, Anja

    2009-01-01

    were represented with recurrent diagnoses of liposarcoma, leiomyosarcoma, and carcinosarcoma. Tumor tissue from eight cases was available for analysis of mismatch-repair (MMR) status using immunohistochemical staining and analysis of microsatellite instability, which revealed MMR defects in six...

  7. Effective inhibition of Rta expression and lytic replication of Kaposi's sarcoma-associated herpesvirus by human RNase P.

    Science.gov (United States)

    Zhu, Jiaming; Trang, Phong; Kim, Kihoon; Zhou, Tianhong; Deng, Hongyu; Liu, Fenyong

    2004-06-15

    Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a nucleic acid-based gene interference approach to knock-down gene expression. Unlike other strategies, such as antisense oligonucleotides, ribozymes, and RNA interference, the RNase P-based technology is unique because a custom-designed EGS molecule can bind to any complementary mRNA sequence and recruit intracellular RNase P for specific degradation of the target mRNA. In this study, we demonstrate that the RNase P-based strategy is effective in blocking gene expression and growth of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), the causative agent of the leading AIDS-associated neoplasms, such as KS and primary-effusion lymphoma. We constructed 2'-O-methyl-modified EGS molecules that target the mRNA encoding KSHV immediate-early transcription activator Rta, and we administered them directly to human primary-effusion lymphoma cells infected with KSHV. A reduction of 90% in Rta expression and a reduction of approximately 150-fold in viral growth were observed in cells treated with a functional EGS. In contrast, a reduction of EGSs are highly effective in inhibiting KSHV gene expression and growth. Exogenous administration of chemically modified EGSs in inducing RNase P-mediated cleavage represents an approach for inhibiting specific gene expression and for treating human diseases, including KSHV-associated tumors.

  8. The Synovial Sarcoma-Associated SS18-SSX2 Fusion Protein Induces Epigenetic Gene (De)Regulation.

    NARCIS (Netherlands)

    Bruijn, D.R.H. de; Allander, S.V.; Dijk, A.H.A.; Willemse, M.P.; Thijssen, J.; Groningen, J.J.M. van; Meltzer, P.S.; Geurts van Kessel, A.H.M.

    2006-01-01

    Fusion of the SS18 and either one of the SSX genes is a hallmark of human synovial sarcoma. The SS18 and SSX genes encode nuclear proteins that exhibit opposite transcriptional activities. The SS18 protein functions as a transcriptional coactivator and is associated with the SWI/SNF complex, whereas

  9. COX-2/PGE2: molecular ambassadors of Kaposi's sarcoma-associated herpes virus oncoprotein-v-FLIP

    Science.gov (United States)

    Sharma-Walia, N; Patel, K; Chandran, K; Marginean, A; Bottero, V; Kerur, N; Paul, A G

    2012-01-01

    Kaposi's sarcoma herpesvirus (KSHV) latent oncoprotein viral FLICE (FADD-like interferon converting enzyme)-like inhibitory protein (v-FLIP) or K13, a potent activator of NF-κB, has well-established roles in KSHV latency and oncogenesis. KSHV-induced COX-2 represents a novel strategy employed by KSHV to promote latency and inflammation/angiogenesis/invasion. Here, we demonstrate that v-FLIP/K13 promotes tumorigenic effects via the induction of host protein COX-2 and its inflammatory metabolite PGE2 in an NF-κB-dependent manner. In addition to our previous studies demonstrating COX-2/PGE2's role in transcriptional regulation of KSHV latency promoter and latent gene expression, the current study adds to the complexity that though LANA-1 (latency associated nuclear antigen) is utilizing COX-2/PGE2 as critical factors for its transcriptional regulation, it is the v-FLIP/K13 gene in the KSHV latency cluster that maintains continuous COX-2/PGE2 levels in the infected cells. We demonstrate that COX-2 inhibition, via its chemical inhibitors (NS-398 or celecoxib), reduced v-FLIP/K13-mediated NF-κB induction, and extracellular matrix (ECM) interaction-mediated signaling, mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) levels, and subsequently downregulated detachment-induced apoptosis (anoikis) resistance. vFLIP expression mediated the secretion of cytokines, and spindle cell differentiation activated the phosphorylation of p38, RSK, FAK, Src, Akt and Rac1-GTPase. The COX-2 inhibition in v-FLIP/K13-HMVECs reduced inflammation and invasion/metastasis-related genes, along with reduced anchorage-independent colony formation via modulating ‘extrinsic' as well as ‘intrinsic' cell death pathways. COX-2 blockade in v-FLIP/K13-HMVEC cells drastically augmented cell death induced by removal of essential growth/survival factors secreted in the microenvironment. Transformed cells obtained from anchorage-independent colonies of COX-2 inhibitor-treated v-FLIP/K13-HMVEC cells expressed lower levels of endothelial–mesenchymal transition genes such as slug, snail and twist, and higher expression of the tumor-suppressor gene, E-cadherin. Taken together, our study provides strong evidences that FDA-approved COX-2 inhibitors have great potential in blocking tumorigenic events linked to KSHV's oncogenic protein v-FLIP/K13. PMID:23552603

  10. Radiation therapy for Kaposi's sarcoma associated with acquired immunodeficiency syndrome. Tokyo Metropolitan Komagome Hospital experience

    Energy Technology Data Exchange (ETDEWEB)

    Ebara, Takeshi [Municipal Kanbara General Hospital, Fujikawa, Shizuoka (Japan); Karasawa, Katsuyuki; Maebayashi, Katsuya; Kurosaki, Hiromasa; Ishikawa, Hitoshi; Kaizu, Toshihide; Tanaka, Yoshiaki; Akagi, Kumiko; Masuda, Gota

    2000-12-01

    Kaposi's sarcoma is frequently found in association with acquired immunodeficiency syndrome (AIDS). We report on radiotherapy for patients with AIDS-related Kaposi's sarcoma at Tokyo Metropolitan Komagome Hospital. Between April 1991 and May 1997, radiotherapy was given to 11 lesions in eight men with AIDS-related Kaposi's sarcoma to relieve their symptoms. The lesions involved the head and neck region, the legs, and the gastrointestinal tract. Radiotherapy was carried out with 4-MV photon through parallel opposed field or high energy electrons. Total doses ranged from 20 to 38 Gy, with a median of 30 Gy, delivered in 2- to 3-Gy fractions. Four patients were given other treatments prior to the radiotherapy. Acute reaction was evaluated according to the modified acute radiation morbidity scoring criteria of the Radiation Therapy Oncology Group (RTOG). Radiotherapy had relieved the symptoms in all patients at completion of this therapy. Lesions that involved the hard palate and vocal cords had completely disappeared. The lesions that received radiotherapy were controlled without symptoms until the patients died. Patients who had the head and neck region treated exhibited severe acute mucosal reaction (at a dose of 30 Gy, there was grade 2 morbidity by modified RTOG criteria, in two patients, and grade 3 in three patients) although the radiation therapy was completed for these patients. Radiotherapy promises a favorable outcome for symptom relief in AIDS-related Kaposi's sarcoma. (author)

  11. Ago HITS-CLIP expands understanding of Kaposi's sarcoma-associated herpesvirus miRNA function in primary effusion lymphomas.

    Directory of Open Access Journals (Sweden)

    Irina Haecker

    Full Text Available KSHV is the etiological agent of Kaposi's sarcoma (KS, primary effusion lymphoma (PEL, and a subset of multicentricCastleman's disease (MCD. The fact that KSHV-encoded miRNAs are readily detectable in all KSHV-associated tumors suggests a potential role in viral pathogenesis and tumorigenesis. MiRNA-mediated regulation of gene expression is a complex network with each miRNA having many potential targets, and to date only few KSHV miRNA targets have been experimentally determined. A detailed understanding of KSHV miRNA functions requires high-through putribonomics to globally analyze putative miRNA targets in a cell type-specific manner. We performed Ago HITS-CLIP to identify viral and cellular miRNAs and their cognate targets in two latently KSHV-infected PEL cell lines. Ago HITS-CLIP recovered 1170 and 950 cellular KSHV miRNA targets from BCBL-1 and BC-3, respectively. Importantly, enriched clusters contained KSHV miRNA seed matches in the 3'UTRs of numerous well characterized targets, among them THBS1, BACH1, and C/EBPβ. KSHV miRNA targets were strongly enriched for genes involved in multiple pathways central for KSHV biology, such as apoptosis, cell cycle regulation, lymphocyte proliferation, and immune evasion, thus further supporting a role in KSHV pathogenesis and potentially tumorigenesis. A limited number of viral transcripts were also enriched by HITS-CLIP including vIL-6 expressed only in a subset of PEL cells during latency. Interestingly, Ago HITS-CLIP revealed extremely high levels of Ago-associated KSHV miRNAs especially in BC-3 cells where more than 70% of all miRNAs are of viral origin. This suggests that in addition to seed match-specific targeting of cellular genes, KSHV miRNAs may also function by hijacking RISCs, thereby contributing to a global de-repression of cellular gene expression due to the loss of regulation by human miRNAs. In summary, we provide an extensive list of cellular and viral miRNA targets representing an important resource to decipher KSHV miRNA function.

  12. Propranolol Decreases Proliferation of Endothelial Cells Transformed by Kaposi's Sarcoma-Associated Herpesvirus and Induces Lytic Viral Gene Expression

    Science.gov (United States)

    Hanson, Ryan S.; Manion, Rory D.

    2015-01-01

    Kaposi's sarcoma (KS) is common in Africa, but economic constraints hinder successful treatment in most patients. Propranolol, a generic β-adrenergic antagonist, decreased proliferation of KS-associated herpesvirus (KSHV)-infected cells. Downregulation of cyclin A2 and cyclin-dependent kinase 1 (CDK1) recapitulated this phenotype. Additionally, propranolol induced lytic gene expression in association with downregulation of CDK6. Thus, propranolol has diverse effects on KSHV-infected cells, and this generic drug has potential as a therapeutic agent for KS. PMID:26269192

  13. Interaction of c-Cbl with myosin IIA regulates Bleb associated macropinocytosis of Kaposi's sarcoma-associated herpesvirus.

    Science.gov (United States)

    Valiya Veettil, Mohanan; Sadagopan, Sathish; Kerur, Nagaraj; Chakraborty, Sayan; Chandran, Bala

    2010-12-23

    KSHV is etiologically associated with Kaposi's sarcoma (KS), an angioproliferative endothelial cell malignancy. Macropinocytosis is the predominant mode of in vitro entry of KSHV into its natural target cells, human dermal microvascular endothelial (HMVEC-d) cells. Although macropinocytosis is known to be a major route of entry for many viruses, the molecule(s) involved in the recruitment and integration of signaling early during macropinosome formation is less well studied. Here we demonstrate that tyrosine phosphorylation of the adaptor protein c-Cbl is required for KSHV induced membrane blebbing and macropinocytosis. KSHV induced the tyrosine phosphorylation of c-Cbl as early as 1 min post-infection and was recruited to the sites of bleb formation. Infection also led to an increase in the interaction of c-Cbl with PI3-K p85 in a time dependent manner. c-Cbl shRNA decreased the formation of KSHV induced membrane blebs and macropinocytosis as well as virus entry. Immunoprecipitation of c-Cbl followed by mass spectrometry identified the interaction of c-Cbl with a novel molecular partner, non-muscle myosin heavy chain IIA (myosin IIA), in bleb associated macropinocytosis. Phosphorylated c-Cbl colocalized with phospho-myosin light chain II in the interior of blebs of infected cells and this interaction was abolished by c-Cbl shRNA. Studies with the myosin II inhibitor blebbistatin demonstrated that myosin IIA is a biologically significant component of the c-Cbl signaling pathway and c-Cbl plays a new role in the recruitment of myosin IIA to the blebs during KSHV infection. Myosin II associates with actin in KSHV induced blebs and the absence of actin and myosin ubiquitination in c-Cbl ShRNA cells suggested that c-Cbl is also responsible for the ubiquitination of these proteins in the infected cells. This is the first study demonstrating the role of c-Cbl in viral entry as well as macropinocytosis, and provides the evidence that a signaling complex containing c-Cbl and myosin IIA plays a crucial role in blebbing and macropinocytosis during viral infection and suggests that targeting c-Cbl could lead to a block in KSHV infection.

  14. Interaction of c-Cbl with myosin IIA regulates Bleb associated macropinocytosis of Kaposi's sarcoma-associated herpesvirus.

    Directory of Open Access Journals (Sweden)

    Mohanan Valiya Veettil

    2010-12-01

    Full Text Available KSHV is etiologically associated with Kaposi's sarcoma (KS, an angioproliferative endothelial cell malignancy. Macropinocytosis is the predominant mode of in vitro entry of KSHV into its natural target cells, human dermal microvascular endothelial (HMVEC-d cells. Although macropinocytosis is known to be a major route of entry for many viruses, the molecule(s involved in the recruitment and integration of signaling early during macropinosome formation is less well studied. Here we demonstrate that tyrosine phosphorylation of the adaptor protein c-Cbl is required for KSHV induced membrane blebbing and macropinocytosis. KSHV induced the tyrosine phosphorylation of c-Cbl as early as 1 min post-infection and was recruited to the sites of bleb formation. Infection also led to an increase in the interaction of c-Cbl with PI3-K p85 in a time dependent manner. c-Cbl shRNA decreased the formation of KSHV induced membrane blebs and macropinocytosis as well as virus entry. Immunoprecipitation of c-Cbl followed by mass spectrometry identified the interaction of c-Cbl with a novel molecular partner, non-muscle myosin heavy chain IIA (myosin IIA, in bleb associated macropinocytosis. Phosphorylated c-Cbl colocalized with phospho-myosin light chain II in the interior of blebs of infected cells and this interaction was abolished by c-Cbl shRNA. Studies with the myosin II inhibitor blebbistatin demonstrated that myosin IIA is a biologically significant component of the c-Cbl signaling pathway and c-Cbl plays a new role in the recruitment of myosin IIA to the blebs during KSHV infection. Myosin II associates with actin in KSHV induced blebs and the absence of actin and myosin ubiquitination in c-Cbl ShRNA cells suggested that c-Cbl is also responsible for the ubiquitination of these proteins in the infected cells. This is the first study demonstrating the role of c-Cbl in viral entry as well as macropinocytosis, and provides the evidence that a signaling complex containing c-Cbl and myosin IIA plays a crucial role in blebbing and macropinocytosis during viral infection and suggests that targeting c-Cbl could lead to a block in KSHV infection.

  15. Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA.

    Science.gov (United States)

    Liu, Dongcheng; Wang, Yan; Yuan, Yan

    2018-01-10

    KSHV lytic replication and constant primary infection of fresh cells are crucial for viral tumorigenicity. Virus-encoded b-Zip family protein K8 plays an important role in viral DNA replication in both viral reactivation and de novo infection. The mechanism underlying the functional role of K8 in the viral life cycle is elusive. Here we report that K8 is a RNA binding protein, which also associates with many proteins including other RNA binding proteins. Many K8-involved protein-protein interactions are mediated by RNA. Using a crosslinking and immunoprecipitation (CLIP) procedure combined with high-throughput sequencing, RNAs that are associated with K8 in BCBL-1 cells were identified, that include both viral (PAN, T1.4, T0.7 and etc.) and cellular (MALAT-1, MRP, 7SK and etc.) RNAs. An RNA-binding motif in K8 was defined, and mutation of the motif abolished the ability of K8 binding to many noncoding RNAs as well as viral DNA replication during de novo infection, suggesting that the K8 functions in viral replication are carried out through RNA association. The function of K8 and associated T1.4 RNA was investigated in details and results showed that T1.4 mediates the binding of K8 with ori-Lyt DNA. T1.4-K8 complex physically bound to KSHV ori-Lyt DNA and recruited other proteins and cofactors to assemble replication complex. Depletion of T1.4 abolished the DNA replication in primary infection. These findings provide mechanistic insights into the role of K8 in coordination with T1.4 RNA in regulating KSHV DNA replication during de novo infection.ImportanceGenome wide analyses of the mammalian transcriptome revealed that a large proportion of sequence previously annotated as noncoding region are actually transcribed and give rise to stable RNAs. Emergence of a large number of noncoding RNAs suggests that functional RNA-protein complexes exampled by ribosome or spliceosome are not ancient relics of the last riboorganism but would be well adapted for regulatory role in biology. K8 has been puzzled by its unique characteristic such as multiple regulatory roles in gene expression and DNA replication without DNA binding capability. This study revealed the mechanism underlying its regulatory role by demonstrating that K8 is an RNA binding protein that binds to DNA and initiate DNA replication in coordination with a noncoding RNA. It is suggested that many of K8 functions, if not all, are carried out through its associated RNAs. Copyright © 2018 American Society for Microbiology.

  16. Use of immunohistochemical staining and electron microscopy to aid in diagnosis of soft tissue sarcomas associated with the fetlock joint in two horses.

    Science.gov (United States)

    Findley, Judith A; Singer, Ellen R; Milner, Peter I; Leeming, Gail H

    2014-05-01

    Soft tissue sarcomas of the equine distal limb associated with joints, sheaths, or bursae have rarely been reported. Accurate diagnosis of these tumors is challenging in both human beings and veterinary species. Immunohistochemical staining and transmission electron microscopy have been used in human beings to reduce misdiagnosis. The current report describes 2 mature horses presenting with lameness and swelling associated with the dorsal aspect of the metacarpo(tarso)phalangeal joint. In both cases, surgical excision was performed with subsequent histological analysis of the masses to determine the tissue of origin. In both cases, immunohistochemical staining and transmission electron microscopy aided the definitive diagnosis of fibrosarcoma associated with the fetlock joints of 2 horses. © 2014 The Author(s).

  17. Histological and immunohistochemical characteristics of undifferentiated small round cell sarcomas associated with CIC-DUX4 and BCOR-CCNB3 fusion genes.

    Science.gov (United States)

    Yamada, Yuichi; Kuda, Masaaki; Kohashi, Kenichi; Yamamoto, Hidetaka; Takemoto, Junkichi; Ishii, Takeaki; Iura, Kunio; Maekawa, Akira; Bekki, Hirofumi; Ito, Takamichi; Otsuka, Hiroshi; Kuroda, Makoto; Honda, Yumi; Sumiyoshi, Shinji; Inoue, Takeshi; Kinoshita, Naoe; Nishida, Atsushi; Yamashita, Kyoko; Ito, Ichiro; Komune, Shizuo; Taguchi, Tomoaki; Iwamoto, Yukihide; Oda, Yoshinao

    2017-04-01

    CIC-DUX4 and BCOR-CCNB3 fusion-gene-associated small round cell sarcomas account for a proportion of pediatric small round cell sarcomas, but their pathological features have not been sufficiently clarified. We reviewed a large number of soft tissue tumors registered at our institution, retrieved the cases of unclassified tumors with a small round cell component, and subjected them to histopathological, immunohistochemical, and gene profile analysis. We reviewed 164 cases of unclassified tumors with a small round cell component and analyzed them by RT-PCR and FISH. Tumors positive for a specific fusion-gene were also subjected to histopathological and immunohistochemical examinations. We identified 16 cases of BCOR-CCNB3/CIC-associated (CIC-DUX4 or CIC gene rearrangement-positive) sarcomas. These included seven BCOR-CCNB3 sarcomas and nine CIC-associated sarcomas. Heterogeneous elements included a myxoid spindle cell component in three BCOR-CCNB3 sarcomas and an epithelioid cell component in two CIC-associated sarcomas (one CIC-DUX4-positive and one CIC-DUX4-negative sarcomas). Mitotic activity was low in both heterogeneous components. By immunohistochemistry, in seven BCOR-CCNB3 sarcomas expression of EMA was positive in two cases, of p63 in three, of CD56 in six, of TLE1 in seven, of NKX2.2 in two, of CCNB3 in seven, and of BCOR in six cases (one case could not be tested for BCOR). In nine cases of CIC-associated sarcoma, CD56 was expressed in five, alpha-smooth muscle actin in one, ERG in three, and CD99, WT1 and TLE1 each in eight cases. Both sarcoma types showed not only a small round cell component, but also a myxoid/epithelioid component with low mitotic activity.

  18. CIB1 synergizes with EphrinA2 to regulate Kaposi's sarcoma-associated herpesvirus macropinocytic entry in human microvascular dermal endothelial cells.

    Directory of Open Access Journals (Sweden)

    Chirosree Bandyopadhyay

    2014-02-01

    Full Text Available KSHV envelope glycoproteins interact with cell surface heparan sulfate and integrins, and activate FAK, Src, PI3-K, c-Cbl, and Rho-GTPase signal molecules in human microvascular dermal endothelial (HMVEC-d cells. c-Cbl mediates the translocation of virus bound α3β1 and αVβ3 integrins into lipid rafts (LRs, where KSHV interacts and activates EphrinA2 (EphA2. EphA2 associates with c-Cbl-myosin IIA and augmented KSHV-induced Src and PI3-K signals in LRs, leading to bleb formation and macropinocytosis of KSHV. To identify the factor(s coordinating the EphA2-signal complex, the role of CIB1 (calcium and integrin binding protein-1 associated with integrin signaling was analyzed. CIB1 knockdown did not affect KSHV binding to HMVEC-d cells but significantly reduced its entry and gene expression. In contrast, CIB1 overexpression increased KSHV entry in 293 cells. Single virus particle infection and trafficking during HMVEC-d cell entry was examined by utilizing DiI (envelope and BrdU (viral DNA labeled virus. CIB1 was associated with KSHV in membrane blebs and in Rab5 positive macropinocytic vesicles. CIB1 knockdown abrogated virus induced blebs, macropinocytosis and virus association with the Rab5 macropinosome. Infection increased the association of CIB1 with LRs, and CIB1 was associated with EphA2 and KSHV entry associated signal molecules such as Src, PI3-K, and c-Cbl. CIB1 knockdown significantly reduced the infection induced EphA2, Src and Erk1/2 activation. Mass spectrometry revealed the simultaneous association of CIB1 and EphA2 with the actin cytoskeleton modulating myosin IIA and alpha-actinin 4 molecules, and CIB1 knockdown reduced EphA2's association with myosin IIA and alpha-actinin 4. Collectively, these studies revealed for the first time that CIB1 plays a role in virus entry and macropinocytosis, and suggested that KSHV utilizes CIB1 as one of the key molecule(s to coordinate and sustain the EphA2 mediated signaling involved in its entry, and CIB1 is an attractive therapeutic target to block KSHV infection.

  19. Effect of extremely low frequency electromagnetic fields (ELF-EMF) on Kaposi's sarcoma-associated herpes virus in BCBL-1 cells.

    Science.gov (United States)

    Pica, Francesca; Serafino, Annalucia; Divizia, Maurizio; Donia, Domenica; Fraschetti, Marzia; Sinibaldi-Salimei, Paola; Giganti, Maria Gabriella; Volpi, Antonio

    2006-04-01

    Association between extremely low frequency electromagnetic fields (ELF-EMF) and human cancers is controversial, and few studies have been conducted on their influence on oncogenic viruses. We studied the effects of 1 mT, 50 Hz sine waves, applied for 24-72 h, on Kaposi's sarcoma (KS)-associated herpesvirus (KSHV or HHV-8) in BCBL-1, a latently infected primary effusion lymphoma (PEL) cell line. ELF-EMF exposure did not affect the growth and viability of BCBL-1 cells, either stimulated or not with TPA. The total amount of KSHV DNA detected in ELF-EMF exposed cultures not stimulated with TPA did not differ from that of the unexposed controls (P = ns). However, in the presence of TPA stimulation, total KSHV DNA content was found higher in ELF-EMF exposed than in control BCBL-1 cultures (P = .024) at 72 h exposure, but not earlier. Viral DNA increase significantly correlated with increased mean fluorescence intensity/cell for the lytic antigen gp K8.1A/B (P EMF exposure consisted mainly of defective viral particles. (c) 2005 Wiley-Liss, Inc.

  20. Herpesviral G protein-coupled receptors activate NFAT to induce tumor formation via inhibiting the SERCA calcium ATPase.

    Science.gov (United States)

    Zhang, Junjie; He, Shanping; Wang, Yi; Brulois, Kevin; Lan, Ke; Jung, Jae U; Feng, Pinghui

    2015-03-01

    G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT) pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi's sarcoma-associated herpesvirus (kGPCR) and cytomegalovirus (US28) shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA), which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of "constitutive" NFAT activation by viral GPCRs.

  1. Herpesviral G protein-coupled receptors activate NFAT to induce tumor formation via inhibiting the SERCA calcium ATPase.

    Directory of Open Access Journals (Sweden)

    Junjie Zhang

    2015-03-01

    Full Text Available G protein-coupled receptors (GPCRs constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi's sarcoma-associated herpesvirus (kGPCR and cytomegalovirus (US28 shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA, which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of "constitutive" NFAT activation by viral GPCRs.

  2. Dicty_cDB: SFH333 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available Kaposi's sarcoma-associated herpes-lik... 39 2e-06 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated...Kaposi's sarcoma-associated herpesvir... 39 5e-05 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated

  3. Dicty_cDB: AFN381 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available Kaposi's sarcoma-associated herpes... 82 6e-14 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated...Kaposi's sarcoma-associated herpes... 81 7e-14 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated

  4. Piracy of PGE2/EP receptor mediated signaling by Kaposi’s sarcoma associated herpes virus (KSHV/HHV-8) for latency gene expression: Strategy of a successful pathogen

    Science.gov (United States)

    Paul, Arun George; Sharma-Walia, Neelam; Kerur, Nagaraj; White, Carl; Chandran, Bala

    2010-01-01

    KSHV is implicated in the pathogenesis of KS, a chronic inflammation associated malignancy. COX-2 and its metabolite PGE2, two pivotal proinflammatory/oncogeneic molecules, are proposed to play roles in the expression of major KSHV latency associated nuclear antigen-1 (LANA-1). Microsomal prostaglandin E2 synthase (mPGES), PGE2 and its receptors (EP1, EP2, EP3, and EP4) were detected in KS lesions with the distinct staining of EP2/EP4 in KS lesions. In latently infected endothelial TIVE-LTC cells, EP receptor antagonists down-regulated LANA-1 expression as well as Ca2+, p-Src, p-PI3K, p-PKCζ/λ, and p-NF-κB, which are also some of the signal molecules proposed to be important in KS pathogenesis. Exogenous PGE2 and EP receptor agonists induced the LANA-1 promoter in 293 cells, and YY1, Sp1, Oct-1, Oct-6, C/EBP and c-Jun transcription factors appear to be involved in this induction. PGE2/EP receptor induced LANA-1 promoter activity was down-regulated significantly by the inhibition of Ca2+, p-Src, p-PI3K, p-PKCζ/λ, and p-NF-κB. These findings implicate the inflammatory PGE2/EP receptors and the associated signal molecules in herpes virus latency and uncover a novel paradigm that demonstrates the evolution of KSHV genome plasticity to utilize inflammatory response for its survival advantage of maintaining latent gene expression. This data also suggests that potential use of anti-COX-2 and anti-EP receptor therapy may not only ameliorate the chronic inflammation associated with KS but could also lead to elimination of the KSHV latent infection and the associated KS lesions. PMID:20388794

  5. Latency-Associated Nuclear Antigen Encoded by Kaposi's Sarcoma-Associated Herpesvirus Interacts with Tat and Activates the Long Terminal Repeat of Human Immunodeficiency Virus Type 1 in Human Cells

    OpenAIRE

    Hyun, Teresa S.; Subramanian, Chitra; Cotter, Murray A.; Robert A. Thomas; Robertson, Erle S.

    2001-01-01

    The latency-associated nuclear antigen (LANA) is constitutively expressed in cells infected with the Kaposi's sarcoma (KS) herpesvirus (KSHV), also referred to as human herpesvirus 8. KSHV is tightly associated with body cavity-based lymphomas (BCBLs) in immunocompromised patients infected with human immunodeficiency virus (HIV). LANA, encoded by open reading frame 73 of KSHV, is one of a small subset of proteins expressed during latent infection and was shown to be important in tethering the...

  6. Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 K-bZIP modulates latency-associated nuclear protein-mediated suppression of lytic origin-dependent DNA synthesis.

    Science.gov (United States)

    Rossetto, Cyprian; Yamboliev, Irena; Pari, Gregory S

    2009-09-01

    The original cotransfection replication assay identified eight human herpesvirus 8 (HHV8)-encoded proteins required for origin-dependent lytic DNA replication. Previously, we demonstrated that under conditions where K-Rta is overexpressed, a K-bZIP knockout bacmid displayed an aberrant subcellular localization pattern for the latency-associated nuclear protein (LANA). Additionally, these same studies demonstrated that K-bZIP interacts with LANA in the absence of K-Rta and that K-bZIP does not directly participate in, but may facilitate, the initiation of lytic DNA synthesis. We developed a modification of the transient cotransfection replication assay wherein both lytic (oriLyt) and latent (terminal repeat) DNA replication are evaluated simultaneously. We now show that LANA represses origin-dependent lytic DNA replication in a dose dependent manner when added to the cotransfection replication assay. This repression was overcome by increasing amounts of a K-bZIP expression plasmid in the cotransfection mixture or by dominant-negative inhibition of the interaction of LANA with K-bZIP by the overexpression of the K-bZIP-LANA binding domain. Chromatin immunoprecipitation assays show that LANA interacts with oriLyt in the absence of K-bZIP expression, suggesting that suppression of lytic replication by LANA is mediated by direct binding. The interaction of K-bZIP with oriLyt was dependent upon the expression of LANA; however, LANA interacted with oriLyt independently of K-bZIP expression. These data suggest that the interaction of LANA with K-bZIP modulates lytic and latent replication and that K-bZIP facilitates lytic DNA replication and modulates the switch from the latent phase of the virus.

  7. Dicty_cDB: SFD486 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 2e-07 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 54 4e-06 BT036468_1( BT036468...Kaposi's sarcoma-associated herpes-lik... 51 3e-05 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated...51 3e-05 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 50 4e-05 CP001071_1748(

  8. Dicty_cDB: SFL145 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 54 2e-06 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 54 2e-06 AF360120_1( AF360120...Kaposi's sarcoma-associated herpes... 54 3e-06 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated...52 1e-05 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 52 1e-05 CP001454_12(

  9. Dicty_cDB: SFJ367 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available Kaposi's sarcoma-associated herpes... 45 0.007 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated...44 0.009 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 44 0.012 AF148805_82(...43 0.026 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 41 0.077 AF360120_1( AF360120

  10. Dicty_cDB: SFC806 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available e-143 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 52 4e-05 AF148805_82( AF148805...Kaposi's sarcoma-associated herpes... 50 2e-04 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated...48 6e-04 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 48 7e-04 AF360120_1( AF360120

  11. Dicty_cDB: SFB257 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available Kaposi's sarcoma-associated herpes... 49 4e-04 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated...45 0.005 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 44 0.008 AL844506_105(...43 0.022 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 42 0.029 AF360120_1( AF360120

  12. Dicty_cDB: SSJ719 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available Value AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 50 1e-04 AF148805_82( AF148805...49 2e-04 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 49 2e-04 AF360120_1( AF360120...Kaposi's sarcoma-associated herpes... 48 4e-04 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated

  13. Dicty_cDB: CHC546 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available e-103 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 2e-04 CR940347_653( CR940347...Kaposi's sarcoma-associated herpes-lik... 45 4e-04 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated...5e-04 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 45 5e-04 (Q54VY3) RecName:

  14. Dicty_cDB: SHH340 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available Kaposi's sarcoma-associated herpes... 65 5e-09 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated...9e-08 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 60 1e-07 ( O76329 ) RecName:

  15. Dicty_cDB: CHQ337 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available Kaposi's sarcoma-associated herpes... 44 0.005 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated...0.008 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 44 0.008 AL034558_21( AL034558

  16. Dicty_cDB: SFJ814 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 3e-05 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 48 3e-04 CR855913_1( CR855913...Kaposi's sarcoma-associated herpes-lik... 47 5e-04 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated

  17. Dicty_cDB: SFH310 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 0.002 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 46 0.003 BT036468_1( BT036468...Kaposi's sarcoma-associated herpes-lik... 44 0.006 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated

  18. Dicty_cDB: SFF501 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available Kaposi's sarcoma-associated herpes... 45 0.006 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated...44 0.010 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 44 0.014 CR855913_1( CR855913

  19. Genetically Engineered Human Islets Protected From CD8-mediated Autoimmune Destruction In Vivo

    NARCIS (Netherlands)

    Zaldumbide, Arnaud; Alkemade, Gonnie; Carlotti, Francoise; Nikolic, Tatjana; Abreu, Joana R. F.; Engelse, Marten A.; Skowera, Anja; de Koning, Eelco J.; Peakman, Mark; Roep, Bart O.; Hoeben, Rob C.; Wiertz, Emmanuel J. H. J.

    Islet transplantation is a promising therapy for type 1 diabetes, but graft function and survival are compromised by recurrent islet autoimmunity. Immunoprotection of islets will be required to improve clinical outcome. We engineered human beta cells to express herpesvirus-encoded immune-evasion

  20. Dicty_cDB: AFM384 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 0.001 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 0.002 BT036468_1( BT036468...0.002 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 45 0.004 AF360120_1( AF360120...45 0.006 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 45 0.007 protein update

  1. Dicty_cDB: AFG142 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 5e-04 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 9e-04 BT036468_1( BT036468...0.001 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 45 0.002 AF360120_1( AF360120...45 0.002 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 45 0.003 protein update

  2. Dicty_cDB: CHC596 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 2e-04 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 45 0.002 (Q54VY3) RecName:...0.003 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 44 0.004 AF360120_1( AF360120...44 0.007 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 44 0.007 protein update

  3. Dicty_cDB: CFG652 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 4e-04 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 7e-04 BT036468_1( BT036468...0.001 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 45 0.002 AF360120_1( AF360120...45 0.002 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 45 0.003 protein update

  4. Dicty_cDB: CHC215 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 4e-04 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 7e-04 BT036468_1( BT036468...0.001 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 45 0.002 AF360120_1( AF360120...45 0.002 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 45 0.003 protein update

  5. Dicty_cDB: CFE142 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 2e-04 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 3e-04 BT036468_1( BT036468...4e-04 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 45 7e-04 AF360120_1( AF360120...45 9e-04 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 45 0.001 protein update

  6. Dicty_cDB: CFG751 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 4e-04 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 7e-04 BT036468_1( BT036468...9e-04 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 45 0.001 AF360120_1( AF360120...45 0.002 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 45 0.002 protein update

  7. Dicty_cDB: SLI306 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 1e-08 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 62 2e-08 AF148805_82( AF148805...62 2e-08 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 60 5e-08 ( O76329 ) RecName:...3e-07 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 57 4e-07 A37102( A37102 ;

  8. Dicty_cDB: CFF674 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 67 7e-10 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 67 1e-09 CP000123_804(...2e-09 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 65 2e-09 AF148805_82( AF148805...3e-08 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 62 3e-08 protein update 2009

  9. Dicty_cDB: AFK694 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 4e-04 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 7e-04 BT036468_1( BT036468...9e-04 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 45 0.002 AF360120_1( AF360120...45 0.002 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 45 0.003 protein update

  10. Dicty_cDB: AFK459 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available e-155 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 45 0.004 (Q54VY3) RecName:...0.007 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 44 0.011 AF148805_82( AF148805...44 0.015 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 43 0.019 (Q23MT7) RecName:...42 0.056 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 40 0.13 protein update

  11. Dicty_cDB: CFH714 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 6e-87 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 54 2e-05 AF148805_82( AF148805...51 8e-05 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 50 1e-04 CR940347_653(...0.001 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 47 0.001 BT036468_1( BT036468...46 0.002 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 45 0.006 protein update

  12. Disease: H00168 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available v K, Dooley CM, Ostergaard E, Kelsh RN, Hansen L, Levesque MP, Vilhelmsen K, Mollgard K, Stemple DL, Rosen...berg T ... TITLE ... Mutations in c10orf11, a melanocyte-differentiation gene, cause

  13. Dicty_cDB: CHO654 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 7e-04 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 44 0.006 AL844504_88( AL844504...44 0.007 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 44 0.007 AC117070_42(

  14. Dicty_cDB: AFN735 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 4e-04 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 7e-04 BT036468_1( BT036468...9e-04 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 45 0.002 AF360120_1( AF360120

  15. Dicty_cDB: SFE858 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 6e-04 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 0.001 BT036468_1( BT036468...0.001 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 45 0.002 AF360120_1( AF360120

  16. Dicty_cDB: CFG784 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 4e-05 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 50 9e-05 AF360120_1( AF360120...49 2e-04 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 49 2e-04 EU930276_1( EU930276

  17. Dicty_cDB: SFI544 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 4e-04 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 44 0.013 AF045453_1( AF045453...0.022 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 42 0.029 AF360120_1( AF360120

  18. Dicty_cDB: CHL705 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 0.001 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 44 0.010 AL844504_88( AL844504...44 0.013 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 44 0.013 AC117070_42(

  19. Dicty_cDB: SFJ324 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 3e-04 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 6e-04 BT036468_1( BT036468...7e-04 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 45 0.001 AF360120_1( AF360120

  20. Dicty_cDB: CHD226 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 0.001 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 0.001 BT036468_1( BT036468...0.001 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 45 0.002 AF360120_1( AF360120

  1. Dicty_cDB: CHI180 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 0.002 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 44 0.013 AL844504_88( AL844504...44 0.017 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 44 0.017 FM992691_90(

  2. Dicty_cDB: SFK701 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 4e-04 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 7e-04 BT036468_1( BT036468...0.001 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 45 0.002 AF360120_1( AF360120

  3. Dicty_cDB: VHG667 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 44 0.002 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 44 0.002 B59254( B59254...0.003 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 43 0.003 protein update 2009

  4. Dicty_cDB: CFF619 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 6e-04 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 0.001 BT036468_1( BT036468...0.001 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 45 0.002 AF360120_1( AF360120

  5. Dicty_cDB: AFJ569 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 7e-05 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 0.001 BC149000_1( BC149000...43 0.014 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 43 0.018 protein update

  6. Dicty_cDB: CHL754 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 9e-13 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 75 1e-12 (Q54VY3) RecName:...4e-12 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 73 8e-12 AF360120_1( AF360120

  7. Dicty_cDB: AFB739 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 51 8e-05 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 50 1e-04 AF360120_1( AF360120...2e-04 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 50 2e-04 U39650_3( U39650

  8. Dicty_cDB: SLI338 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 0.012 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 42 0.016 EU930308_1( EU930308...42 0.021 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 41 0.036 AY166677_1( AY166677

  9. Dicty_cDB: CHQ169 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 44 0.002 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 42 0.006 AF063866_46(...41 0.011 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 41 0.011 AF360120_1( AF360120

  10. Dicty_cDB: SFK893 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 64 8e-09 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 64 1e-08 AY166677_1( AY166677...4e-08 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 62 5e-08 L04159_1( L04159

  11. Dicty_cDB: AFL401 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 50 2e-04 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 46 0.002 (Q54VY3) RecName:...0.014 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 44 0.014 BT036468_1( BT036468

  12. Dicty_cDB: AFE349 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 1e-04 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 47 0.001 ( O76329 ) RecName:...45 0.003 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 45 0.004 (Q54VY3) RecName:

  13. Dicty_cDB: CHO428 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 7e-04 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 44 0.006 AL844504_88( AL844504...44 0.007 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 44 0.007 AC117070_42(

  14. Dicty_cDB: VHF146 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 62 3e-08 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 61 7e-08 AF360120_1( AF360120...9e-08 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 59 2e-07 (Q86KL1) RecName:...3e-07 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 58 4e-07 BC091989_1( BC091989

  15. Dicty_cDB: AFN863 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 46 0.003 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 44 0.010 BC149000_1( BC149000...0.029 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 42 0.038 ( O76329 ) RecName:...42 0.050 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 42 0.050 AP008210_2195(

  16. Dicty_cDB: SHH312 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 71 8e-11 U93872_83( U93872 |pid:none) Kaposi's sarcoma-associated herpesvir... 69 3e-10 AF360120_1( AF360120...3e-10 AF305694_1( AF305694 |pid:none) Kaposi's sarcoma-associated herpes... 67 9e-10 AC116957_105( AC116957...66 3e-09 U52064_1( U52064 |pid:none) Kaposi's sarcoma-associated herpes-lik... 65 4e-09 L08068_1( L08068...4e-09 AF192756_1( AF192756 |pid:none) Kaposi's sarcoma-associated herpes... 64 8e-09 protein update 2009

  17. Two distinct gamma-2 herpesviruses in African green monkeys: a second gamma-2 herpesvirus lineage among old world primates?

    NARCIS (Netherlands)

    Greensill, J.; Sheldon, J. A.; Renwick, N. M.; Beer, B. E.; Norley, S.; Goudsmit, J.; Schulz, T. F.

    2000-01-01

    Primate gamma-2 herpesviruses (rhadinoviruses) have so far been found in humans (Kaposi's sarcoma-associated herpesvirus [KSHV], also called human herpesvirus 8), macaques (Macaca spp.) (rhesus rhadinovirus [RRV] and retroperitoneal fibromatosis herpesvirus [RFHV]), squirrel monkeys (Saimiri

  18. Characterization of the putative replisome organizer of the lactococcal bacteriophage r1t

    NARCIS (Netherlands)

    Zuniga, M; Franke-Fayard, B; Venema, G; Kok, J; Nauta, A

    2002-01-01

    Analysis of the nucleotide sequence of the genome of the lactococcal bacteriophage r1t showed that it may encode at least two proteins involved in DNA replication. On the basis of its similarity with the G38P protein encoded by the Bacillus subtilis phage SPP1, the product of orf11 (Pro11) is

  19. GenBank blastx search result: AK061938 [KOME

    Lifescience Database Archive (English)

    Full Text Available multiple cutaneous and mucosal) (TIE2, VMCM, TIE-2, VMCM1), the C9orf11 gene for chromosome 9 open reading f...ome 9p21.1-21.3 Contains the 3' end of the TEK gene for tyrosine kinase, endothelial (venous malformations,

  20. Functional properties of Virus-Encoded and Virus-Regulated 7TM Receptors

    DEFF Research Database (Denmark)

    Spiess, Katja; Rosenkilde, Mette Marie

    2014-01-01

    -herpesvirus-encoded BILF1 receptors, the human cytomegalovirus (HCMV)-encoded US28 receptor and the Epstein-Barr virus (EBV)-regulated EBI2 (or GPR183), 2) the tissue tropism and virus-dissemination properties, exemplified by the murine CMV-encoded M33, and 3) the tumorigenic properties, exemplified...... by the human herpesvirus 8 (HHV8)-encoded ORF74, HCMV-US28 and EBV-BILF1. Given the general high “druggability” of 7TM receptors, and the recent progress in the understanding of in particular immune evasive functions of the virus-exploited 7TM receptors, we put a special emphasis on the progress of novel anti...

  1. Identification, Characterization, and Application of the Replicon Region of the Halophilic Temperate Sphaerolipovirus SNJ1

    OpenAIRE

    Wang, Yuchen; Sima, Linshan; Lv, Jie; Huang, Suiyuan; Liu, Ying; Wang, Jiao; Krupovic, Mart; Chen, Xiangdong

    2016-01-01

    The temperate haloarchaeal virus SNJ1 displays lytic and lysogenic life cycles. During the lysogenic cycle, the virus resides in its host, Natrinema sp. strain J7-1, in the form of an extrachromosomal circular plasmid, pHH205. In this study, a 3.9-kb region containing seven predicted genes organized in two operons was identified as the minimal replicon of SNJ1. Only RepA, encoded by open reading frame 11-12 (ORF11-12), was found to be essential for replication, and its expression increased du...

  2. Isolated Kaposi Sarcoma of the Tonsil: A Case Report and Review of the Scientific Literature

    Science.gov (United States)

    Pittore, Barbara; Pelagatti, Carlo Loris; Deiana, Francesco; Ortu, Francesco; Maricosu, Elena; Cossu, Sergio; Sotgiu, Giovanni

    2015-01-01

    Kaposi sarcoma is a tumour caused by human herpes virus 8, also known as Kaposi sarcoma-associated herpes virus. It usually affects the skin and oral mucosa; however, it can also sometimes affect the lungs, the liver, the stomach, the bowel, and lymph nodes. Several body sites may be affected simultaneously. The involvement of the tonsils is rare. We described an isolated localization of Kaposi's sarcoma of the right tonsil in a HIV-positive patient. PMID:25755902

  3. Isolated Kaposi Sarcoma of the Tonsil: A Case Report and Review of the Scientific Literature

    Directory of Open Access Journals (Sweden)

    Barbara Pittore

    2015-01-01

    Full Text Available Kaposi sarcoma is a tumour caused by human herpes virus 8, also known as Kaposi sarcoma-associated herpes virus. It usually affects the skin and oral mucosa; however, it can also sometimes affect the lungs, the liver, the stomach, the bowel, and lymph nodes. Several body sites may be affected simultaneously. The involvement of the tonsils is rare. We described an isolated localization of Kaposi’s sarcoma of the right tonsil in a HIV-positive patient.

  4. Inhibition of the phosphatidylinositol 3-kinase-Akt pathway enhances gamma-2 herpesvirus lytic replication and facilitates reactivation from latency

    OpenAIRE

    Peng, Li; Wu, Ting-Ting; Tchieu, Jason H.; Feng, Jun; Brown, Helen J.; Feng, Jiaying; Li, Xudong; Qi, Jing; Deng, Hongyu; Vivanco, Igor; Mellinghoff, Ingo K.; Jamieson, Christina; Sun, Ren

    2010-01-01

    Cellular signalling pathways are critical in regulating the balance between latency and lytic replication of herpesviruses. Here, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway on replication of two gamma-2 herpesviruses, murine gammaherpesvirus-68 (MHV-68) and human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV). We found that de novo infection of MHV-68 induced PI3K-dependent Akt activation and the lytic replication of MHV-68 was enhan...

  5. Rhadinoviral interferon regulatory factor homologues.

    Science.gov (United States)

    Koch, Sandra; Schulz, Thomas F

    2017-07-26

    Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV8) is a gammaherpesvirus and the etiological agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman disease. The KSHV genome contains genes for a unique group of proteins with homology to cellular interferon regulatory factors, termed viral interferon regulatory factors (vIRFs). This review will give an overview over the oncogenic, antiapoptotic and immunomodulatory characteristics of KSHV and related vIRFs.

  6. Mutations in the C-terminal region affect subcellular localization of crucian carp herpesvirus (CaHV) GPCR.

    Science.gov (United States)

    Wang, Jun; Gui, Lang; Chen, Zong-Yan; Zhang, Qi-Ya

    2016-08-01

    G protein-coupled receptors (GPCRs) are known as seven transmembrane domain receptors and consequently can mediate diverse biological functions via regulation of their subcellular localization. Crucian carp herpesvirus (CaHV) was recently isolated from infected fish with acute gill hemorrhage. CaHV GPCR of 349 amino acids (aa) was identified based on amino acid identity. A series of variants with truncation/deletion/substitution mutation in the C-terminal (aa 315-349) were constructed and expressed in fathead minnow (FHM) cells. The roles of three key C-terminal regions in subcellular localization of CaHV GPCR were determined. Lysine-315 (K-315) directed the aggregation of the protein preferentially at the nuclear side. Predicted N-myristoylation site (GGGWTR, aa 335-340) was responsible for punctate distribution in periplasm or throughout the cytoplasm. Predicted phosphorylation site (SSR, aa 327-329) and GGGWTR together determined the punctate distribution in cytoplasm. Detection of organelles localization by specific markers showed that the protein retaining K-315 colocalized with the Golgi apparatus. These experiments provided first evidence that different mutations of CaHV GPCR C-terminals have different affects on the subcellular localization of fish herpesvirus-encoded GPCRs. The study provided valuable information and new insights into the precise interactions between herpesvirus and fish cells, and could also provide useful targets for antiviral agents in aquaculture.

  7. Proteins of purified Epstein-Barr virus.

    Science.gov (United States)

    Johannsen, Eric; Luftig, Micah; Chase, Michael R; Weicksel, Steve; Cahir-McFarland, Ellen; Illanes, Diego; Sarracino, David; Kieff, Elliott

    2004-11-16

    Mature Epstein-Barr virus (EBV) was purified from the culture medium of infected lymphocytes made functionally conditional for Zta activation of lytic replication by an in-frame fusion with a mutant estrogen receptor. Proteins in purified virus preparations were separated by gradient gel electrophoresis and trypsin-digested; peptides were then analyzed by tandem hydrophobic chromatography, tandem MS sequencing, and MS scans. Potential peptides were matched with EBV and human gene ORFs. Mature EBV was mostly composed of homologues of proteins previously found in a herpes virion. However, EBV homologues to herpes simplex virus capsid-associated or tegument components UL7 (BBRF2), UL14 (BGLF3), and EBV BFRF1 were not significantly detected. Instead, probable tegument components included the EBV and gamma-herpesvirus-encoded BLRF2, BRRF2, BDLF2 and BKRF4 proteins. Actin was also a major tegument protein, and cofilin, tubulin, heat shock protein 90, and heat shock protein 70 were substantial components. EBV envelope glycoprotein gp350 was highly abundant, followed by glycoprotein gH, intact and furin-cleaved gB, gM, gp42, gL, gp78, gp150, and gN. BILF1 (gp64) and proteins associated with latent EBV infection were not detected in virions.

  8. [Large B-cell lymphoma coexisting with Castleman's disease. Report of one case].

    Science.gov (United States)

    Avilés-Salas, Alejandro; Candelaria, Myrna; Sevilla-Lizcano, Diana Brisa; Burgos, Sebastián

    2017-07-01

    We report a 73-year-old female patient with Castleman's disease coexistent with large B cell type non-Hodgkin's lymphoma in a right axillary lymphadenopathy. An excisional biopsy was performed: microscopically, the lymph node revealed the presence of numerous plasma cells and small lymphoid cells characteristic of Castleman's disease. An analysis of another portion of the specimen revealed lymphoid cells with large abnormal nuclei gathered locally that were CDD 79+, CD 38+ and MUM-1+ as well as positive for Kaposi sarcoma-associated herpesvirus and negative for Epstein Barr virus encoded RNA-1 (EBER).

  9. Cloning and Analysis of microRNAs Encoded by the Primate γ-Herpesvirus Rhesus Monkey Rhadinovirus

    OpenAIRE

    Schäfer, Alexandra; Cai, Xuezhong; Bilello, John P.; Desrosiers, Ronald C; Cullen, Bryan R.

    2007-01-01

    Several pathogenic human herpesviruses have recently been shown to express virally encoded microRNAs in infected cells. Although the function of these microRNAs is largely unknown, they are hypothesized to play a role in mediating viral replication by down-regulating cellular mRNAs encoding antiviral factors. Here, we report the cloning and analysis of microRNAs encoded by Rhesus Monkey Rhadinovirus (RRV), an animal virus model for the pathogenic human γ-herpesvirus Kaposi’s Sarcoma-Associate...

  10. Interference with the Autophagic Process as a Viral Strategy to Escape from the Immune Control: Lesson from Gamma Herpesviruses

    Directory of Open Access Journals (Sweden)

    Roberta Santarelli

    2015-01-01

    Full Text Available We summarized the most recent findings on the role of autophagy in antiviral immune response. We described how viruses have developed strategies to subvert the autophagic process. A particular attention has been given to Epstein-Barr and Kaposi’s sarcoma associated Herpesvirus, viruses studied for many years in our laboratory. These two viruses belong to γ-Herpesvirus subfamily and are associated with several human cancers. Besides the effects on the immune response, we have described how autophagy subversion by viruses may also concur to the enhancement of their replication and to viral tumorigenesis.

  11. Prostate Mass as a First Manifestation of Myeloid Sarcoma in a Patient With an Occult Acute Myeloid Leukemia-A Case Report.

    Science.gov (United States)

    Horvat, Natally; Sergio Brito Panizza, Pedro; Perego Costa, Frederico; Cesar Cavalcanti Viana, Publio

    2017-09-01

    Myeloid sarcoma (MS) with either primary or secondary prostate involvement is extremely rare. Its diagnostic is particularly challenging when prostate lesion precedes the systemic manifestation of a myeloproliferative disorder. We report such a case in a 44-year-old man with a prostate mass as a first manifestation of myeloid sarcoma associated with acute myeloid leukemia. The diagnosis of lymphoproliferative disorder was suspected in the prostate magnetic resonance imaging, and myeloid sarcoma was confirmed after transrectal ultrasound-guided prostate core biopsy. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Oncolytic virus immunotherapy for melanoma.

    Science.gov (United States)

    Dharmadhikari, Neal; Mehnert, Janice M; Kaufman, Howard L

    2015-03-01

    Melanoma is a type of skin cancer arising from melanocytes and is increasing in incidence. Although complete surgical excision of early stage lesions may be curative, metastatic melanoma continues to be a major therapeutic challenge. Advances in understanding the molecular pathways that promote tumorigenesis and the interactions between melanoma cells and the immune system have resulted in the approval of several newly targeted agents and immunotherapy strategies for the treatment of advanced disease. Oncolytic virus immunotherapy is a new approach that uses native or attenuated live viruses to selectively kill melanoma cells and induce systemic tumor-specific immune responses. A variety of viruses are now in clinical development with the attenuated oncolytic herpesvirus encoding granulocyte-macrophage colony stimulating factor, known as talimogene laherparepvec, recently demonstrating an improvement in durable response rate in patients with advanced melanoma compared with granulocyte-macrophage colony stimulating factor alone. A major advantage of talimogene laherparepvec and related agents is the limited toxicity and ability to use each individual tumor as a source of antigen to generate a highly specific antitumor immune response. These agents are easily administered in the out-patient setting and may be a reasonable option for patients with limited metastatic tumor burden, those with a good performance status and without extensive prior treatment, and in those who cannot tolerate more difficult therapeutic regimens. Further investigation into the impact on overall survival as monotherapy and combination of oncolytic virus immunotherapy with other forms of immunotherapy merit high priority for further clinical application of these novel agents for the treatment of melanoma and perhaps other cancers as well.

  13. Cyclin-dependent kinase-like function is shared by the beta- and gamma- subset of the conserved herpesvirus protein kinases.

    Directory of Open Access Journals (Sweden)

    Chad V Kuny

    2010-09-01

    Full Text Available The UL97 protein of human cytomegalovirus (HCMV, or HHV-5 (human herpesvirus 5, is a kinase that phosphorylates the cellular retinoblastoma (Rb tumor suppressor and lamin A/C proteins that are also substrates of cellular cyclin-dependent kinases (Cdks. A functional complementation assay has further shown that UL97 has authentic Cdk-like activity. The other seven human herpesviruses each encode a kinase with sequence and positional homology to UL97. These UL97-homologous proteins have been termed the conserved herpesvirus protein kinases (CHPKs to distinguish them from other human herpesvirus-encoded kinases. To determine if the Cdk-like activities of UL97 were shared by all of the CHPKs, we individually expressed epitope-tagged alleles of each protein in human Saos-2 cells to test for Rb phosphorylation, human U-2 OS cells to monitor nuclear lamina disruption and lamin A phosphorylation, or S. cerevisiae cdc28-13 mutant cells to directly assay for Cdk function. We found that the ability to phosphorylate Rb and lamin A, and to disrupt the nuclear lamina, was shared by all CHPKs from the beta- and gamma-herpesvirus families, but not by their alpha-herpesvirus homologs. Similarly, all but one of the beta and gamma CHPKs displayed bona fide Cdk activity in S. cerevisiae, while the alpha proteins did not. Thus, we have identified novel virally-encoded Cdk-like kinases, a nomenclature we abbreviate as v-Cdks. Interestingly, we found that other, non-Cdk-related activities reported for UL97 (dispersion of promyelocytic leukemia protein nuclear bodies (PML-NBs and disruption of cytoplasmic or nuclear aggresomes showed weak conservation among the CHPKs that, in general, did not segregate to specific viral families. Therefore, the genomic and evolutionary conservation of these kinases has not been fully maintained at the functional level. Our data indicate that these related kinases, some of which are targets of approved or developmental antiviral drugs

  14. Varicella-Zoster Virus Expresses Multiple Small Noncoding RNAs.

    Science.gov (United States)

    Markus, Amos; Golani, Linoy; Ojha, Nishant Kumar; Borodiansky-Shteinberg, Tatiana; Kinchington, Paul R; Goldstein, Ronald S

    2017-12-15

    Many herpesviruses express small noncoding RNAs (sncRNAs), including microRNAs (miRNAs), that may play roles in regulating lytic and latent infections. None have yet been reported in varicella-zoster virus (VZV; also known as human herpesvirus 3 [HHV-3]). Here we analyzed next-generation sequencing (NGS) data for small RNAs in VZV-infected fibroblasts and human embryonic stem cell-derived (hESC) neurons. Two independent bioinformatics analyses identified more than 20 VZV-encoded 20- to 24-nucleotide RNAs, some of which are predicted to have stem-loop precursors potentially representing miRNAs. These sequences are perfectly conserved between viruses from three clades of VZV. One NGS-identified sequence common to both bioinformatics analyses mapped to the repeat regions of the VZV genome, upstream of the predicted promoter of the immediate early gene open reading frame 63 (ORF63). This miRNA candidate was detected in each of 3 independent biological repetitions of NGS of RNA from fibroblasts and neurons productively infected with VZV using TaqMan quantitative PCR (qPCR). Importantly, transfected synthetic RNA oligonucleotides antagonistic to the miRNA candidate significantly enhanced VZV plaque growth rates. The presence of 6 additional small noncoding RNAs was also verified by TaqMan qPCR in productively infected fibroblasts and ARPE19 cells. Our results show VZV, like other human herpesviruses, encodes several sncRNAs and miRNAs, and some may regulate infection of host cells. IMPORTANCE Varicella-zoster virus is an important human pathogen, with herpes zoster being a major health issue in the aging and immunocompromised populations. Small noncoding RNAs (sncRNAs) are recognized as important actors in modulating gene expression, and this study demonstrates the first reported VZV-encoded sncRNAs. Many are clustered to a small genomic region, as seen in other human herpesviruses. At least one VZV sncRNA was expressed in productive infection of neurons and fibroblasts

  15. Pseudorabies virus infected porcine epithelial cell line generates a diverse set of host microRNAs and a special cluster of viral microRNAs.

    Directory of Open Access Journals (Sweden)

    Yi-Quan Wu

    Full Text Available Pseudorabies virus (PRV belongs to Alphaherpesvirinae subfamily that causes huge economic loss in pig industry worldwide. It has been recently demonstrated that many herpesviruses encode microRNAs (miRNAs, which play crucial roles in viral life cycle. However, the knowledge about PRV-encoded miRNAs is still limited. Here, we report a comprehensive analysis of both viral and host miRNA expression profiles in PRV-infected porcine epithelial cell line (PK-15. Deep sequencing data showed that the ∼4.6 kb intron of the large latency transcript (LLT functions as a primary microRNA precursor (pri-miRNA that encodes a cluster of 11 distinct miRNAs in the PRV genome, and 209 known and 39 novel porcine miRNAs were detected. Viral miRNAs were further confirmed by stem-loop RT-PCR and northern blot analysis. Intriguingly, all of these viral miRNAs exhibited terminal heterogeneity both at the 5' and 3' ends. Seven miRNA genes produced mature miRNAs from both arms and two of the viral miRNA genes showed partially overlapped in their precursor regions. Unexpectedly, a terminal loop-derived small RNA with high abundance and one special miRNA offset RNA (moRNA were processed from a same viral miRNA precursor. The polymorphisms of viral miRNAs shed light on the complexity of host miRNA-processing machinery and viral miRNA-regulatory mechanism. The swine genes and PRV genes were collected for target prediction of the viral miRNAs, revealing a complex network formed by both host and viral genes. GO enrichment analysis of host target genes suggests that PRV miRNAs are involved in complex cellular pathways including cell death, immune system process, metabolic pathway, indicating that these miRNAs play significant roles in virus-cells interaction of PRV and its hosts. Collectively, these data suggest that PRV infected epithelial cell line generates a diverse set of host miRNAs and a special cluster of viral miRNAs, which might facilitate PRV replication in cells.

  16. A New Megastigmane Sesquiterpenoid from Zanthoxylum Schinifolium Sieb. et Zucc

    Directory of Open Access Journals (Sweden)

    Linzhen Hu

    2016-03-01

    Full Text Available Zanthoxylum schinifolium Sieb. et Zucc. (Rutaceae, a dioecious shrub with hooked prickly branches, has been used as folk medicine for the treatment of the common cold, stomach ache, diarrhea, and jaundice in China, Korea, and Japan. In our phytochemical investigations on this genus, a new megastigmane sesquiterpenoid, which is referred to as schinifolenol A (1, was isolated from Z. schinifolium. The stereochemistry was characterized via the analyses of extensive spectra. The absolute configuration was established by the application of a modified Mosher’s experiment and assisted by a time-dependent density functional theory (TD-DFT on calculated electronic circular dichroism (ECD. Bioactivity screenings showed that compound 1 exhibited a safe hypotoxicity and a better selectivity on anti-Kaposi’s sarcoma associated herpes virus (KSHV.

  17. A New Megastigmane Sesquiterpenoid from Zanthoxylum Schinifolium Sieb. et Zucc.

    Science.gov (United States)

    Hu, Linzhen; Wang, Kongchao; Wang, Zhenzhen; Liu, Junjun; Wang, Kaiping; Zhang, Jinwen; Luo, Zengwei; Xue, Yongbo; Zhang, Yu; Zhang, Yonghui

    2016-03-19

    Zanthoxylum schinifolium Sieb. et Zucc. (Rutaceae), a dioecious shrub with hooked prickly branches, has been used as folk medicine for the treatment of the common cold, stomach ache, diarrhea, and jaundice in China, Korea, and Japan. In our phytochemical investigations on this genus, a new megastigmane sesquiterpenoid, which is referred to as schinifolenol A (1), was isolated from Z. schinifolium. The stereochemistry was characterized via the analyses of extensive spectra. The absolute configuration was established by the application of a modified Mosher's experiment and assisted by a time-dependent density functional theory (TD-DFT) on calculated electronic circular dichroism (ECD). Bioactivity screenings showed that compound 1 exhibited a safe hypotoxicity and a better selectivity on anti-Kaposi's sarcoma associated herpes virus (KSHV).

  18. Co-infections and Pathogenesis of KSHV-Associated Malignancies

    Directory of Open Access Journals (Sweden)

    Suhani eThakker

    2016-02-01

    Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV, also known as human herpes virus 8 (HHV-8 is one of the several carcinogenic viruses that infect humans. KSHV infection has been implicated in the development of Kaposi’s sarcoma (KS, primary effusion lymphoma (PEL, and multicentric Castleman’s Disease (MCD. While KSHV infection is necessary for the development of KSHV associated malignancies, it is not sufficient to induce tumoriegenesis. Evidently, other co-factors are essential for the progression of KSHV induced malignancies. One of the most important co-factors, necessary for the progression of KSHV induced tumors, is immune suppression that frequently arises during co-infection with HIV and also by other immune suppressants. In this mini-review, we discuss the roles of co-infection with HIV and other pathogens on KSHV infection and pathogenesis.

  19. Glycosylation of KSHV Encoded vGPCR Functions in Its Signaling and Tumorigenicity

    Directory of Open Access Journals (Sweden)

    Hui Wu

    2015-03-01

    Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV is a tumor virus and the etiologic agent of Kaposi’s Sarcoma (KS. KSHV G protein-coupled receptor (vGPCR is an oncogene that is implicated in malignancies associated with KHSV infection. In this study, we show that vGPCR undergoes extensive N-linked glycosylation within the extracellular domains, specifically asparagines 18, 22, 31 and 202. An immunofluorescence assay demonstrates that N-linked glycosylation are necessary for vGPCR trafficking to the cellular membrane. Employing vGPCR mutants whose glycosylation sites were ablated, we show that these vGPCR mutants failed to activate downstream signaling in cultured cells and were severely impaired to induce tumor formation in the xenograph nude mouse model. These findings support the conclusion that glycosylation is critical for vGPCR tumorigenesis and imply that chemokine regulation at the plasma membrane is crucial for vGPCR mediated signaling.

  20. AKTivation of the PI3K/AKT/mTOR signaling pathway by KSHV

    Directory of Open Access Journals (Sweden)

    Aadra P Bhatt

    2013-01-01

    Full Text Available As an obligate intracellular parasite, the Kaposi sarcoma-associated herpesvirus (KSHV relies on host cell machinery to meet its needs for survival, viral replication, production, and dissemination of progeny virions. KSHV is a ɣ-herpesvirus that is associated with three different malignancies: Kaposi sarcoma (KS, and two B cell lymphoproliferative disorders, primary effusion lymphoma (PEL and multicentric Castleman disease (MCD. KSHV viral proteins modulate cellular phosphatidylinositol-3-kinase (PI3K/AKT/mammalian target of rapamycin (mTOR signaling pathway, which is a ubiquitous pathway that also controls B lymphocyte proliferation and development. We review the mechanisms by which KSHV manipulates the PI3K/AKT/mTOR pathway, with a specific focus on B cells.

  1. Two New Bioactive α-Pyrones from Hypericum japonicum

    Directory of Open Access Journals (Sweden)

    Linzhen Hu

    2016-04-01

    Full Text Available Hypericum japonicum (Guttiferae, a type of annual or perennial herb, has been historically applied to cure infectious hepatitis, acute and chronic hepatitis, gastrointestinal disorder, and internal hemorrhage. In our successive studies on the genus Hypericum, two new α-pyrones termed japopyrones A and B (1 and 2 were isolated from H. japonicum. Their structures and absolute configurations were established by the comprehensive analyses of spectroscopic data, the application of the Single-crystal X-ray diffraction structural analysis, and the experimental electronic circular dichroism (ECD spectra. Bioactivity screenings suggested that compound 2 possessed the potential inhibition efficacy on lytic replication of Kaposi’s sarcoma associated herpesvirus (KSHV with an IC50 29.46 μM and a selective index of higher than 6.79, respectively.

  2. Pre-micro RNA signatures delineate stages of endothelial cell transformation in Kaposi sarcoma.

    Directory of Open Access Journals (Sweden)

    Andrea J O'Hara

    2009-04-01

    Full Text Available MicroRNAs (miRNA have emerged as key regulators of cell lineage differentiation and cancer. We used precursor miRNA profiling by a novel real-time QPCR method (i to define progressive stages of endothelial cell transformation cumulating in Kaposi sarcoma (KS and (ii to identify specific miRNAs that serve as biomarkers for tumor progression. We were able to compare primary patient biopsies to well-established culture and mouse tumor models. Loss of mir-221 and gain of mir-15 expression demarked the transition from merely immortalized to fully tumorigenic endothelial cells. Mir-140 and Kaposi sarcoma-associated herpesvirus viral miRNAs increased linearly with the degree of transformation. Mir-24 emerged as a biomarker specific for KS.

  3. Using Proteomics to Identify Viral microRNA-Regulated Genes | Center for Cancer Research

    Science.gov (United States)

    Kaposi sarcoma is a soft tissue malignancy that affects the skin, the mucous membranes, the lymph nodes and other organs of individuals with compromised immune systems. It is caused by infection with human herpesvirus-8 also known as Kaposi sarcoma-associated herpesvirus or KSHV. The herpesvirus family is unique in that it is the only viral family currently known to express multiple microRNAs (miRNAs); KSHV produces 12 pre-miRNAs, which are processed into at least 25 mature miRNAs. While their functions are not well understood, these miRNAs may be a way for the virus to alter the host immune response without producing proteins that could be recognized and targeted by the immune system. Joseph Ziegelbauer, Ph.D., in CCR’s HIV and AIDS Malignancy Branch, and his colleagues set out to identify human targets of KSHV miRNAs and to understand their functional importance.

  4. The use of monoclonal antibodies to treat Castleman's disease.

    Science.gov (United States)

    Robey, Rebecca C; Mletzko, Salvinia; Colley, Charlotte; Balachandran, Kirsty; Bower, Mark

    2014-01-01

    Multicentric Castleman's disease (MCD) is a rare lymphoproliferative disorder presenting with heterogeneous clinical features and with a complex etiology. MCD incidence is increased in people living with HIV/AIDS when it is causally associated with Kaposi's sarcoma-associated herpes virus (KSHV). HIV-seronegative individuals present with either idiopathic or KSHV-associated MCD. Central to MCD pathology is altered expression and signaling of IL-6, which promotes B-cell proliferation and causes systemic manifestations. KSHV encodes a viral homolog of human IL-6, accounting for its role in MCD, while recent evidence shows an association between IL-6 receptor polymorphisms and idiopathic MCD. The increased understanding of mechanisms underlying the pathogenesis of MCD has guided the use of new monoclonal antibody therapies for treating this complex disorder.

  5. KSHV-encoded viral interferon regulatory factor 4 (vIRF4) interacts with IRF7 and inhibits interferon alpha production.

    Science.gov (United States)

    Hwang, Sung-Woo; Kim, DongIk; Jung, Jae U; Lee, Hye-Ra

    2017-05-06

    Before an infection can be completely established, the host immediately turns on the innate immune system through activating the interferon (IFN)-mediated antiviral pathway. Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes a unique antagonistic mechanism of type I IFN-mediated host antiviral immunity by incorporating four viral interferon regulatory factors (vIRF1-4). Herein, we characterized novel immune evasion strategies of vIRF4 to inhibit the IRF7-mediated IFN-α production. KSHV vIRF4 specifically interacts with IRF7, resulting in inhibition of IRF7 dimerization and ultimately suppresses IRF7-mediated activation of type I IFN. These results suggest that each of the KSHV vIRFs, including vIRF4, subvert IFN-mediated anti-viral response via different mechanisms. Therefore, it is indicated that KSHV vIRFs are indeed a crucial immunomodulatory component of their life cycles. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Non-detection of human herpesvirus 8 (HHV-8) DNA in HHV-8-seropositive blood donors from three Brazilian regions.

    Science.gov (United States)

    Levi, José Eduardo; Nascimento, Maria Claudia; Sumita, Laura Masami; de Souza, Vanda Akico Ueda Fick; Freire, Wilton S; Mayaud, Philippe; Pannuti, Claudio S

    2011-01-01

    Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is the etiologic agent of all forms of Kaposi's sarcoma, primary effusion lymphoma and the plasmablastic cell variant of multicentric Castleman disease. In endemic areas of sub-Saharan Africa, blood transfusions have been associated with a substantial risk of HHV-8 transmission. By contrast, several studies among healthy blood donors from North America have failed to detect HHV-8 DNA in samples of seropositive individuals. In this study, using a real-time PCR assay, we investigated the presence of HHV-8 DNA in whole-blood samples of 803 HHV-8 blood donors from three Brazilian states (São Paulo, Amazon, Bahia) who tested positive for HHV-8 antibodies, in a previous multicenter study. HHV-8 DNA was not detected in any sample. Our findings do not support the introduction of routine HHV-8 screening among healthy blood donors in Brazil. (WC = 140).

  7. KSHV cell attachment sites revealed by ultra sensitive tyramide signal amplification (TSA) localize to membrane microdomains that are up-regulated on mitotic cells.

    Science.gov (United States)

    Garrigues, H Jacques; Rubinchikova, Yelena E; Rose, Timothy M

    2014-03-01

    Cell surface structures initiating attachment of Kaposi's sarcoma-associated herpesvirus (KSHV) were characterized using purified hapten-labeled virions visualized by confocal microscopy with a sensitive fluorescent enhancement using tyramide signal amplification (TSA). KSHV attachment sites were present in specific cellular domains, including actin-based filopodia, lamellipodia, ruffled membranes, microvilli and intercellular junctions. Isolated microdomains were identified on the dorsal surface, which were heterogeneous in size with a variable distribution that depended on cellular confluence and cell cycle stage. KSHV binding domains ranged from scarce on interphase cells to dense and continuous on mitotic cells, and quantitation of bound virus revealed a significant increase on mitotic compared to interphase cells. KSHV also bound to a supranuclear domain that was distinct from microdomains in confluent and interphase cells. These results suggest that rearrangement of the cellular membrane during mitosis induces changes in cell surface receptors implicated in the initial attachment stage of KSHV entry. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Discovery of a Novel Bat Gammaherpesvirus.

    Science.gov (United States)

    Host, Kurtis M; Damania, Blossom

    2016-01-01

    Zoonosis is the leading cause of emerging infectious diseases. In a recent article, R. S. Shabman et al. (mSphere 1[1]:e00070-15, 2016, http://dx.doi.org/10.1128/mSphere.00070-15) report the identification of a novel gammaherpesvirus in a cell line derived from the microbat Myotis velifer incautus. This is the first report on a replicating, infectious gammaherpesvirus from bats. The new virus is named bat gammaherpesvirus 8 (BGHV8), also known as Myotis gammaherpesvirus 8, and is able to infect multiple cell lines, including those of human origin. Using next-generation sequencing technology, the authors constructed a full-length annotated genomic map of BGHV8. Phylogenetic analysis of several genes from BGHV8 revealed similarity to several mammalian gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV).

  9. Susceptibility of KSHV-Infected PEL Cell Lines to the Human Complement System.

    Science.gov (United States)

    Yoo, Seung-Min; Jeon, Hyungtaek; Lee, Suhyuk; Lee, Myung-Shin

    2016-03-01

    Pleural effusion lymphoma (PEL) is a rare B-cell lymphoma that has a very poor prognosis with a median survival time of around 6 months. PEL is caused by Kaposi's sarcoma-associated herpesvirus, and is often co-infected with the Epstein Barr virus. The complement system is fundamental in the innate immune system against pathogen invasion and tumor development. In the present study, we investigated the activation of the complement system in PEL cells using human serum complements. Interestingly, two widely used PEL cell lines, BCP-1 and BCBL-1, showed different susceptibility to the complement system, which may be due to CD46 expression on their cell membranes. Complement activation did not induce apoptosis but supported cell survival considerably. Our results demonstrated the susceptibility of PEL to the complement system and its underlying mechanisms, which would provide insight into understanding the pathogenesis of PEL.

  10. RNA N6-adenosine methylation (m6A) steers epitranscriptomic control of herpesvirus replication.

    Science.gov (United States)

    Ye, Fengchun

    2017-01-01

    Latency is a hallmark of all herpesviruses, during which the viral genomes are silenced through DNA methylation and suppressive histone modifications. When latent herpesviruses reactivate to undergo productive lytic replication, the suppressive epigenetic marks are replaced with active ones to allow for transcription of viral genes. Interestingly, by using Kaposi's sarcoma-associated herpesvirus (KSHV) as a model, we recently demonstrated that the newly transcribed viral RNAs are also subjected to post-transcriptional N6-adenosine methylation (m6A). Blockade of this post-transcriptional event abolishes viral protein expression and halts virion production. We found that m6A modification controls RNA splicing, stability, and protein translation to regulate viral lytic gene expression and replication. Thus, our finding for the first time reveals a critical role of this epitranscriptomic mechanism in the control of herpesviral replication, which shall shed lights on development of novel strategies for the control of herpesviral infection.

  11. [Association of Kaposi sarcoma--multiple myeloma. A new case].

    Science.gov (United States)

    Cohen, J D; Thomas, E; Garnier, N; Hellier, I; Durand, L; Guilhou, J J; Baldet, P; Blotman, F

    2000-11-01

    Kaposi's disease is an angiogenic multifocal cancer process that has several forms, namely Mediterranean, African, HIV-associated, and secondary to a preexisting immunodepressive state (hematological disorder, corticosteroid therapy, immunodepressive treatment). Whatever its form, Kaposi's sarcoma is probably associated with a chronic viral human herpes type 8 infection (HHV8). This virus has been implicated in the pathogenesis of multiple myeloma (17 cases recorded to date). In the present study, a further case of Kaposi's sarcoma associated with multiple myeloma has been reported. However, Epstein-Barr virus, cytomegalovirus, hepatitis B and C, HIV and HHV8 serologies were negative. Radiotherapy on the lower limbs was initiated. It is concluded that HHV8 does not appear to play a pathogenic role in cases of multiple myeloma, given the rarity of the association between Kaposi's sarcoma/multiple myeloma/HHV8.

  12. Lipoxins: nature's way to resolve inflammation

    Directory of Open Access Journals (Sweden)

    Chandrasekharan JA

    2015-09-01

    Full Text Available Jayashree A Chandrasekharan, Neelam Sharma-Walia HM Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USAAbstract: An effective host defense mechanism involves inflammation to eliminate pathogens from the site of infection, followed by the resolution of inflammation and the restoration of tissue homeostasis. Lipoxins are endogenous anti-inflammatory, pro-resolving molecules that play a vital role in reducing excessive tissue injury and chronic inflammation. In this review, the mechanisms of action of lipoxins at the site of inflammation and their interaction with other cellular signaling molecules and transcription factors are discussed. Emphasis has also been placed on immune modulatory role(s of lipoxins. Lipoxins regulate components of both the innate and adaptive immune systems including neutrophils, macrophages, T-, and B-cells. Lipoxins also modulate levels of various transcription factors such as nuclear factor κB, activator protein-1, nerve growth factor-regulated factor 1A binding protein 1, and peroxisome proliferator activated receptor γ and control the expression of many inflammatory genes. Since lipoxins and aspirin-triggered lipoxins have clinical relevance, we discuss their important role in clinical research to treat a wide range of diseases like inflammatory disorders, renal fibrosis, cerebral ischemia, and cancer. A brief overview of lipoxins in viral malignancies and viral pathogenesis especially the unexplored role of lipoxins in Kaposi's sarcoma-associated herpes virus biology is also presented. Keywords: lipoxins, epi-lipoxins, inflammation, pro-resolving, aspirin-triggered lipoxins, cyclooxygenases, lipoxygenases, therapeutic potential, transcription factors, Kaposi's sarcoma-associated herpes virus

  13. Calcium spirulan derived from Spirulina platensis inhibits herpes simplex virus 1 attachment to human keratinocytes and protects against herpes labialis.

    Science.gov (United States)

    Mader, Julia; Gallo, Antonio; Schommartz, Tim; Handke, Wiebke; Nagel, Claus-Henning; Günther, Patrick; Brune, Wolfram; Reich, Kristian

    2016-01-01

    Chronic infections with herpes simplex virus (HSV) type 1 are highly prevalent in populations worldwide and cause recurrent oral lesions in up to 40% of infected subjects. We investigated the antiviral activity of a defined Spirulina platensis microalga extract and of purified calcium spirulan (Ca-SP), a sulfated polysaccharide contained therein. The inhibitory effects of HSV-1 were assessed by using a plaque reduction assay and quantitative PCR in a susceptible mammalian epithelial cell line and confirmed in human keratinocytes. Time-of-addition and attachment experiments and fluorescence detection of the HSV-1 tegument protein VP16 were used to analyze the mechanism of HSV-1 inhibition. Effects of Ca-SP on Kaposi sarcoma-associated herpesvirus/human herpes virus 8 replication and uptake of the ORF45 tegument protein were tested in human retinal pigment epithelial cells. In an observational trial the prophylactic effects of topically applied Ca-SP were compared with those of systemic and topical nucleoside analogues in 198 volunteers with recurrent herpes labialis receiving permanent lip makeup. Ca-SP inhibited HSV-1 infection in vitro with a potency at least comparable to that of acyclovir by blocking viral attachment and penetration into host cells. Ca-SP also inhibited entry of Kaposi sarcoma-associated herpesvirus/human herpes virus 8. In the clinical model of herpes exacerbation, the prophylactic effect of a Ca-SP and microalgae extract containing cream was superior to that of acyclovir cream. These data indicate a potential clinical use of Ca-SP containing Spirulina species extract for the prophylactic treatment of herpes labialis and suggest possible activity of Ca-SP against infections caused by other herpesviruses. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  14. Mutational Analysis of Oculocutaneous Albinism: A Compact Review

    Directory of Open Access Journals (Sweden)

    Balu Kamaraj

    2014-01-01

    Full Text Available Oculocutaneous albinism (OCA is an autosomal recessive disorder caused by either complete lack of or a reduction of melanin biosynthesis in the melanocytes. The OCA1A is the most severe type with a complete lack of melanin production throughout life, while the milder forms OCA1B, OCA2, OCA3, and OCA4 show some pigment accumulation over time. Mutations in TYR, OCA2, TYRP1, and SLC45A2 are mainly responsible for causing oculocutaneous albinism. Recently, two new genes SLC24A5 and C10orf11 are identified that are responsible to cause OCA6 and OCA7, respectively. Also a locus has been mapped to the human chromosome 4q24 region which is responsible for genetic cause of OCA5. In this paper, we summarized the clinical and molecular features of OCA genes. Further, we reviewed the screening of pathological mutations of OCA genes and its molecular mechanism of the protein upon mutation by in silico approach. We also reviewed TYR (T373K, N371Y, M370T, and P313R, OCA2 (R305W, TYRP1 (R326H and R356Q mutations and their structural consequences at molecular level. It is observed that the pathological genetic mutations and their structural and functional significance of OCA genes will aid in development of personalized medicine for albinism patients.

  15. Agrobacterium rhizogenes pRi8196 T-DNA: mapping and DNA sequence of functions involved in mannopine synthesis and hairy root differentiation.

    Science.gov (United States)

    Hansen, G; Larribe, M; Vaubert, D; Tempé, J; Biermann, B J; Montoya, A L; Chilton, M D; Brevet, J

    1991-01-01

    This paper presents the map and DNA sequence analysis of pRi8196 transferred DNA (T-DNA) genes encoding root-inducing and mannopine synthesis functions. A canonical 24-base-pair border repeat as well as two "pseudoborders" are present at the functional right T-DNA border. To the left of this border are homologs of the mas1' and mas2' genes of TR pRiA4. Next to these are five open reading frames (ORFs) homologous to ORFs 10-14 of TL of pRiA4. ORFs 10-12 (rolA, rolB, and rolC) are less related to their pRiA4 homologs than are the other large ORFs analyzed here. In contrast to T-DNA genes of pRiA4, pRi8196 T-DNA ORFs 11 and 12 (rolB and rolC) are sufficient to induce hairy roots on carrot disks. Images PMID:1909028

  16. Mutational analysis of oculocutaneous albinism: a compact review.

    Science.gov (United States)

    Kamaraj, Balu; Purohit, Rituraj

    2014-01-01

    Oculocutaneous albinism (OCA) is an autosomal recessive disorder caused by either complete lack of or a reduction of melanin biosynthesis in the melanocytes. The OCA1A is the most severe type with a complete lack of melanin production throughout life, while the milder forms OCA1B, OCA2, OCA3, and OCA4 show some pigment accumulation over time. Mutations in TYR, OCA2, TYRP1, and SLC45A2 are mainly responsible for causing oculocutaneous albinism. Recently, two new genes SLC24A5 and C10orf11 are identified that are responsible to cause OCA6 and OCA7, respectively. Also a locus has been mapped to the human chromosome 4q24 region which is responsible for genetic cause of OCA5. In this paper, we summarized the clinical and molecular features of OCA genes. Further, we reviewed the screening of pathological mutations of OCA genes and its molecular mechanism of the protein upon mutation by in silico approach. We also reviewed TYR (T373K, N371Y, M370T, and P313R), OCA2 (R305W), TYRP1 (R326H and R356Q) mutations and their structural consequences at molecular level. It is observed that the pathological genetic mutations and their structural and functional significance of OCA genes will aid in development of personalized medicine for albinism patients.

  17. Increasing the complexity: new genes and new types of albinism.

    Science.gov (United States)

    Montoliu, Lluís; Grønskov, Karen; Wei, Ai-Hua; Martínez-García, Mónica; Fernández, Almudena; Arveiler, Benoît; Morice-Picard, Fanny; Riazuddin, Saima; Suzuki, Tamio; Ahmed, Zubair M; Rosenberg, Thomas; Li, Wei

    2014-01-01

    Albinism is a rare genetic condition globally characterized by a number of specific deficits in the visual system, resulting in poor vision, in association with a variable hypopigmentation phenotype. This lack or reduction in pigment might affect the eyes, skin, and hair (oculocutaneous albinism, OCA), or only the eyes (ocular albinism, OA). In addition, there are several syndromic forms of albinism (e.g. Hermansky-Pudlak and Chediak-Higashi syndromes, HPS and CHS, respectively) in which the described hypopigmented and visual phenotypes coexist with more severe pathological alterations. Recently, a locus has been mapped to the 4q24 human chromosomal region and thus represents an additional genetic cause of OCA, termed OCA5, while the gene is eventually identified. In addition, two new genes have been identified as causing OCA when mutated: SLC24A5 and C10orf11, and hence designated as OCA6 and OCA7, respectively. This consensus review, involving all laboratories that have reported these new genes, aims to update and agree upon the current gene nomenclature and types of albinism, while providing additional insights from the function of these new genes in pigment cells. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Homozygosity mapping in albinism patients using a novel panel of 13 STR markers inside the nonsyndromic OCA genes: introducing 5 novel mutations.

    Science.gov (United States)

    Khordadpoor-Deilamani, Faravareh; Akbari, Mohammad Taghi; Karimipoor, Morteza; Javadi, Gholam Reza

    2016-05-01

    Albinism is a heterogeneous genetic disorder of melanin synthesis that results in hypopigmented hair, skin and eyes. It is associated with decreased visual acuity, nystagmus, strabismus and photophobia. Six genes are known to be involved in nonsyndromic oculocutaneous albinism (OCA). In this study, we aimed to find the disease causing mutations in albinism patients using homozygosity mapping. Twenty three unrelated patients with nonsyndromic OCA or autosomal recessive ocular albinism were recruited in this study. All of the patients' parents had consanguineous marriage and all were screened for TYR mutations previously. At first, we performed homozygosity mapping using fluorescently labeled primers to amplify a novel panel of 13 STR markers inside the OCA genes and then the screened loci in each family were studied using PCR and cycle sequencing methods. We found five mutations including three mutations in OCA2, one mutation in SLC45A2 and one mutation in C10ORF11 genes, all of which were novel. In cases where the disease causing mutations are identical by descent due to a common ancestor, these STR markers can enable us to screen for the responsible genes.

  19. Ophthalmo-genetic analysis of Pakistani patients with nonsyndromic oculocutaneous albinism through whole exome sequencing.

    Science.gov (United States)

    Gul, Hadia; Ali, Muhammad Zeeshan; Khan, Ejazullah; Zubair, Muhammad; Badar, Muhammad; Khan, Saadullah; Shah, Abdul Haleem; Khan, Muzammil Ahmad

    2017-05-01

    Oculocutaneous albinism (OCA) is a disorder of defective melanin biosynthesis that is characterized by hypo-pigmentation of skin, hair and retinal pigment epithelium. Phenotypically, OCA patients exhibit white milky skin, whitish to golden hair and deterioration of retinal cells. Until recently, genetic studies have reported seven causative genes (TYR, TYRP1, OCA2, SLC45A2, SLC24A2, C10ORF11 and MCIR) and an uncharacterized OCA5 locus. Herein we present the medico-genetic study of three Pakistani patients inheriting autosomal recessive OCA. Whole exome sequencing, followed by Sanger DNA sequencing for segregation analysis, revealed recurrent mutations c.346C>T (p.Arg116*) and c.1255G>A (p.Gly419Arg) (family A and B respectively) in TYR gene, while the patient from family C did not reveal any known gene mutation, which suggests the involvement of some novel genetic factor. It is the first report of mapping c.346C>T mutation in a Pakistani patient. Our study further extends the evidence of genetic hotspots regions in TYR gene causing OCA in Pakistani population.

  20. MC1R gene variants involvement in human OCA phenotype

    Directory of Open Access Journals (Sweden)

    Saleha Shamim

    2016-01-01

    Full Text Available Oculocutaneous albinism (OCA is a genetic disorder of melanin synthesis that results in hypopigmentation in hair, skin and eyes. OCA has been reported in individuals from all ethnic backgrounds but it is more common among those with Europeans ancestry. OCA is heterogeneous group of disorders and seven types of OCA are caused by mutations in TYR (OCA1, OCA2 (OCA2, TYRP1 (OCA3, SLC45A2 (OCA4, SLC24A5 (OCA6 and C10oRF11 (OCA7 genes. However, MC1R gene variants have been reported that modify OCA2 phenotype but the knowledge about the function ofMC1R gene in melanogenesis, and genotype-phenotype association, in case of OCA, is limited. In this review article we present a comprehensive description of classification of OCA, role of MSH-R in melanin synthesis, the sequence variations in MC1R and their association with OCA. This review will enhance our understanding of MC1R gene variants involved in human OCA2 phenotype.

  1. An uncultivated crenarchaeota contains functional bacteriochlorophyll a synthase.

    Science.gov (United States)

    Meng, Jun; Wang, Fengping; Wang, Feng; Zheng, Yanping; Peng, Xiaotong; Zhou, Huaiyang; Xiao, Xiang

    2009-01-01

    A fosmid clone 37F10 containing an archaeal 16S rRNA gene was screened out from a metagenomic library of Pearl River sediment, southern China. Sequence analysis of the 35 kb inserted fragment of 37F10 found that it contains a single 16S rRNA gene belonging to Miscellaneous Crenarchaeotal Group (MCG) and 36 open reading frames (ORFs). One ORF (orf11) encodes putative bacteriochlorophyll a synthase (bchG) gene. Bacteriochlorophyll a synthase gene has never been reported in a member of the domain Archaea, in accordance with the fact that no (bacterio)-chlorophyll has ever been detected in any cultivated archaea. The putative archaeal bchG (named as ar-bchG) was cloned and heterologously expressed in Escherichia coli. The protein was found to be capable of synthesizing bacteriochlorophyll a by esterification of bacteriochlorophyllide a with phytyl diphosphate or geranylgeranyl diphosphate. Furthermore, phylogenetic analysis clearly indicates that the ar-bchG diverges before the bacterial bchGs. Our results for the first time demonstrate that a key and functional enzyme for bacteriochlorophyll a biosynthesis does exist in Archaea.

  2. Functional metagenomics reveals novel β-galactosidases not predictable from gene sequences.

    Science.gov (United States)

    Cheng, Jiujun; Romantsov, Tatyana; Engel, Katja; Doxey, Andrew C; Rose, David R; Neufeld, Josh D; Charles, Trevor C

    2017-01-01

    The techniques of metagenomics have allowed researchers to access the genomic potential of uncultivated microbes, but there remain significant barriers to determination of gene function based on DNA sequence alone. Functional metagenomics, in which DNA is cloned and expressed in surrogate hosts, can overcome these barriers, and make important contributions to the discovery of novel enzymes. In this study, a soil metagenomic library carried in an IncP cosmid was used for functional complementation for β-galactosidase activity in both Sinorhizobium meliloti (α-Proteobacteria) and Escherichia coli (γ-Proteobacteria) backgrounds. One β-galactosidase, encoded by six overlapping clones that were selected in both hosts, was identified as a member of glycoside hydrolase family 2. We could not identify ORFs obviously encoding possible β-galactosidases in 19 other sequenced clones that were only able to complement S. meliloti. Based on low sequence identity to other known glycoside hydrolases, yet not β-galactosidases, three of these ORFs were examined further. Biochemical analysis confirmed that all three encoded β-galactosidase activity. Lac36W_ORF11 and Lac161_ORF7 had conserved domains, but lacked similarities to known glycoside hydrolases. Lac161_ORF10 had neither conserved domains nor similarity to known glycoside hydrolases. Bioinformatic and structural modeling implied that Lac161_ORF10 protein represented a novel enzyme family with a five-bladed propeller glycoside hydrolase domain. By discovering founding members of three novel β-galactosidase families, we have reinforced the value of functional metagenomics for isolating novel genes that could not have been predicted from DNA sequence analysis alone.

  3. Niclosamide inhibits lytic replication of Epstein-Barr virus by disrupting mTOR activation.

    Science.gov (United States)

    Huang, Lu; Yang, Mengtian; Yuan, Yan; Li, Xiaojuan; Kuang, Ersheng

    2017-02-01

    Infection with the oncogenic γ-herpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause several severe malignancies in humans. Inhibition of the lytic replication of EBV and KSHV eliminates the reservoir of persistent infection and transmission, consequently preventing the occurrence of diseases from the sources of infection. Antiviral drugs are limited in controlling these viral infectious diseases. Here, we demonstrate that niclosamide, an old anthelmintic drug, inhibits mTOR activation during EBV lytic replication. Consequently, niclosamide effectively suppresses EBV lytic gene expression, viral DNA lytic replication and virion production in EBV-infected lymphoma cells and epithelial cells. Niclosamide exhibits cytotoxicity toward lymphoma cells and induces irreversible cell cycle arrest in lytically EBV-infected cells. The ectopic overexpression of mTOR reverses the inhibition of niclosamide in EBV lytic replication. Similarly, niclosamide inhibits KSHV lytic replication. Thus, we conclude that niclosamide is a promising candidate for chemotherapy against the acute occurrence and transmission of infectious diseases of oncogenic γ-herpesviruses. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Evasion of innate cytosolic DNA sensing by a gammaherpesvirus facilitates establishment of latent infection1

    Science.gov (United States)

    Sun, Chenglong; Schattgen, Stefan A.; Pisitkun, Prapaporn; Jorgensen, Joan P.; Hilterbrand, Adam T.; Wang, Lucas J.; West, John A.; Hansen, Kathrine; Horan, Kristy A.; Jakobsen, Martin R.; O'Hare, Peter; Adler, Heiko; Sun, Ren; Ploegh, Hidde L.; Damania, Blossom; Upton, Jason W.; Fitzgerald, Katherine A.; Paludan, Søren R.

    2014-01-01

    Herpesviruses are DNA viruses harboring the capacity to establish lifelong latent-recurrent infections. There is currently limited knowledge on viruses targeting the innate DNA sensing pathway and also on how the innate system impacts on the latent reservoir of herpesvirus infections. Here we report that murine gammaherpesvirus MHV68, in contrast to alpha- and beta-herpesviruses, induce very limited innate immune responses through DNA-stimulated pathways, which correspondingly played only a minor role in control of MHV68 infections in vivo. Similarly, Kaposi's sarcoma-associated herpesvirus also did not stimulate immune signaling through the DNA sensing pathways. Interestingly, a MHV68 mutant lacking the deubiquitinase (DUB) activity, embedded within the large tegument protein ORF64, gained the capacity to stimulate the DNA-activated STING pathway. We found that ORF64 targeted a step in the DNA-activated pathways upstream of the bifurcation into the STING and AIM2 pathways, and lack of the ORF64 DUB was associated with impaired delivery of viral DNA to the nucleus, which instead localized to the cytoplasm. Correspondingly, the ORF64 DUB active site mutant virus exhibited impaired ability to establish latent infection in wild type but not STING-deficient mice. Thus, gammaherpesviruses evade immune activation by the cytosolic DNA sensing pathway, which in the MHV68 model facilitates establishment of infections. PMID:25595793

  5. Human herpesvirus 8 in primary effusion lymphoma in an HIV-seronegative male. A case report.

    Science.gov (United States)

    Munichor, Mariana; Cohen, Hector; Sarid, Ronit; Manov, Irena; Iancu, Theodore C

    2004-01-01

    AIDS-related body cavity-based lymphoma, or primary effusion lymphoma (PEL), is a distinct clinicopathologic entity that occurs predominantly in immunosuppressed patients infected with human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. Although it rarely occurs in human immunodeficiency virus (HIV)-negative patients, we report such a case here. A 74-year-old male, who was HIV and Epstein-Barr virus (EBV) negative, was admitted to the hospital with dyspnea and chest pain. Chest radiography and computed tomography showed right pleural effusion. Cytologic analysis of the pleural effusion revealed a high grade lymphoma with round nuclei, prominent nucleoli and abundant cytoplasm. Polymerase chain reaction performed on the pleural effusion was positive for HHV-8 and negative for EBV. On molecular studies, the immunoglobulin heavy and kappa light chains were rearranged. Flow cytometry revealed a hyperploid fraction with DNA index of 1.29 expressing CD30. Immunostaining for HHV-8 from a cell block was positive. Electron microscopy revealed lymphomalike cells, many in various stages of apoptosis, with large nucleoli and clusters of viruslike particles in the nucleoplasm. A firm diagnosis of PEL can be established by the examination of cells from the lymphomatous effusion by a combination of cytology, molecular genetics, phenotypic features, immunostaining and electron microscopy. To our knowledge, this is the first case in which immunostaining for anti-HHV-8 monoclonal antibodies was used to support the diagnosis.

  6. Kaposi sarcoma herpes virus latency associated nuclear antigen protein release the G2/M cell cycle blocks by modulating ATM/ATR mediated checkpoint pathway.

    Directory of Open Access Journals (Sweden)

    Amit Kumar

    Full Text Available The Kaposi's sarcoma-associated herpesvirus infects the human population and maintains latency stage of viral life cycle in a variety of cell types including cells of epithelial, mesenchymal and endothelial origin. The establishment of latent infection by KSHV requires the expression of an unique repertoire of genes among which latency associated nuclear antigen (LANA plays a critical role in the replication of the viral genome. LANA regulates the transcription of a number of viral and cellular genes essential for the survival of the virus in the host cell. The present study demonstrates the disruption of the host G2/M cell cycle checkpoint regulation as an associated function of LANA. DNA profile of LANA expressing human B-cells demonstrated the ability of this nuclear antigen in relieving the drug (Nocodazole induced G2/M checkpoint arrest. Caffeine suppressed nocodazole induced G2/M arrest indicating involvement of the ATM/ATR. Notably, we have also shown the direct interaction of LANA with Chk2, the ATM/ATR signalling effector and is responsible for the release of the G2/M cell cycle block.

  7. Human I-mfa domain proteins specifically interact with KSHV LANA and affect its regulation of Wnt signaling-dependent transcription

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    Kusano, Shuichi, E-mail: skusano@m2.kufm.kagoshima-u.ac.jp [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Eizuru, Yoshito [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)

    2010-06-04

    Kaposi's sarcoma-associated herpes virus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein has been reported to interact with glycogen synthase kinase 3{beta} (GSK-3{beta}) and to negatively regulate its activity, leading to stimulation of GSK-3{beta}-dependent {beta}-catenin degradation. We show here that the I-mfa domain proteins, HIC (human I-mfa domain-containing protein) and I-mfa (inhibitor of MyoD family a), interacted in vivo with LANA through their C-terminal I-mfa domains. This interaction affected the intracellular localization of HIC, inhibited the LANA-dependent transactivation of a {beta}-catenin-regulated reporter construct, and decreased the level of the LANA.GSK-3{beta} complex. These data reveal for the first time that I-mfa domain proteins interact with LANA and negatively regulate LANA-mediated activation of Wnt signaling-dependent transcription by inhibiting the formation of the LANA.GSK-3{beta} complex.

  8. A Unique Case of Malignant Pleuropericardial Effusion: HHV-8-Unrelated PEL-Like Lymphoma—A Case Report and Review of the Literature

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    Farhan Mohammad

    2014-01-01

    Full Text Available Primary effusion lymphoma (PEL or body cavity lymphoma is a rare type of extra nodal lymphoma of B-cell origin that presents as lymphomatous effusion(s without any nodal enlargement or tumor masses. It belongs to the group of AIDS related non-Hodgkin’s lymphomas. First described in 1996 in HIV infected individuals who were coinfected with Kaposi’s sarcoma-associated herpesvirus (KSHV or HHV-8 virus, it was included as a separate entity in WHO classification of tumors of hematopoietic and lymphoid tissue in the year 2001. The definition included association with HHV-8 virus as a mandatory diagnostic criterion. However, cases were later reported where PEL-like disease process was diagnosed in HHV-8 negative patients. This was eventually recognized as a rare but distinct entity termed as “HHV-8-unrelated PEL-like lymphoma”. Herein, we are reporting a case of an elderly patient who presented with a large pleuropericardial effusion and was eventually diagnosed with this entity. Till date, only around 50 cases of HHV-8-unrelated PEL-like lymphoma have been reported and our case being EBV, HIV, and Hepatitis C negative makes it very unique and rare occurrence. We are also presenting a review of relevant literature focused mainly on comparing outcomes in patients treated with and without chemotherapy.

  9. KSHV-encoded CC chemokine vMIP-III is a CCR4 agonist, stimulates angiogenesis, and selectively chemoattracts TH2 cells.

    Science.gov (United States)

    Stine, J T; Wood, C; Hill, M; Epp, A; Raport, C J; Schweickart, V L; Endo, Y; Sasaki, T; Simmons, G; Boshoff, C; Clapham, P; Chang, Y; Moore, P; Gray, P W; Chantry, D

    2000-02-15

    Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 3 genes that are homologous to cellular chemokines. vMIP-III, the product of open reading frame K4.1, is the most distantly related to human chemokines and has yet to be characterized. We have examined the interaction of vMIP-III with chemokine receptors, its expression in KS lesions, and its in ovo angiogenic properties. We show expression of vMIP-III in KS lesions and demonstrate the stimulation of angiogenesis by this chemokine, like vMIP-I and vMIP-II, in the chick chorioallantoic membrane assay. vMIP-III does not block human immunodeficiency virus entry through the coreceptors CCR3, CCR5, or CXCR4. However, vMIP-III is an agonist for the cellular chemokine receptor CCR4. CCR4 is expressed by TH2-type T cells. Consistent with this, vMIP-III preferentially chemoattracts this cell type. Because of these biologic properties and because it is expressed in KS lesions, vMIP-III may play an important role in the pathobiology of KS. (Blood. 2000;95:1151-1157)

  10. Nonsulfated, cinnamic acid-based lignins are potent antagonists of HSV-1 entry into cells.

    Science.gov (United States)

    Thakkar, Jay N; Tiwari, Vaibhav; Desai, Umesh R

    2010-05-10

    In an effort to discover macromolecular mimetics of heparan sulfate (HS), we previously designed sulfated lignins (Raghuraman et al. Biomacromolecules 2007, 8, 1759-1763). To probe the relevance of sulfate groups of HS in viral entry, lignins completely devoid of sulfate moieties, and yet possessing an electrostatic surface equivalent to that of HS, were designed. Two carboxylated lignins based on a 4-hydroxy cinnamic acid scaffold were synthesized using enzymatic oxidative coupling in high yields, fractionated according to their sizes, and tested in cellular assays of herpes simplex virus-1 (HSV-1) infection. The two carboxylated lignins were found to not only inhibit HSV-1 entry into mammalian cells (IC(50) = 8-56 nM), but were more potent than sulfated lignins. In addition, shorter carboxylated lignins were found to be as active as the longer chains, suggesting that structural features, in addition to carboxylate groups, may be important. It can be expected that carboxylated lignins also antagonize the entry of other enveloped viruses, for example, HIV-1, Kaposi's sarcoma-associated herpes virus, and hepatitis C virus, that utilize HS to gain entry into cells. The results present major opportunities for developing lignin-based antiviral formulations for topical use.

  11. Host entry by gamma-herpesviruses--lessons from animal viruses?

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    Gillet, Laurent; Frederico, Bruno; Stevenson, Philip G

    2015-12-01

    The oncogenicity of gamma-herpesviruses (γHVs) motivates efforts to control them and their persistence makes early events key targets for intervention. Human γHVs are often assumed to enter naive hosts orally and infect B cells directly. However, neither assumption is supported by direct evidence, and vaccination with the Epstein-Barr virus (EBV) gp350, to block virion binding to B cells, failed to reduce infection rates. Thus, there is a need to re-evaluate assumptions about γHV host entry. Given the difficulty of analysing early human infections, potentially much can be learned from animal models. Genomic comparisons argue that γHVs colonized mammals long before humans speciation, and so that human γHVs are unlikely to differ dramatically in behaviour from those of other mammals. Murid Herpesvirus-4 (MuHV-4), which like EBV and the Kaposi's Sarcoma-associated Herpesvirus (KSHV) persists in memory B cells, enters new hosts via olfactory neurons and exploits myeloid cells to spread. Integrating these data with existing knowledge of human and veterinary γHVs suggests a new model of host entry, with potentially important implications for infection control. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Animal herpesviruses and their zoonotic potential for cross-species infection

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    Grzegorz Woźniakowski

    2015-05-01

    Full Text Available Herpesviruses of humans and animals cause severe diseases that influence not only the health and epidemiological status but are also economically important in the context of food production. The members of Herpesviridae are host specific agents that also share many properties that potentially make them capable of crossing the species barriers. The objective of presented review paper was to summarize the relationship between herpesviruses of animals and humans and their zoonotic potential. In humans, the most epidemiologically important herpesviruses are represented by Human herepesvirus-1 and Human herpesvirus-2, which are commonly known as herpes simplex virus type 1 and 2, varicella-zooster virus (VZV, Epstein-Barr virus (EBV, Kaposi’s Sarcoma-associated herpesvirus (KSHV, cytomegalovirus (CMV, as well as Human herpesviruses: HHV-6A, HHV-6B, and HHV-7. However, in terms of the potential to cross the species barrier, there are a few herpesviruses, including B virus disease (CeHV-1, Marek’s disease virus (MDV, Equid herpesvirus-1 (EHV-1 or pseudorabies virus (PRV, which are potentially able to infect different hosts. To summarize, in advantageous conditions the host specific herpesviruses may pose a threat for public health but also may exert a negative impact on the economical aspects of animal production. The most probable of these are zoonotic infections caused by B virus disease; however, close contact between infected animal hosts and humans may lead to transmission and replication of other Herpesviridae members.

  13. LANA-Mediated Recruitment of Host Polycomb Repressive Complexes onto the KSHV Genome during De Novo Infection.

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    Zsolt Toth

    2016-09-01

    Full Text Available One of the hallmarks of the latent phase of Kaposi's sarcoma-associated herpesvirus (KSHV infection is the global repression of lytic viral gene expression. Following de novo KSHV infection, the establishment of latency involves the chromatinization of the incoming viral genomes and recruitment of the host Polycomb repressive complexes (PRC1 and PRC2 to the promoters of lytic genes, which is accompanied by the inhibition of lytic genes. However, the mechanism of how PRCs are recruited to the KSHV episome is still unknown. Utilizing a genetic screen of latent genes in the context of KSHV genome, we identified the latency-associated nuclear antigen (LANA to be responsible for the genome-wide recruitment of PRCs onto the lytic promoters following infection. We found that LANA initially bound to the KSHV genome right after infection and subsequently recruited PRCs onto the viral lytic promoters, thereby repressing lytic gene expression. Furthermore, both the DNA and chromatin binding activities of LANA were required for the binding of LANA to the KSHV promoters, which was necessary for the recruitment of PRC2 to the lytic promoters during de novo KSHV infection. Consequently, the LANA-knockout KSHV could not recruit PRCs to its viral genome upon de novo infection, resulting in aberrant lytic gene expression and dysregulation of expression of host genes involved in cell cycle and proliferation pathways. In this report, we demonstrate that KSHV LANA recruits host PRCs onto the lytic promoters to suppress lytic gene expression following de novo infection.

  14. A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells

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    Ina Ersing

    2017-05-01

    Full Text Available Epstein-Barr virus (EBV replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication.

  15. Coupled transcriptome and proteome analysis of human lymphotropic tumor viruses: insights on the detection and discovery of viral genes

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    Dresang Lindsay R

    2011-12-01

    Full Text Available Abstract Background Kaposi's sarcoma-associated herpesvirus (KSHV and Epstein-Barr virus (EBV are related human tumor viruses that cause primary effusion lymphomas (PEL and Burkitt's lymphomas (BL, respectively. Viral genes expressed in naturally-infected cancer cells contribute to disease pathogenesis; knowing which viral genes are expressed is critical in understanding how these viruses cause cancer. To evaluate the expression of viral genes, we used high-resolution separation and mass spectrometry coupled with custom tiling arrays to align the viral proteomes and transcriptomes of three PEL and two BL cell lines under latent and lytic culture conditions. Results The majority of viral genes were efficiently detected at the transcript and/or protein level on manipulating the viral life cycle. Overall the correlation of expressed viral proteins and transcripts was highly complementary in both validating and providing orthogonal data with latent/lytic viral gene expression. Our approach also identified novel viral genes in both KSHV and EBV, and extends viral genome annotation. Several previously uncharacterized genes were validated at both transcript and protein levels. Conclusions This systems biology approach coupling proteome and transcriptome measurements provides a comprehensive view of viral gene expression that could not have been attained using each methodology independently. Detection of viral proteins in combination with viral transcripts is a potentially powerful method for establishing virus-disease relationships.

  16. KSHV Targeted Therapy: An Update on Inhibitors of Viral Lytic Replication

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    Natacha Coen

    2014-11-01

    Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV is the causative agent of Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castleman’s disease. Since the discovery of KSHV 20 years ago, there is still no standard treatment and the management of virus-associated malignancies remains toxic and incompletely efficacious. As the majority of tumor cells are latently infected with KSHV, currently marketed antivirals that target the virus lytic cycle have shown inconsistent results in clinic. Nevertheless, lytic replication plays a major role in disease progression and virus dissemination. Case reports and retrospective studies have pointed out the benefit of antiviral therapy in the treatment and prevention of KSHV-associated diseases. As a consequence, potent and selective antivirals are needed. This review focuses on the anti-KSHV activity, mode of action and current status of antiviral drugs targeting KSHV lytic cycle. Among these drugs, different subclasses of viral DNA polymerase inhibitors and compounds that do not target the viral DNA polymerase are being discussed. We also cover molecules that target cellular kinases, as well as the potential of new drug targets and animal models for antiviral testing.

  17. Chromatinization of the KSHV Genome During the KSHV Life Cycle

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    Uppal, Timsy [Department of Microbiology and Immunology, School of Medicine, University of Nevada, 1664 N Virginia Street, MS 320, Reno, NV 89557 (United States); Jha, Hem C. [Department of Microbiology and the Tumor Virology Program of the Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, 201E Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104 (United States); Verma, Subhash C. [Department of Microbiology and Immunology, School of Medicine, University of Nevada, 1664 N Virginia Street, MS 320, Reno, NV 89557 (United States); Robertson, Erle S., E-mail: erle@mail.med.upenn.edu [Department of Microbiology and the Tumor Virology Program of the Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, 201E Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104 (United States)

    2015-01-14

    Kaposi’s sarcoma-associated herpesvirus (KSHV) belongs to the gamma herpesvirus family and is the causative agent of various lymphoproliferative diseases in humans. KSHV, like other herpesviruses, establishes life-long latent infection with the expression of a limited number of viral genes. Expression of these genes is tightly regulated by both the viral and cellular factors. Recent advancements in identifying the expression profiles of viral transcripts, using tilling arrays and next generation sequencing have identified additional coding and non-coding transcripts in the KSHV genome. Determining the functions of these transcripts will provide a better understanding of the mechanisms utilized by KSHV in altering cellular pathways involved in promoting cell growth and tumorigenesis. Replication of the viral genome is critical in maintaining the existing copies of the viral episomes during both latent and lytic phases of the viral life cycle. The replication of the viral episome is facilitated by viral components responsible for recruiting chromatin modifying enzymes and replication factors for altering the chromatin complexity and replication initiation functions, respectively. Importantly, chromatin modification of the viral genome plays a crucial role in determining whether the viral genome will persist as latent episome or undergo lytic reactivation. Additionally, chromatinization of the incoming virion DNA, which lacks chromatin structure, in the target cells during primary infection, helps in establishing latent infection. Here, we discuss the recent advancements on our understating of KSHV genome chromatinization and the consequences of chromatin modifications on viral life cycle.

  18. Chromatinization of the KSHV Genome During the KSHV Life Cycle

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    Timsy Uppal

    2015-01-01

    Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV belongs to the gamma herpesvirus family and is the causative agent of various lymphoproliferative diseases in humans. KSHV, like other herpesviruses, establishes life-long latent infection with the expression of a limited number of viral genes. Expression of these genes is tightly regulated by both the viral and cellular factors. Recent advancements in identifying the expression profiles of viral transcripts, using tilling arrays and next generation sequencing have identified additional coding and non-coding transcripts in the KSHV genome. Determining the functions of these transcripts will provide a better understanding of the mechanisms utilized by KSHV in altering cellular pathways involved in promoting cell growth and tumorigenesis. Replication of the viral genome is critical in maintaining the existing copies of the viral episomes during both latent and lytic phases of the viral life cycle. The replication of the viral episome is facilitated by viral components responsible for recruiting chromatin modifying enzymes and replication factors for altering the chromatin complexity and replication initiation functions, respectively. Importantly, chromatin modification of the viral genome plays a crucial role in determining whether the viral genome will persist as latent episome or undergo lytic reactivation. Additionally, chromatinization of the incoming virion DNA, which lacks chromatin structure, in the target cells during primary infection, helps in establishing latent infection. Here, we discuss the recent advancements on our understating of KSHV genome chromatinization and the consequences of chromatin modifications on viral life cycle.

  19. A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants

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    Vaibhav Jain

    2016-02-01

    Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV encodes 12 viral microRNAs (miRNAs that are expressed during latency. Research into KSHV miRNA function has suffered from a lack of genetic systems to study viral miRNA mutations in the context of the viral genome. We used the Escherichia coli Red recombination system together with a new bacmid background, BAC16, to create mutants for all known KSHV miRNAs. The specific miRNA deletions or mutations and the integrity of the bacmids have been strictly quality controlled using PCR, restriction digestion, and sequencing. In addition, stable viral producer cell lines based on iSLK cells have been created for wildtype KSHV, for 12 individual miRNA knock-out mutants (ΔmiR-K12-1 through -12, and for mutants deleted for 10 of 12 (ΔmiR-cluster or all 12 miRNAs (ΔmiR-all. NGS, in combination with SureSelect technology, was employed to sequence the entire latent genome within all producer cell lines. qPCR assays were used to verify the expression of the remaining viral miRNAs in a subset of mutants. Induction of the lytic cycle leads to efficient production of progeny viruses that have been used to infect endothelial cells. Wt BAC16 and miR mutant iSLK producer cell lines are now available to the research community.

  20. Prevalence and risk factors of gammaherpesvirus infection in domestic cats in Central Europe.

    Science.gov (United States)

    Ertl, Reinhard; Korb, Melanie; Langbein-Detsch, Ines; Klein, Dieter

    2015-09-17

    Gammaherpesviruses (GHVs) are a large group of dsDNA viruses that can infect humans and several animal species. The two human GHVs, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are known for their oncogenic properties in individuals with immunodeficiency. Recently, the first feline GHV, Felis catus gammaherpesvirus 1 (FcaGHV1) was discovered and frequently found in domestic cats in Australia, Singapore and the USA. FcaGHV1 is more likely to be detected in cats co-infected with the feline immunodeficiency virus (FIV). The prevalence of FcaGHV1 in pet cats from Germany and Austria was 16.2 % (95 % CI = 12.38-20.02). The odds for GHV infection were greater for FIV positive (OR = 4.5), male (OR = 13.32) and older (OR = 2.36) cats. Furthermore, FcaGHV1 viral loads were significantly higher in FIV-infected cats compared to matched controls. GHV infections are common in domestic cats in Central Europe. The worldwide distribution of FcaGHV1 can be assumed. A potential role as a co-factor in FIV-induced pathogeneses is supported.

  1. KSHV MicroRNAs Repress Tropomyosin 1 and Increase Anchorage-Independent Growth and Endothelial Tube Formation.

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    Philippe Kieffer-Kwon

    Full Text Available Kaposi's sarcoma (KS is characterized by highly vascularized spindle-cell tumors induced after infection of endothelial cells by Kaposi's sarcoma-associated herpesvirus (KSHV. In KS tumors, KSHV expresses only a few latent proteins together with 12 pre-microRNAs. Previous microarray and proteomic studies predicted that multiple splice variants of the tumor suppressor protein tropomyosin 1 (TPM1 were targets of KSHV microRNAs. Here we show that at least two microRNAs of KSHV, miR-K2 and miR-K5, repress protein levels of specific isoforms of TPM1. We identified a functional miR-K5 binding site in the 3' untranslated region (UTR of one TPM1 isoform. Furthermore, the inhibition or loss of miR-K2 or miR-K5 restores expression of TPM1 in KSHV-infected cells. TPM1 protein levels were also repressed in KSHV-infected clinical samples compared to uninfected samples. Functionally, miR-K2 increases viability of unanchored human umbilical vein endothelial cells (HUVEC by inhibiting anoikis (apoptosis after cell detachment, enhances tube formation of HUVECs, and enhances VEGFA expression. Taken together, KSHV miR-K2 and miR-K5 may facilitate KSHV pathogenesis.

  2. Inhibition of the phosphatidylinositol 3-kinase-Akt pathway enhances gamma-2 herpesvirus lytic replication and facilitates reactivation from latency.

    Science.gov (United States)

    Peng, Li; Wu, Ting-Ting; Tchieu, Jason H; Feng, Jun; Brown, Helen J; Feng, Jiaying; Li, Xudong; Qi, Jing; Deng, Hongyu; Vivanco, Igor; Mellinghoff, Ingo K; Jamieson, Christina; Sun, Ren

    2010-02-01

    Cellular signalling pathways are critical in regulating the balance between latency and lytic replication of herpesviruses. Here, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway on replication of two gamma-2 herpesviruses, murine gammaherpesvirus-68 (MHV-68) and human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV). We found that de novo infection of MHV-68 induced PI3K-dependent Akt activation and the lytic replication of MHV-68 was enhanced by inhibiting the PI3K-Akt pathway with both chemical inhibitors and RNA interference technology. Inhibiting the activity of Akt using Akt inhibitor VIII also facilitated the reactivation of KSHV from latency. Both lytic replication and latency depend on the activity of viral transactivator RTA and we further show that the activity of RTA is increased by reducing Akt1 expression. The data suggest that the PI3K-Akt pathway suppresses the activity of RTA and thereby contributes to the maintenance of viral latency and promotes tumorigenesis.

  3. Frequency of Certain Established Risk Factors in Soft Tissue Sarcomas in Adults: A Prospective Descriptive Study of 658 Cases

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    Nicolas Penel

    2008-01-01

    Full Text Available Soft tissue sarcomas are rare tumours with infrequent identified aetiological factors. Several genetic syndromes as well as previous radiation therapy and/or chronic lymphoedema have been suspected to predispose to some soft tissue sarcomas. Between January 1997 and September 2005, we carried out a prospective descriptive study to estimate the frequency of some particular etiological factors among 658 patients with soft tissue sarcomas. Sarcomas associated with a clinically identified genetic disease represent 2.8% out of all cases (95%CI: 1.5–3.8%. Most of these cases (14/19 are related to Recklinghausen neurofibromatosis. Radiation-induced sarcomas represent 3.3% out of all cases (95%CI: 1.7–5.1%. Most of these cases (9/22 are related to prior breast cancer treatment. We had observed only 1 case of Stewart-Treves syndrome. Liposarcoma, the most frequent histological subtype observed, is not associated with any particular aetiological entity. Finally, most of the adult soft tissue sarcomas are not related to any classical clinically identified genetic disease or previous radiation therapy and/or chronic lymphoedema risk factors. Frequency of underlying genetic syndrome which may predispose to soft tissue sarcomas could be higher than previously reported.

  4. Viral Causes of Lymphoma: The History of Epstein-Barr Virus and Human T-Lymphotropic Virus 1

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    Daniel Esau

    2017-09-01

    Full Text Available In 1964, Epstein, Barr, and Achong published a report outlining their discovery of viral particles in lymphoblasts isolated from a patient with Burkitt lymphoma. The Epstein-Barr virus (EBV was the first human cancer virus to be described, and its discovery paved the way for further investigations into the oncogenic potential of viruses. In the decades following the discovery of EBV, multinational research efforts led to the discovery of further viral causes of various human cancers. Lymphomas are perhaps the cancer type that is most closely associated with oncogenic viruses: infection with EBV, human T-lymphotropic virus 1 (HTLV-1, human immunodeficiency virus (HIV, Kaposi sarcoma-associated herpesvirus/human herpesvirus 8, and hepatitis C virus have all been associated with lymphomagenesis. Lymphomas have also played an important role in the history of oncoviruses, as both the first human oncovirus (EBV and the first human retrovirus (HTLV-1 were discovered through isolates taken from patients with unique lymphoma syndromes. The history of the discovery of these 2 key oncoviruses is presented here, and their impact on further medical research, using the specific example of HIV research, is briefly discussed.

  5. Viral Causes of Lymphoma: The History of Epstein-Barr Virus and Human T-Lymphotropic Virus 1.

    Science.gov (United States)

    Esau, Daniel

    2017-01-01

    In 1964, Epstein, Barr, and Achong published a report outlining their discovery of viral particles in lymphoblasts isolated from a patient with Burkitt lymphoma. The Epstein-Barr virus (EBV) was the first human cancer virus to be described, and its discovery paved the way for further investigations into the oncogenic potential of viruses. In the decades following the discovery of EBV, multinational research efforts led to the discovery of further viral causes of various human cancers. Lymphomas are perhaps the cancer type that is most closely associated with oncogenic viruses: infection with EBV, human T-lymphotropic virus 1 (HTLV-1), human immunodeficiency virus (HIV), Kaposi sarcoma-associated herpesvirus/human herpesvirus 8, and hepatitis C virus have all been associated with lymphomagenesis. Lymphomas have also played an important role in the history of oncoviruses, as both the first human oncovirus (EBV) and the first human retrovirus (HTLV-1) were discovered through isolates taken from patients with unique lymphoma syndromes. The history of the discovery of these 2 key oncoviruses is presented here, and their impact on further medical research, using the specific example of HIV research, is briefly discussed.

  6. Proteomic characterization of murid herpesvirus 4 extracellular virions.

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    Sarah Vidick

    Full Text Available Gammaherpesvirinae, such as the human Epstein-Barr virus (EBV and the Kaposi's sarcoma associated herpesvirus (KSHV are highly prevalent pathogens that have been associated with several neoplastic diseases. As EBV and KSHV are host-range specific and replicate poorly in vitro, animal counterparts such as Murid herpesvirus-4 (MuHV-4 have been widely used as models. In this study, we used MuHV-4 in order to improve the knowledge about proteins that compose gammaherpesviruses virions. To this end, MuHV-4 extracellular virions were isolated and structural proteins were identified using liquid chromatography tandem mass spectrometry-based proteomic approaches. These analyses allowed the identification of 31 structural proteins encoded by the MuHV-4 genome which were classified as capsid (8, envelope (9, tegument (13 and unclassified (1 structural proteins. In addition, we estimated the relative abundance of the identified proteins in MuHV-4 virions by using exponentially modified protein abundance index analyses. In parallel, several host proteins were found in purified MuHV-4 virions including Annexin A2. Although Annexin A2 has previously been detected in different virions from various families, its role in the virion remains controversial. Interestingly, despite its relatively high abundance in virions, Annexin A2 was not essential for the growth of MuHV-4 in vitro. Altogether, these results extend previous work aimed at determining the composition of gammaherpesvirus virions and provide novel insights for understanding MuHV-4 biology.

  7. Sequencing of bovine herpesvirus 4 v.test strain reveals important genome features

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    Gillet Laurent

    2011-08-01

    Full Text Available Abstract Background Bovine herpesvirus 4 (BoHV-4 is a useful model for the human pathogenic gammaherpesviruses Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus. Although genome manipulations of this virus have been greatly facilitated by the cloning of the BoHV-4 V.test strain as a Bacterial Artificial Chromosome (BAC, the lack of a complete genome sequence for this strain limits its experimental use. Methods In this study, we have determined the complete sequence of BoHV-4 V.test strain by a pyrosequencing approach. Results The long unique coding region (LUR consists of 108,241 bp encoding at least 79 open reading frames and is flanked by several polyrepetitive DNA units (prDNA. As previously suggested, we showed that the prDNA unit located at the left prDNA-LUR junction (prDNA-G differs from the other prDNA units (prDNA-inner. Namely, the prDNA-G unit lacks the conserved pac-2 cleavage and packaging signal in its right terminal region. Based on the mechanisms of cleavage and packaging of herpesvirus genomes, this feature implies that only genomes bearing left and right end prDNA units are encapsulated into virions. Conclusions In this study, we have determined the complete genome sequence of the BAC-cloned BoHV-4 V.test strain and identified genome organization features that could be important in other herpesviruses.

  8. Coupled Transcriptome and Proteome Analysis of Human Lymphotropic Tumor Viruses: Insights on the Detection and Discovery of Viral Genes

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    Dresang, Lindsay R.; Teuton, Jeremy R.; Feng, Huichen; Jacobs, Jon M.; Camp, David G.; Purvine, Samuel O.; Gritsenko, Marina A.; Li, Zhihua; Smith, Richard D.; Sugden, Bill; Moore, Patrick S.; Chang, Yuan

    2011-12-20

    Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are related human tumor viruses that cause primary effusion lymphomas (PEL) and Burkitt's lymphomas (BL), respectively. Viral genes expressed in naturally-infected cancer cells contribute to disease pathogenesis; knowing which viral genes are expressed is critical in understanding how these viruses cause cancer. To evaluate the expression of viral genes, we used high-resolution separation and mass spectrometry coupled with custom tiling arrays to align the viral proteomes and transcriptomes of three PEL and two BL cell lines under latent and lytic culture conditions. Results The majority of viral genes were efficiently detected at the transcript and/or protein level on manipulating the viral life cycle. Overall the correlation of expressed viral proteins and transcripts was highly complementary in both validating and providing orthogonal data with latent/lytic viral gene expression. Our approach also identified novel viral genes in both KSHV and EBV, and extends viral genome annotation. Several previously uncharacterized genes were validated at both transcript and protein levels. Conclusions This systems biology approach coupling proteome and transcriptome measurements provides a comprehensive view of viral gene expression that could not have been attained using each methodology independently. Detection of viral proteins in combination with viral transcripts is a potentially powerful method for establishing virus-disease relationships.

  9. Herpesviruses shape tumour microenvironment through exosomal transfer of viral microRNAs.

    Directory of Open Access Journals (Sweden)

    Ohad Yogev

    2017-08-01

    Full Text Available Metabolic changes within the cell and its niche affect cell fate and are involved in many diseases and disorders including cancer and viral infections. Kaposi's sarcoma-associated herpesvirus (KSHV is the etiological agent of Kaposi's sarcoma (KS. KSHV latently infected cells express only a subset of viral genes, mainly located within the latency-associated region, among them 12 microRNAs. Notably, these miRNAs are responsible for inducing the Warburg effect in infected cells. Here we identify a novel mechanism enabling KSHV to manipulate the metabolic nature of the tumour microenvironment. We demonstrate that KSHV infected cells specifically transfer the virus-encoded microRNAs to surrounding cells via exosomes. This flow of genetic information results in a metabolic shift toward aerobic glycolysis in the surrounding non-infected cells. Importantly, this exosome-mediated metabolic reprogramming of neighbouring cells supports the growth of infected cells, thereby contributing to viral fitness. Finally, our data show that this miRNA transfer-based regulation of cell metabolism is a general mechanism used by other herpesviruses, such as EBV, as well as for the transfer of non-viral onco-miRs. This exosome-based crosstalk provides viruses with a mechanism for non-infectious transfer of genetic material without production of new viral particles, which might expose them to the immune system. We suggest that viruses and cancer cells use this mechanism to shape a specific metabolic niche that will contribute to their fitness.

  10. KSHV/HHV-8 and HIV infection in Kaposi's sarcoma development

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    Kaaya Ephata

    2007-02-01

    Full Text Available Abstract Kaposi's sarcoma (KS is a highly and abnormally vascularized tumor-like lesion affecting the skin, lymphnodes and viscera, which develops from early inflammatory stages of patch/plaque to late, nodular tumors composed predominant of spindle cells (SC. These SC are infected with the Kaposi's sarcoma-associated herpesvirus or human herpesvirus-8 (KSHV/HHV-8. KS is promoted during HIV infection by various angiogenic and pro-inflammatory factors including HIV-Tat. The latency associated nuclear antigen type 1 (LANA-1 protein is well expressed in SC, highly immunogenic and considered important in the generation and maintenance of HHV-8 associated malignancies. Various studies favour an endothelial origin of the KS SC, expressing "mixed" lymphatic and vascular endothelial cell markers, possibly representing hybrid phenotypes of endothelial cells (EC. A significant number of SC during KS development are apparently not HHV8 infected, which heterogeneity in viral permissiveness may indicate that non-infected SC may continuously be recruited in to the lesion from progenitor cells and locally triggered to develop permissiveness to HHV8 infection. In the present study various aspects of KS pathogenesis are discussed, focusing on the histopathological as well as cytogenetic and molecular genetic changes in KS.

  11. Viruses and human cancer: from detection to causality.

    Science.gov (United States)

    Sarid, Ronit; Gao, Shou-Jiang

    2011-06-28

    The study of cancer is incomplete without taking into consideration of tumorigenic viruses. Initially, searches for human cancer viruses were fruitless despite an expansion of our knowledge in the same period concerning acute-transforming retroviruses in animals. However, over the last 40 years, we have witnessed rapid progress in the tumor virology field. Currently, acknowledged human cancer viruses include Epstein-Barr virus, hepatitis B virus, hepatitis C virus, high-risk human papilloma viruses, human T-cell lymphotropic virus type 1 and Kaposi's sarcoma-associated herpesvirus. Extensive epidemiological and mechanistic studies have led to the development of novel preventive and therapeutic approaches for managing some of these infections and associated cancers. In addition, recent advances in molecular technologies have enabled the discovery of a new potential human tumor virus, Merkel cell polyomavirus, but its association with cancer remains to be validated. It is anticipated that in the next few decades many additional human cancer viruses will be discovered and the mechanisms underlying viral oncogenesis delineated. Thus, it can be expected that better tools for preventing and treating virus-associated cancer will be available in the near future. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  12. Virus infection and human cancer: an overview.

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    Schiller, John T; Lowy, Douglas R

    2014-01-01

    It is now estimated that approximately 10 % of worldwide cancers are attributable to viral infection, with the vast majority (>85 %) occurring in the developing world. Oncogenic viruses include various classes of DNA and RNA viruses and induce cancer by a variety of mechanisms. A unifying theme is that cancer develops in a minority of infected individuals and only after chronic infection of many years duration. The viruses associated with the greatest number of cancer cases are the human papillomaviruses (HPVs), which cause cervical cancer and several other epithelial malignancies, and the hepatitis viruses HBV and HCV, which are responsible for the majority of hepatocellular cancer. Other oncoviruses include Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpes virus (KSHV), human T-cell leukemia virus (HTLV-I), and Merkel cell polyomavirus (MCPyV). Identification of the infectious cause has led to several interventions that may reduce the risk of developing these tumors. These include preventive vaccines against HBV and HPV, HPV-based testing for cervical cancer screening, anti-virals for the treatment of chronic HBV and HCV infection, and screening the blood supply for the presence of HBV and HCV. Successful efforts to identify additional oncogenic viruses in human cancer may lead to further insight into etiology and pathogenesis as well as to new approaches for therapeutic and prophylactic intervention.

  13. Paraneoplastic vasculitis associated to pelvic chondrosarcoma: a case report

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    Arvinius Camilla

    2016-01-01

    Full Text Available Vasculopathic syndromes have been associated with hematological and solid organ malignancies. The pathogenesis of these syndromes remains largely unknown and there are no biologic markers identified. Whether it is or is not a paraneoplastic syndrome is under discussion, the close temporal relationship of cancer and vasculitis suggests that these vasculitides are a paraneoplastic condition. We report a case of a 45-year-old female patient with pelvic chondrosarcoma who underwent surgical treatment and started to present visual loss, systemic inflammatory response syndrome (SRIS, cardiac insufficiency, hepatosplenomegaly, cholestasis as well as pulmonary bleeding suggesting a sarcoma-associated vasculitis. All antibodies were negative as in secondary vasculitis. After corticoideal therapy the vasculitis resolved and at 3-year follow-up the patient had not showed any further medical complications or recurrences of the vasculitis. The parallel evolution of the vasculitis and the solid tumor combined with the resolution of the vasculitis after corticotherapy enhances the likelihood of a paraneoplastic vasculitis associated with a chondrosarcoma according to literature review.

  14. Simian Homologues of Human Gamma-2 and Betaherpesviruses in Mandrill and Drill Monkeys

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    Lacoste, Vincent; Mauclere, Philippe; Dubreuil, Guy; Lewis, John; Georges-Courbot, Marie-Claude; Rigoulet, Jacques; Petit, Thierry; Gessain, Antoine

    2000-01-01

    Recent serological and molecular surveys of different primate species allowed the characterization of several Kaposi's sarcoma-associated herpesvirus (KSHV) homologues in macaques, African green monkeys, chimpanzees, and gorillas. Identification of these new primate rhadinoviruses revealed the existence of two distinct genogroups, called RV1 and RV2. Using a degenerate consensus primer PCR method for the herpesvirus DNA polymerase gene, the presence of KSHV homologues has been investigated in two semi-free-ranging colonies of eight drill (Mandrillus leucophaeus), five mandrill (Mandrillus sphinx), and two hybrid (Mandrillus leucophaeus-Mandrillus sphinx) monkeys, living in Cameroon and Gabon, Central Africa. This search revealed the existence of not only two distinct KSHV homologues, each one belonging to one of the two rhadinovirus genogroups, but also of two new betaherpesvirus sequences, one being close to cytomegaloviruses and the other being related to human herpesviruses 6 and 7 (HHV-6 and -7). The latter viruses are the first simian HHV-6 and -7 homologues identified to date. These data show that mandrill and drill monkeys are the hosts of at least four novel distinct herpesviruses. Moreover, mandrills, like macaques and African green monkeys, harbor also two distinct gamma-2 herpesviruses, thus strongly suggesting that a second gamma-2 herpesvirus, belonging to the RV2 genogroup, may exist in humans. PMID:11090203

  15. Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX.

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    Marta Maria Gaglia

    2015-12-01

    Full Text Available Many viruses express factors that reduce host gene expression through widespread degradation of cellular mRNA. An example of this class of proteins is the mRNA-targeting endoribonuclease SOX from the gamma-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV. Previous studies indicated that cleavage of messenger RNAs (mRNA by SOX occurs at specific locations defined by the sequence of the target RNA, which is at odds with the down-regulation of a large portion of cellular transcripts. In this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify SOX cleavage sites across the mRNA transcriptome. These data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. This degenerate element is well represented in both human and KSHV mRNA, and its presence correlates with RNA destabilization by SOX. This represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables RNA cleavage at specific locations without restricting the range of targets. Furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity.

  16. An RNA element in human interleukin 6 confers escape from degradation by the gammaherpesvirus SOX protein.

    Science.gov (United States)

    Hutin, Stephanie; Lee, Yeon; Glaunsinger, Britt A

    2013-04-01

    Several viruses express factors to silence host gene expression via widespread mRNA degradation. This phenotype is the result of the coordinated activity of the viral endonuclease SOX and the cellular RNA degradation enzyme Xrn1 during lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection. While most cellular transcripts are highly downregulated, a subset of host mRNA escapes turnover via unknown mechanisms. One of the most prominent escapees is the interleukin 6 (IL-6) mRNA, which accumulates robustly during KSHV lytic infection and is not subjected to SOX-induced degradation. Here we reveal that the IL-6 mRNA contains a dominant, cis-acting ∼100-nucleotide element within its 3' untranslated region (UTR) that renders it directly refractory to cleavage by SOX. This element specifically interacts with a cellular protein complex both in SOX-transfected cells and in KSHV-infected B cells. Using a directed RNA pulldown approach, we identified two components of this complex to be the AU-rich element (ARE) binding proteins AUF1 and HuR. Depletion of these proteins significantly reduced the protective capacity of the IL-6 RNA element in SOX-expressing cells. These findings suggest that SOX activity may be directly counteracted by select RNA regulatory complexes and reveal a novel mechanism contributing to the robust expression of IL-6 during KSHV replication.

  17. Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX

    Science.gov (United States)

    Gaglia, Marta Maria; Rycroft, Chris H.; Glaunsinger, Britt A.

    2015-01-01

    Many viruses express factors that reduce host gene expression through widespread degradation of cellular mRNA. An example of this class of proteins is the mRNA-targeting endoribonuclease SOX from the gamma-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV). Previous studies indicated that cleavage of messenger RNAs (mRNA) by SOX occurs at specific locations defined by the sequence of the target RNA, which is at odds with the down-regulation of a large portion of cellular transcripts. In this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify SOX cleavage sites across the mRNA transcriptome. These data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. This degenerate element is well represented in both human and KSHV mRNA, and its presence correlates with RNA destabilization by SOX. This represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables RNA cleavage at specific locations without restricting the range of targets. Furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity. PMID:26646420

  18. Aberrant herpesvirus-induced polyadenylation correlates with cellular messenger RNA destruction.

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    Yeon J Lee

    2009-05-01

    Full Text Available Regulation of messenger RNA (mRNA stability plays critical roles in controlling gene expression, ensuring transcript fidelity, and allowing cells to respond to environmental cues. Unregulated enhancement of mRNA turnover could therefore dampen cellular responses to such signals. Indeed, several herpesviruses instigate widespread destruction of cellular mRNAs to block host gene expression and evade immune detection. Kaposi's sarcoma-associated herpesvirus (KSHV promotes this phenotype via the activity of its viral SOX protein, although the mechanism of SOX-induced mRNA turnover has remained unknown, given its apparent lack of intrinsic ribonuclease activity. Here, we report that KSHV SOX stimulates cellular transcriptome turnover via a unique mechanism involving aberrant polyadenylation. Transcripts in SOX-expressing cells exhibit extended poly(A polymerase II-generated poly(A tails and polyadenylation-linked mRNA turnover. SOX-induced polyadenylation changes correlate with its RNA turnover function, and inhibition of poly(A tail formation blocks SOX activity. Both nuclear and cytoplasmic poly(A binding proteins are critical cellular cofactors for SOX function, the latter of which undergoes striking nuclear relocalization by SOX. SOX-induced mRNA turnover therefore represents both a novel mechanism of host shutoff as well as a new model system to probe the regulation of poly(A tail-stimulated mRNA turnover in mammalian cells.

  19. Host transcript accumulation during lytic KSHV infection reveals several classes of host responses.

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    Sanjay Chandriani

    Full Text Available Lytic infection by Kaposi's sarcoma-associated herpesvirus (KSHV is associated with an extensive shutoff of host gene expression, mediated chiefly by accelerated mRNA turnover due to expression of the viral SOX protein. We have previously identified a small number of host mRNAs that can escape SOX-mediated degradation. Here we present a detailed, transcriptome-wide analysis of host shutoff, with careful microarray normalization to allow rigorous determination of the magnitude and extent of transcript loss. We find that the extent of transcript reduction represents a continuum of susceptibilities of transcripts to virus-mediated shutoff. Our results affirm that the levels of over 75% of host transcripts are substantially reduced during lytic infection, but also show that another approximately 20% of cellular mRNAs declines only slightly (less than 2-fold during the course of infection. Approximately 2% of examined cellular genes are strongly upregulated during lytic infection, most likely due to transcriptional induction of mRNAs that display intrinsic SOX-resistance.

  20. Evolutionarily conserved herpesviral protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Even Fossum

    2009-09-01

    Full Text Available Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV and Kaposi's sarcoma-associated herpesvirus (KSHV. In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1, murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H, and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species.

  1. Anti-CD20 Monoclonal Antibody Treatment of Human Herpesvirus 8-Associated, Body Cavity-Based Lymphoma with an Unusual Phenotype in a Human Immunodeficiency Virus-Negative Patient

    Science.gov (United States)

    Pérez, Celeste L.; Rudoy, Silvia

    2001-01-01

    Human herpesvirus 8 (HHV-8), or Kaposi's sarcoma-associated herpesvirus, is a gammaherpesvirus first detected in Kaposi's sarcoma tumor cells and subsequently in primary effusion lymphoma (PEL) tumor cells and peripheral blood mononuclear cells from PEL patients. PEL has been recognized as an individual nosologic entity based on its distinctive features and consistent association with HHV-8 infection. PEL is an unusual form of body cavity-based B-cell lymphoma (BCBL). It occurs predominantly in human immunodeficiency virus (HIV)-positive patients but occasionally also in elderly HIV-negative patients. We describe a case of PEL, with ascites, bilateral pleural effusions, and a small axillary lymphadenopathy, in a 72-year-old HIV-negative man. PCR performed on a lymph node specimen and in liquid effusion was positive for HHV-8 and negative for Epstein-Barr virus. The immunophenotype of the neoplastic cells was B CD19+ CD20+ CD22+ with coexpression of CD10 and CD23 and with clonal kappa light chain rearrangement. The patient was treated with Rituximab, a chimeric (human-mouse) anti-CD20 monoclonal antibody. Thirteen months later, the patient continued in clinical remission. This is the first report of an HHV-8-associated BCBL in an HIV-negative patient in Argentina. PMID:11527816

  2. Anti-viral treatment and cancer control.

    Science.gov (United States)

    Shih, Wei-Liang; Fang, Chi-Tai; Chen, Pei-Jer

    2014-01-01

    Hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), and Epstein-Barr virus (EBV) contribute to about 10-15 % global burden of human cancers. Conventional chemotherapy or molecular target therapies have been used to treat virus-associated cancers. However, a more proactive approach would be the use of antiviral treatment to suppress or eliminate viral infections to prevent the occurrence of cancer in the first place. Antiviral treatments against chronic HBV and HCV infections have achieved this goal, with significant reduction in the incidence of hepatocellular carcinoma in treated patients. Antiviral treatments for EBV, Kaposi's sarcoma-associated herpesvirus (KSHV), and human T-cell lymphotropic virus type 1 (HTLV-1) had limited success in treating refractory EBV-associated lymphoma and post-transplant lymphoproliferative disorder, KSHV-associated Kaposi's sarcoma in AIDS patients, and HTLV-1-associated acute, chronic, and smoldering subtypes of adult T-cell lymphoma, respectively. Therapeutic HPV vaccine and RNA-interference-based therapies for treating HPV-associated cervical cancers also showed some encouraging results. Taken together, antiviral therapies have yielded promising results in cancer prevention and treatment. More large-scale studies are necessary to confirm the efficacy of antiviral therapy. Further investigation for more effective and convenient antiviral regimens warrants more attention.

  3. Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes

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    Rahayu, Retno; Ohsaki, Eriko [Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Omori, Hiroko [Central Instrumentation Laboratory Research Institute for Microbial Diseases (BIKEN), Osaka University, Osaka 565-0871 (Japan); Ueda, Keiji, E-mail: kueda@virus.med.osaka-u.ac.jp [Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan)

    2016-09-15

    In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy–electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres, and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division. - Highlights: • This is the first report showing LANA dots on mitotic chromosomes by fluorescent microscopy followed by electron microscopy. • LANA dots localized randomly on condensed chromosomes other than centromere/pericentromere and telomere/peritelomre. • Cellular mitotic checkpoint should not be always involved in the segregation of KSHV genomes in the latency.

  4. Non-Detection of Human Herpesvirus 8 (HHV-8) DNA in HHV-8-Seropositive Blood Donors from Three Brazilian Regions

    Science.gov (United States)

    Levi, José Eduardo; Nascimento, Maria Claudia; Sumita, Laura Masami; de Souza, Vanda Akico Ueda Fick; Freire, Wilton S.; Mayaud, Philippe; Pannuti, Claudio S.

    2011-01-01

    Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is the etiologic agent of all forms of Kaposi's sarcoma, primary effusion lymphoma and the plasmablastic cell variant of multicentric Castleman disease. In endemic areas of sub-Saharan Africa, blood transfusions have been associated with a substantial risk of HHV-8 transmission. By contrast, several studies among healthy blood donors from North America have failed to detect HHV-8 DNA in samples of seropositive individuals. In this study, using a real-time PCR assay, we investigated the presence of HHV-8 DNA in whole-blood samples of 803 HHV-8 blood donors from three Brazilian states (São Paulo, Amazon, Bahia) who tested positive for HHV-8 antibodies, in a previous multicenter study. HHV-8 DNA was not detected in any sample. Our findings do not support the introduction of routine HHV-8 screening among healthy blood donors in Brazil. (WC = 140). PMID:21858163

  5. Manipulation of the host cell membrane by human γ-herpesviruses EBV and KSHV for pathogenesis.

    Science.gov (United States)

    Wei, Fang; Zhu, Qing; Ding, Ling; Liang, Qing; Cai, Qiliang

    2016-10-01

    The cell membrane regulates many physiological processes including cellular communication, homing and metabolism. It is therefore not surprising that the composition of the host cell membrane is manipulated by intracellular pathogens. Among these, the human oncogenic herpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) exploit the host cell membrane to avoid immune surveillance and promote viral replication. Accumulating evidence has shown that both EBV and KSHV directly encode several similar membrane-associated proteins, including receptors and receptor-specific ligands (cytokines and chemokines), to increase virus fitness in spite of host antiviral immune responses. These proteins are expressed individually at different phases of the EBV/KSHV life cycle and employ various mechanisms to manipulate the host cell membrane. In recent decades, much effort has been made to address how these membrane-based signals contribute to viral tumorigenesis. In this review, we summarize and highlight the recent understanding of how EBV and KSHV similarly manipulate host cell membrane signals, particularly how remodeling of the cell membrane allows EBV and KSHV to avoid host antiviral immune responses and favors their latent and lytic infection.

  6. Proteomic screening of human targets of viral microRNAs reveals functions associated with immune evasion and angiogenesis.

    Directory of Open Access Journals (Sweden)

    Amelia M Gallaher

    Full Text Available Kaposi's sarcoma (KS is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV. The virus expresses unique microRNAs (miRNAs, but the targets and functions of these miRNAs are not completely understood. In order to identify human targets of viral miRNAs, we measured protein expression changes caused by multiple KSHV miRNAs using pulsed stable labeling with amino acids in cell culture (pSILAC in primary endothelial cells. This led to the identification of multiple human genes that are repressed at the protein level, but not at the miRNA level. Further analysis also identified that KSHV miRNAs can modulate activity or expression of upstream regulatory factors, resulting in suppressed activation of a protein involved in leukocyte recruitment (ICAM1 following lysophosphatidic acid treatment, as well as up-regulation of a pro-angiogenic protein (HIF1α, and up-regulation of a protein involved in stimulating angiogenesis (HMOX1. This study aids in our understanding of miRNA mechanisms of repression and miRNA contributions to viral pathogenesis.

  7. Haploid genetic screens identify an essential role for PLP2 in the downregulation of novel plasma membrane targets by viral E3 ubiquitin ligases.

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    Richard T Timms

    Full Text Available The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2, a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

  8. Tumour viruses and innate immunity.

    Science.gov (United States)

    Hopcraft, Sharon E; Damania, Blossom

    2017-10-19

    Host cells sense viral infection through pattern recognition receptors (PRRs), which detect pathogen-associated molecular patterns (PAMPs) and stimulate an innate immune response. PRRs are localized to several different cellular compartments and are stimulated by viral proteins and nucleic acids. PRR activation initiates signal transduction events that ultimately result in an inflammatory response. Human tumour viruses, which include Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus, human papillomavirus, hepatitis C virus, hepatitis B virus, human T-cell lymphotropic virus type 1 and Merkel cell polyomavirus, are detected by several different PRRs. These viruses engage in a variety of mechanisms to evade the innate immune response, including downregulating PRRs, inhibiting PRR signalling, and disrupting the activation of transcription factors critical for mediating the inflammatory response, among others. This review will describe tumour virus PAMPs and the PRRs responsible for detecting viral infection, PRR signalling pathways, and the mechanisms by which tumour viruses evade the host innate immune system.This article is part of the themed issue 'Human oncogenic viruses'. © 2017 The Author(s).

  9. Nuclease escape elements protect messenger RNA against cleavage by multiple viral endonucleases.

    Science.gov (United States)

    Muller, Mandy; Glaunsinger, Britt A

    2017-08-01

    During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, the viral endonu- clease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs, including the transcript encoding interleukin-6 (IL-6), escape SOX-induced cleavage. IL-6 escape is mediated through a 3' UTR RNA regulatory element that overrides the SOX targeting mechanism. Here, we reveal that this protective RNA element functions to broadly restrict cleavage by a range of homologous and non-homologous viral endonucleases. However, it does not impede cleavage by cellular endonucleases. The IL-6 protective sequence may be representative of a larger class of nuclease escape elements, as we identified a similar protective element in the GADD45B mRNA. The IL-6 and GADD45B-derived elements display similarities in their sequence, putative structure, and several associated RNA binding proteins. However, the overall composition of their ribonucleoprotein complexes appears distinct, leading to differences in the breadth of nucleases restricted. These findings highlight how RNA elements can selectively control transcript abundance in the background of widespread virus-induced mRNA degradation.

  10. Animal herpesviruses and their zoonotic potential for cross-species infection

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    Grzegorz Woźniakowski

    2015-05-01

    Full Text Available Herpesviruses of humans and animals cause severe diseases that influence not only the health and epidemiological status but are also economically important in the context of food production. The members of[i] Herpesviridae[/i] are host specific agents that also share many properties that potentially make them capable of crossing the species barriers. The objective of presented review paper was to summarize the relationship between herpesviruses of animals and humans and their zoonotic potential. In humans, the most epidemiologically important herpesviruses are represented by Human herepesvirus-1 and Human herpesvirus-2, which are commonly known as herpes simplex virus type 1 and 2, varicella-zooster virus (VZV, Epstein-Barr virus (EBV, Kaposi’s Sarcoma-associated herpesvirus (KSHV, cytomegalovirus (CMV, as well as Human herpesviruses: HHV-6A, HHV-6B, and HHV-7. However, in terms of the potential to cross the species barrier, there are a few herpesviruses, including B virus disease (CeHV-1, Marek’s disease virus (MDV, Equid herpesvirus-1 (EHV-1 or pseudorabies virus (PRV, which are potentially able to infect different hosts. To summarize, in advantageous conditions the host specific herpesviruses may pose a threat for public health but also may exert a negative impact on the economical aspects of animal production. The most probable of these are zoonotic infections caused by B virus disease; however, close contact between infected animal hosts and humans may lead to transmission and replication of other [i]Herpesviridae[/i] members.

  11. Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses.

    Science.gov (United States)

    Pentland, Ieisha; Parish, Joanna L

    2015-07-06

    All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF) is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV) utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions.

  12. Insights into the broad cellular effects of nelfinavir and the HIV protease inhibitors supporting their role in cancer treatment and prevention.

    Science.gov (United States)

    Gantt, Soren; Casper, Corey; Ambinder, Richard F

    2013-09-01

    The development of HIV protease inhibitors more than two decades ago heralded a new era in HIV care, changing the infection from universally fatal to chronic but controllable. With the widespread use of protease inhibitors, there was a reduction in the incidence and mortality of HIV-associated malignancies. Studies later found these drugs to have promising direct antitumor effects. Protease inhibitors have a wide range of effects on several cellular pathways that are important for tumorigenesis and independent of inhibition of the HIV protease, including reducing angiogenesis and cell invasion, inhibition of the Akt pathway, induction of autophagy, and promotion of apoptosis. Among protease inhibitors, nelfinavir appears to have the most potent and broad antineoplastic activities, and also affects replication of the oncogenic herpesviruses Kaposi sarcoma-associated herpesvirus and Epstein-Barr virus. Nelfinavir is being studied for the prevention and treatment of a wide range of malignancies in persons with and without HIV infection. Nelfinavir and other protease inhibitors are well tolerated, oral drugs that have promising antitumor properties, and may prove to play an important role in the prevention and treatment of several cancers. Additional insights into protease inhibitors' mechanisms of action may lead to the development of novel cancer chemotherapy agents.

  13. G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV.

    Science.gov (United States)

    Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P; Robertson, Erle S; Schildkraut, Carl L; Verma, Subhash C

    2016-05-05

    Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Expansion of effector memory TCR Vbeta4+ CD8+ T cells is associated with latent infection-mediated resistance to transplantation tolerance.

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    Stapler, Dale; Lee, Eun D; Selvaraj, Saranya A; Evans, Andrew G; Kean, Leslie S; Speck, Samuel H; Larsen, Christian P; Gangappa, Shivaprakash

    2008-03-01

    Therapies that control largely T cell-dependent allograft rejection in humans also possess the undesirable effect of impairing T cell function, leaving transplant recipients susceptible to opportunistic viruses. Prime among these opportunists are the ubiquitous herpesviruses. To date, studies are lacking that address the effect of viruses that establish a true latent state on allograft tolerance or the effect of tolerance protocols on the immune control of latent viruses. By using a mixed chimerism-based tolerance-induction protocol, we found that mice undergoing latent infection with gammaHV68, a murine gamma-herpesvirus closely related to human gamma-herpesviruses such as EBV and Kaposi's sarcoma-associated herpesvirus, significantly resist tolerance to allografts. Limiting the degree of virus reactivation or innate immune response did not reconstitute chimerism in latently infected mice. However, gammaHV68-infected mice showed increased frequency of CD8+ T cell alloreactivity and, interestingly, expansion of virus-induced, alloreactive, "effector/effector memory" TCR Vbeta4+CD8+ T cells driven by the gammaHV68-M1 gene was associated with resistance to tolerance induction in studies using gammaHV68-M1 mutant virus. These results define the viral gene and immune cell types involved in latent infection-mediated resistance to allograft tolerance and underscore the influence of latent herpesviruses on allograft survival.

  15. AIDS-related lymphomas: from pathogenesis to pathology.

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    Carbone, Antonino; Gloghini, Annunziata

    2005-09-01

    Human immunodeficiency virus (HIV)-associated lymphomas include: (1) lymphomas also occurring, although sporadically, in the absence of HIV infection. The vast majority of these lymphomas are high-grade B-cell lymphomas: Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) with centroblastic (CB) features and DLBCL with immunoblastic (IBL) features; (2) unusual lymphomas occurring more specifically in HIV-positive patients and include two rare entities, namely 'primary effusion lymphoma' (PEL) and 'plasmablastic lymphoma' of the oral cavity. The pathological heterogeneity of acquired immunodeficiency syndrome-associated non-Hodgkin's lymphomas (AIDS-NHL) reflects the heterogeneity of their associated molecular lesions. In AIDS-BL, the molecular lesions involve activation of cMYC, inactivation of P53, and infection with Epstein-Barr virus (EBV). AIDS-IBL infected with EBV are characterised by frequent expression of latent membrane protein 1--an EBV oncoprotein. The biological heterogeneity of AIDS-NHL is highlighted by their histogenetic differences. Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV8)-associated lymphomas, which often develop in persons with advanced AIDS, present predominantly as PEL. KSHV/HHV8 has also been recently detected in solid extracavitary-based lymphomas. The KSHV/HHV8-associated solid lymphomas are (1) unusual lymphomas that occur more specifically in HIV-positive patients; (2) extracavitary and arise in nodal and/or extranodal sites; and (3) histologically, they usually display a PEL-like morphology and plasma cell-related phenotype.

  16. Systematic analysis of a xenograft mice model for KSHV+ primary effusion lymphoma (PEL.

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    Lu Dai

    Full Text Available Kaposi's sarcoma-associated herpesvirus is the causative agent of primary effusion lymphoma (PEL, which arises preferentially in the setting of infection with human immunodeficiency virus (HIV. Even with standard cytotoxic chemotherapy, PEL continues to cause high mortality rates, requiring the development of novel therapeutic strategies. PEL xenograft models employing immunodeficient mice have been used to study the in vivo effects of a variety of therapeutic approaches. However, it remains unclear whether these xenograft models entirely reflect clinical presentations of KSHV(+ PEL, especially given the recent description of extracavitary solid tumor variants arising in patients. In addition, effusion and solid tumor cells propagated in vivo exhibit unique biology, differing from one another or from their parental cell lines propagated through in vitro culture. Therefore, we used a KSHV(+ PEL/BCBL-1 xenograft model involving non-obese diabetic/severe-combined immunodeficient (NOD/SCID mice, and compared characteristics of effusion and solid tumors with their parent cell culture-derived counterparts. Our results indicate that although this xenograft model can be used for study of effusion and solid lymphoma observed in patients, tumor cells in vivo display unique features to those passed in vitro, including viral lytic gene expression profile, rate of solid tumor development, the host proteins and the complex of tumor microenvironment. These items should be carefully considered when the xenograft model is used for testing novel therapeutic strategies against KSHV-related lymphoma.

  17. KSHV ORF57, a Protein of Many Faces

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    Vladimir Majerciak

    2015-02-01

    Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV ORF57 protein (also known as mRNA transcript accumulation (Mta is a potent posttranscriptional regulator essential for the efficient expression of KSHV lytic genes and productive KSHV replication. ORF57 possesses numerous activities that promote the expression of viral genes, including the three major functions of enhancement of RNA stability, promotion of RNA splicing, and stimulation of protein translation. The multifunctional nature of ORF57 is driven by its ability to interact with an array of cellular cofactors. These interactions are required for the formation of ORF57-containing ribonucleoprotein complexes at specific binding sites in the target transcripts, referred as Mta-responsive elements (MREs. Understanding of the ORF57 protein conformation has led to the identification of two structurally-distinct domains within the ORF57 polypeptide: an unstructured intrinsically disordered N-terminal domain and a structured α-helix-rich C-terminal domain. The distinct structures of the domains serve as the foundation for their unique binding affinities: the N-terminal domain mediates ORF57 interactions with cellular cofactors and target RNAs, and the C-terminal domain mediates ORF57 homodimerization. In addition, each domain has been found to contribute to the stability of ORF57 protein in infected cells by counteracting caspase- and proteasome-mediated degradation pathways. Together, these new findings provide insight into the function and biological properties of ORF57 in the KSHV life cycle and pathogenesis.

  18. BST2/Tetherin enhances entry of human cytomegalovirus.

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    Kasinath Viswanathan

    2011-11-01

    Full Text Available Interferon-induced BST2/Tetherin prevents budding of vpu-deficient HIV-1 by tethering mature viral particles to the plasma membrane. BST2 also inhibits release of other enveloped viruses including Ebola virus and Kaposi's sarcoma associated herpesvirus (KSHV, indicating that BST2 is a broadly acting antiviral host protein. Unexpectedly however, recovery of human cytomegalovirus (HCMV from supernatants of BST2-expressing human fibroblasts was increased rather than decreased. Furthermore, BST2 seemed to enhance viral entry into cells since more virion proteins were released into BST2-expressing cells and subsequent viral gene expression was elevated. A significant increase in viral entry was also observed upon induction of endogenous BST2 during differentiation of the pro-monocytic cell line THP-1. Moreover, treatment of primary human monocytes with siRNA to BST2 reduced HCMV infection, suggesting that BST2 facilitates entry of HCMV into cells expressing high levels of BST2 either constitutively or in response to exogenous stimuli. Since BST2 is present in HCMV particles we propose that HCMV entry is enhanced via a reverse-tethering mechanism with BST2 in the viral envelope interacting with BST2 in the target cell membrane. Our data suggest that HCMV not only counteracts the well-established function of BST2 as inhibitor of viral egress but also employs this anti-viral protein to gain entry into BST2-expressing hematopoietic cells, a process that might play a role in hematogenous dissemination of HCMV.

  19. Molecularly targeted therapy for Kaposi's sarcoma in a kidney transplant patient: case report, "what worked and what did not"

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    Correa-Rotter Ricardo

    2007-03-01

    Full Text Available Abstract Background Imatinib is a tyrosine-kinase inhibitor; for which there is limited information regarding its effects on AIDS Kaposi's sarcoma and none in patients with transplant-associated Kaposi's sarcoma. Sirolimus, an immunosuppressive drug used for kidney transplant, exhibits antiangiogenic activity related to impaired production of VEGF (vascular endothelial growth factor, clinical benefit has been reported in Kaposi's sarcoma associated with renal graft. Case Presentation Here we report a case of an 80 year old male, who developed Kaposi's Sarcoma nine months after receiving a living non-related donor kidney transplant at age 74. Three years after treatment with different chemotherapeutic agents for progressive cutaneous Kaposi's Sarcoma with no visceral involvement, he was prescribed Imatinib (200 mg/day for two weeks followed by 400 mg/day after four weeks of treatment he developed anasarca, further progression of KS and agranulocytosis. Imatinib was discontinued and there was significant clinical recovery. One year later his immunosuppressive therapy was changed to Sirolimus and regression of the Kaposi's sarcoma occurred. Conclusion The lack of benefit and severe toxicity associated with the use of Imatinib in this patient should alert clinicians of potentially adverse consequence of its use in patients with transplant associated Kaposi's sarcoma. On the other hand the positive response seen in this patient to Sirolimus even after a long evolution of Kaposi's sarcoma, multiple chemotherapy regimens and extensive cutaneous disease further suggest it therapeutical utility for transplant associated Kaposi's sarcoma.

  20. Integrated systems biology analysis of KSHV latent infection reveals viral induction and reliance on peroxisome mediated lipid metabolism.

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    Sychev, Zoi E; Hu, Alex; DiMaio, Terri A; Gitter, Anthony; Camp, Nathan D; Noble, William S; Wolf-Yadlin, Alejandro; Lagunoff, Michael

    2017-03-01

    Kaposi's Sarcoma associated Herpesvirus (KSHV), an oncogenic, human gamma-herpesvirus, is the etiological agent of Kaposi's Sarcoma the most common tumor of AIDS patients world-wide. KSHV is predominantly latent in the main KS tumor cell, the spindle cell, a cell of endothelial origin. KSHV modulates numerous host cell-signaling pathways to activate endothelial cells including major metabolic pathways involved in lipid metabolism. To identify the underlying cellular mechanisms of KSHV alteration of host signaling and endothelial cell activation, we identified changes in the host proteome, phosphoproteome and transcriptome landscape following KSHV infection of endothelial cells. A Steiner forest algorithm was used to integrate the global data sets and, together with transcriptome based predicted transcription factor activity, cellular networks altered by latent KSHV were predicted. Several interesting pathways were identified, including peroxisome biogenesis. To validate the predictions, we showed that KSHV latent infection increases the number of peroxisomes per cell. Additionally, proteins involved in peroxisomal lipid metabolism of very long chain fatty acids, including ABCD3 and ACOX1, are required for the survival of latently infected cells. In summary, novel cellular pathways altered during herpesvirus latency that could not be predicted by a single systems biology platform, were identified by integrated proteomics and transcriptomics data analysis and when correlated with our metabolomics data revealed that peroxisome lipid metabolism is essential for KSHV latent infection of endothelial cells.

  1. Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa [version 3; referees: 2 approved

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    Érica L. Fonseca

    2015-11-01

    Full Text Available The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF, a short 5’ untranslated region (UTR and a 3’ recombination site (attC. Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-blaGES-1/aacA4 gene cassette array, which harbours a fused gene cassette represented by blaGES-1/aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR in order to assess the transcription mechanism of blaGES-1/aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR were performed to detect the free circular forms of gcu14, blaGES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette blaGES-1/aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW, had a higher transcription level than blaGES-1/aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream blaGES-1/aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-blaGES-1/aacA4 free circular forms, but not to blaGES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription

  2. Evolution of cocirculating varicella-zoster virus genotypes during a chickenpox outbreak in Guinea-Bissau.

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    Depledge, Daniel P; Gray, Eleanor R; Kundu, Samit; Cooray, Samantha; Poulsen, Anja; Aaby, Peter; Breuer, Judith

    2014-12-01

    Varicella-zoster virus (VZV), a double-stranded DNA alphaherpesvirus, is associated with seasonal outbreaks of varicella in nonimmunized populations. Little is known about whether these outbreaks are associated with a single or multiple viral genotypes and whether new mutations rapidly accumulate during transmission. Here, we take advantage of a well-characterized population cohort in Guinea-Bissau and produce a unique set of 23 full-length genome sequences, collected over 7 months from eight households. Comparative sequence analysis reveals that four distinct genotypes cocirculated among the population, three of which were present during the first week of the outbreak, although no patients were coinfected, which indicates that exposure to infectious virus from multiple sources is common during VZV outbreaks. Transmission of VZV was associated with length polymorphisms in the R1 repeat region and the origin of DNA replication. In two cases, these were associated with the formation of distinct lineages and point to the possible coevolution of these loci, despite the lack of any known functional link in VZV or related herpesviruses. We show that these and all other sequenced clade 5 viruses possess a distinct R1 repeat motif that increases the acidity of an ORF11p protein domain and postulate that this has either arisen or been lost following divergence of the major clades. Thus, sequencing of whole VZV genomes collected during an outbreak has provided novel insights into VZV biology, transmission patterns, and (recent) natural history. VZV is a highly infectious virus and the causative agent of chickenpox and shingles, the latter being particularly associated with the risk of painful complications. Seasonal outbreaks of chickenpox are very common among young children, yet little is known about the dynamics of the virus during person-to-person to transmission or whether multiple distinct viruses seed and/or cocirculate during an outbreak. In this study, we have

  3. Efflux pump genes of the resistance-nodulation-division family in Burkholderia cenocepacia genome

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    Manina Giulia

    2006-07-01

    Full Text Available Abstract Background Burkholderia cenocepacia is recognized as opportunistic pathogen that can cause lung infections in cystic fibrosis patients. A hallmark of B. cenocepacia infections is the inability to eradicate the organism because of multiple intrinsic antibiotic resistance. As Resistance-Nodulation-Division (RND efflux systems are responsible for much of the intrinsic multidrug resistance in Gram-negative bacteria, this study aims to identify RND genes in the B. cenocepacia genome and start to investigate their involvement into antimicrobial resistance. Results Genome analysis and homology searches revealed 14 open reading frames encoding putative drug efflux pumps belonging to RND family in B. cenocepacia J2315 strain. By reverse transcription (RT-PCR analysis, it was found that orf3, orf9, orf11, and orf13 were expressed at detectable levels, while orf10 appeared to be weakly expressed in B. cenocepacia. Futhermore, orf3 was strongly induced by chloramphenicol. The orf2 conferred resistance to fluoroquinolones, tetraphenylphosphonium, streptomycin, and ethidium bromide when cloned and expressed in Escherichia coli KAM3, a strain lacking the multidrug efflux pump AcrAB. The orf2-overexpressing E. coli also accumulate low concentrations of ethidium bromide, which was restored to wild type level in the presence of CCCP, an energy uncoupler altering the energy of the drug efflux pump. Conclusion The 14 RND pumps gene we have identified in the genome of B. cenocepacia suggest that active efflux could be a major mechanism underlying antimicrobial resistance in this microorganism. We have characterized the ORF2 pump, one of these 14 potential RND efflux systems. Its overexpression in E. coli conferred resistance to several antibiotics and to ethidium bromide but it remains to be determined if this pump play a significant role in the antimicrobial intrinsic resistance of B. cenocepacia. The characterization of antibiotic efflux pumps in B

  4. Identification, Characterization, and Application of the Replicon Region of the Halophilic Temperate Sphaerolipovirus SNJ1.

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    Wang, Yuchen; Sima, Linshan; Lv, Jie; Huang, Suiyuan; Liu, Ying; Wang, Jiao; Krupovic, Mart; Chen, Xiangdong

    2016-07-15

    The temperate haloarchaeal virus SNJ1 displays lytic and lysogenic life cycles. During the lysogenic cycle, the virus resides in its host, Natrinema sp. strain J7-1, in the form of an extrachromosomal circular plasmid, pHH205. In this study, a 3.9-kb region containing seven predicted genes organized in two operons was identified as the minimal replicon of SNJ1. Only RepA, encoded by open reading frame 11-12 (ORF11-12), was found to be essential for replication, and its expression increased during the lytic cycle. Sequence analysis suggested that RepA is a distant homolog of HUH endonucleases, a superfamily that includes rolling-circle replication initiation proteins from various viruses and plasmids. In addition to RepA, two genetic elements located within both termini of the 3.9-kb replicon were also required for SNJ1 replication. SNJ1 genome and SNJ1 replicon-based shuttle vectors were present at 1 to 3 copies per chromosome. However, the deletion of ORF4 significantly increased the SNJ1 copy number, suggesting that the product of ORF4 is a negative regulator of SNJ1 abundance. Shuttle vectors based on the SNJ1 replicon were constructed and validated for stable expression of heterologous proteins, both in J7 derivatives and in Natrinema pallidum JCM 8980(T), suggesting their broad applicability as genetic tools for Natrinema species. Archaeal viruses exhibit striking morphological diversity and unique gene content. In this study, the minimal replicon of the temperate haloarchaeal virus SNJ1 was identified. A number of ORFs and genetic elements controlling virus genome replication, maintenance, and copy number were characterized. In addition, based on the replicon, a novel expression shuttle vector has been constructed and validated for protein expression and purification in Natrinema sp. CJ7 and Natrinema pallidum JCM 8980(T) This study not only provided mechanistic and functional insights into SNJ1 replication but also led to the development of useful genetic

  5. The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence.

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    Li, Shijun; Tan, Min; Juillard, Franceline; Ponnusamy, Rajesh; Correia, Bruno; Simas, J Pedro; Carrondo, Maria A; McVey, Colin E; Kaye, Kenneth M

    2015-11-20

    Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. A computational profiling of changes in gene expression and transcription factors induced by vFLIP K13 in primary effusion lymphoma.

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    Vasu Punj

    Full Text Available Infection with Kaposi's sarcoma associated herpesvirus (KSHV has been linked to the development of primary effusion lymphoma (PEL, a rare lymphoproliferative disorder that is characterized by loss of expression of most B cell markers and effusions in the body cavities. This unique clinical presentation of PEL has been attributed to their distinctive plasmablastic gene expression profile that shows overexpression of genes involved in inflammation, adhesion and invasion. KSHV-encoded latent protein vFLIP K13 has been previously shown to promote the survival and proliferation of PEL cells. In this study, we employed gene array analysis to characterize the effect of K13 on global gene expression in PEL-derived BCBL1 cells, which express negligible K13 endogenously. We demonstrate that K13 upregulates the expression of a number of NF-κB responsive genes involved in cytokine signaling, cell death, adhesion, inflammation and immune response, including two NF-κB subunits involved in the alternate NF-κB pathway, RELB and NFKB2. In contrast, CD19, a B cell marker, was one of the genes downregulated by K13. A comparison with K13-induced genes in human vascular endothelial cells revealed that although there was a considerable overlap among the genes induced by K13 in the two cell types, chemokines genes were preferentially induced in HUVEC with few exceptions, such as RANTES/CCL5, which was induced in both cell types. Functional studies confirmed that K13 activated the RANTES/CCL5 promoter through the NF-κB pathway. Taken collectively, our results suggest that K13 may contribute to the unique gene expression profile, immunophenotype and clinical presentation that are characteristics of KSHV-associated PEL.

  7. Pyrrolidinium fullerene induces apoptosis by activation of procaspase-9 via suppression of Akt in primary effusion lymphoma.

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    Watanabe, Tadashi; Nakamura, Shigeo; Ono, Toshiya; Ui, Sadaharu; Yagi, Syota; Kagawa, Hiroki; Watanabe, Hisami; Ohe, Tomoyuki; Mashino, Tadahiko; Fujimuro, Masahiro

    2014-08-15

    Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin's B-cell lymphoma and is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. In general, PEL cells are derived from post-germinal center B-cells and are infected with KSHV. To evaluate potential novel anti-tumor compounds against KSHV-associated PEL, seven water-soluble fullerene derivatives were evaluated as potential drug candidates for the treatment of PEL. Herein, we discovered a pyrrolidinium fullerene derivative, 1,1,1',1'-tetramethyl [60]fullerenodipyrrolidinium diiodide, which induced apoptosis of PEL cells via a novel mechanism, the caspase-9 activation by suppressing the caspase-9 phosphorylation, causing caspase-9 inactivation. Pyrrolidinium fullerene treatment reduced significantly the viability of PEL cells compared with KSHV-uninfected lymphoma cells, and induced the apoptosis of PEL cells by activating caspase-9 via procaspase-9 cleavage. Pyrrolidinium fullerene additionally reduced the Ser473 phosphorylation of Akt and Ser196 of procaspase-9. Ser473-phosphorylated Akt (i.e., activated Akt) phosphorylates Ser196 in procaspase-9, causing inactivation of procaspase-9. We also demonstrated that Akt inhibitors suppressed the proliferation of PEL cells compared with KSHV-uninfected cells. Our data therefore suggest that Akt activation is essential for cell survival in PEL and a pyrrolidinium fullerene derivative induced apoptosis by activating caspase-9 via suppression of Akt in PEL cells. In addition, we evaluated whether pyrrolidinium fullerene in combination with the HSP90 inhibitor (geldanamycin; GA) or valproate, potentiated the cytotoxic effects on PEL cells. Compared to treatment with pyrrolidinium fullerene alone, the addition of low-concentration GA or valproate enhanced the cytotoxic activity of pyrrolidinium fullerene. These results indicate that pyrrolidinium fullerene could be used as a novel therapy for the treatment of PEL

  8. A Critical Role of Glutamine and Asparagine γ-Nitrogen in Nucleotide Biosynthesis in Cancer Cells Hijacked by an Oncogenic Virus

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    Ying Zhu

    2017-08-01

    Full Text Available While glutamine is a nonessential amino acid that can be synthesized from glucose, some cancer cells primarily depend on glutamine for their growth, proliferation, and survival. Numerous types of cancer also depend on asparagine for cell proliferation. The underlying mechanisms of the glutamine and asparagine requirement in cancer cells in different contexts remain unclear. In this study, we show that the oncogenic virus Kaposi’s sarcoma-associated herpesvirus (KSHV accelerates the glutamine metabolism of glucose-independent proliferation of cancer cells by upregulating the expression of numerous critical enzymes, including glutaminase 2 (GLS2, glutamate dehydrogenase 1 (GLUD1, and glutamic-oxaloacetic transaminase 2 (GOT2, to support cell proliferation. Surprisingly, cell crisis is rescued only completely by supplementation with asparagine but minimally by supplementation with α-ketoglutarate, aspartate, or glutamate upon glutamine deprivation, implying an essential role of γ-nitrogen in glutamine and asparagine for cell proliferation. Specifically, glutamine and asparagine provide the critical γ-nitrogen for purine and pyrimidine biosynthesis, as knockdown of four rate-limiting enzymes in the pathways, including carbamoylphosphate synthetase 2 (CAD, phosphoribosyl pyrophosphate amidotransferase (PPAT, and phosphoribosyl pyrophosphate synthetases 1 and 2 (PRPS1 and PRPS2, respectively, suppresses cell proliferation. These findings indicate that glutamine and asparagine are shunted to the biosynthesis of nucleotides and nonessential amino acids from the tricarboxylic acid (TCA cycle to support the anabolic proliferation of KSHV-transformed cells. Our results illustrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway.

  9. Cancer risk in persons with HIV/AIDS in India: a review and future directions for research

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    Bhatia Kishor

    2009-03-01

    Full Text Available Abstract Background India has a large and evolving HIV epidemic. Little is known about cancer risk in Indian persons with HIV/AIDS (PHA but risk is thought to be low. Methods To describe the state of knowledge about cancer patterns in Indian PHA, we reviewed reports from the international and Indian literature. Results As elsewhere, non-Hodgkin lymphomas dominate the profile of recognized cancers, with immunoblastic/large cell diffuse lymphoma being the most common type. Hodgkin lymphoma is proportionally increased, perhaps because survival with AIDS is truncated by fatal infections. In contrast, Kaposi sarcoma is rare, in association with an apparently low prevalence of Kaposi sarcoma-associated herpesvirus. If confirmed, the reasons for the low prevalence need to be understood. Cervical, anal, vulva/vaginal and penile cancers all appear to be increased in PHA, based on limited data. The association may be confounded by sexual behaviors that transmit both HIV and human papillomavirus. Head and neck tumor incidence may also be increased, an important concern since these tumors are among the most common in India. Based on limited evidence, the increase is at buccal/palatal sites, which are associated with tobacco and betel nut chewing rather than human papillomavirus. Conclusion With improving care of HIV and better management of infections, especially tuberculosis, the longer survival of PHA in India will likely increase the importance of cancer as a clinical problem in India. With the population's geographic and social diversity, India presents unique research opportunities that can be embedded in programs targeting HIV/AIDS and other public health priorities.

  10. BET-Inhibitors Disrupt Rad21-Dependent Conformational Control of KSHV Latency.

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    Horng-Shen Chen

    2017-01-01

    Full Text Available Kaposi's Sarcoma-associated Herpesvirus (KSHV establishes stable latent infection in B-lymphocytes and pleural effusion lymphomas (PELs. During latency, the viral genome persists as an epigenetically constrained episome with restricted gene expression programs. To identify epigenetic regulators of KSHV latency, we screened a focused small molecule library containing known inhibitors of epigenetic factors. We identified JQ1, a Bromodomain and Extended Terminal (BET protein inhibitor, as a potent activator of KSHV lytic reactivation from B-cells carrying episomal KSHV. We validated that JQ1 and other BET inhibitors efficiently stimulated reactivation of KSHV from latently infected PEL cells. We found that BET proteins BRD2 and BRD4 localize to several regions of the viral genome, including the LANA binding sites within the terminal repeats (TR, as well as at CTCF-cohesin sites in the latent and lytic control regions. JQ1 did not disrupt the interaction of BRD4 or BRD2 with LANA, but did reduce the binding of LANA with KSHV TR. We have previously demonstrated a cohesin-dependent DNA-loop interaction between the latent and lytic control regions that restrict expression of ORF50/RTA and ORF45 immediate early gene transcripts. JQ1 reduced binding of cohesin subunit Rad21 with the CTCF binding sites in the latency and lytic control regions. JQ1 also reduced DNA-loop interaction between latent and lytic control regions. These findings implicate BET proteins BRD2 and BRD4 in the maintenance of KSHV chromatin architecture during latency and reveal BET inhibitors as potent activators of KSHV reactivation from latency.

  11. Kaposi’s Sarcoma Herpesvirus Genome Persistence

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    Franceline Juillard

    2016-08-01

    Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV has an etiologic role in Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castleman’s disease. These diseases are most common in immunocompromised individuals, especially those with AIDS. Similar to all herpesviruses, KSHV infection is lifelong. KSHV infection in tumor cells is primarily latent, with only a small subset of cells undergoing lytic infection. During latency, the KSHV genome persists as a multiple copy, extrachromosomal episome in the nucleus. In order to persist in proliferating tumor cells, the viral genome replicates once per cell cycle and then segregates to daughter cell nuclei. KSHV only expresses several genes during latent infection. Prominent among these genes, is the latency-associated nuclear antigen (LANA. LANA is responsible for KSHV genome persistence and also exerts transcriptional regulatory effects. LANA mediates KSHV DNA replication and in addition, is responsible for segregation of replicated genomes to daughter nuclei. LANA serves as a molecular tether, bridging the viral genome to mitotic chromosomes to ensure that KSHV DNA reaches progeny nuclei. N-terminal LANA attaches to mitotic chromosomes by binding histones H2A/H2B at the surface of the nucleosome. C-terminal LANA binds specific KSHV DNA sequence and also has a role in chromosome attachment. In addition to the essential roles of N- and C-terminal LANA in genome persistence, internal LANA sequence is also critical for efficient episome maintenance. LANA’s role as an essential mediator of virus persistence makes it an attractive target for inhibition in order to prevent or treat KSHV infection and disease.

  12. SUMO and KSHV Replication

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    Chang, Pei-Ching [Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 112, Taiwan (China); Kung, Hsing-Jien, E-mail: hkung@nhri.org.tw [Institute for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan (China); Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616 (United States); UC Davis Cancer Center, University of California, Davis, CA 95616 (United States); Division of Molecular and Genomic Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan (China)

    2014-09-29

    Small Ubiquitin-related MOdifier (SUMO) modification was initially identified as a reversible post-translational modification that affects the regulation of diverse cellular processes, including signal transduction, protein trafficking, chromosome segregation, and DNA repair. Increasing evidence suggests that the SUMO system also plays an important role in regulating chromatin organization and transcription. It is thus not surprising that double-stranded DNA viruses, such as Kaposi’s sarcoma-associated herpesvirus (KSHV), have exploited SUMO modification as a means of modulating viral chromatin remodeling during the latent-lytic switch. In addition, SUMO regulation allows the disassembly and assembly of promyelocytic leukemia protein-nuclear bodies (PML-NBs), an intrinsic antiviral host defense, during the viral replication cycle. Overcoming PML-NB-mediated cellular intrinsic immunity is essential to allow the initial transcription and replication of the herpesvirus genome after de novo infection. As a consequence, KSHV has evolved a way as to produce multiple SUMO regulatory viral proteins to modulate the cellular SUMO environment in a dynamic way during its life cycle. Remarkably, KSHV encodes one gene product (K-bZIP) with SUMO-ligase activities and one gene product (K-Rta) that exhibits SUMO-targeting ubiquitin ligase (STUbL) activity. In addition, at least two viral products are sumoylated that have functional importance. Furthermore, sumoylation can be modulated by other viral gene products, such as the viral protein kinase Orf36. Interference with the sumoylation of specific viral targets represents a potential therapeutic strategy when treating KSHV, as well as other oncogenic herpesviruses. Here, we summarize the different ways KSHV exploits and manipulates the cellular SUMO system and explore the multi-faceted functions of SUMO during KSHV’s life cycle and pathogenesis.

  13. γ-Herpesvirus load as surrogate marker of early death in HIV-1 lymphoma patients submitted to high dose chemotherapy and autologous peripheral blood stem cell transplantation.

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    Pratesi, Chiara; Zanussi, Stefania; Tedeschi, Rosamaria; Bortolin, Maria Teresa; Talamini, Renato; Rupolo, Maurizio; Scaini, Chiara; Basaglia, Giancarlo; Di Maso, Matteo; Mazzucato, Mario; Zanet, Ernesto; Tirelli, Umberto; Michieli, Mariagrazia; Carbone, Antonino; De Paoli, Paolo

    2015-01-01

    Autologous stem cell transplantation (ASCT) is a feasible procedure for human immunodeficiency virus-1 (HIV-1) lymphoma patients, whose underlying disease and intrinsic HIV-1- and ASCT-associated immunodeficiency might increase the risk for γ-herpesvirus load persistence and/or reactivation. We evaluated this hypothesis by investigating the levels of Epstein-Barr virus (EBV)- and Kaposi sarcoma-associated herpesvirus (KSHV)-DNA levels in the peripheral blood of 22 HIV-1-associated lymphoma patients during ASCT, highlighting their relationship with γ-herpesvirus lymphoma status, immunological parameters, and clinical events. EBV-DNA was detected in the pre-treatment plasma and peripheral blood mononuclear cells (PBMCs) of 12 (median 12,135 copies/mL) and 18 patients (median 417 copies/10(6) PBMCs), respectively; the values in the two compartments were correlated (r = 0.77, p = 0.0001). Only EBV-positive lymphomas showed detectable levels of plasma EBV-DNA. After debulking chemotherapy, plasma EBV-DNA was associated with lymphoma chemosensitivity (p = 0.03) and a significant higher mortality risk by multivariate Cox analysis adjusted for EBV-lymphoma status (HR, 10.46, 95% CI, 1.11-98.32, p = 0.04). After infusion, EBV-DNA was detectable in five EBV-positive lymphoma patients who died within six months. KSHV-DNA load was positive in only one patient, who died from primary effusion lymphoma. Fluctuations in levels of KSHV-DNA reflected the patient's therapy and evolution of his underlying lymphoma. Other γ-herpesvirus-associated malignancies, such as multicentric Castleman disease and Kaposi sarcoma, or end-organ complications after salvage treatment were not found. Overall, these findings suggest a prognostic and predictive value of EBV-DNA and KSHV-DNA, the monitoring of which could be a simple, complementary tool for the management of γ-herpesvirus-positive lymphomas in HIV-1 patients submitted to ASCT.

  14. Molecular biology of human herpesvirus 8: novel functions and virus-host interactions implicated in viral pathogenesis and replication.

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    Cousins, Emily; Nicholas, John

    2014-01-01

    Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is the second identified human gammaherpesvirus. Like its relative Epstein-Barr virus, HHV-8 is linked to B-cell tumors, specifically primary effusion lymphoma and multicentric Castleman's disease, in addition to endothelial-derived KS. HHV-8 is unusual in its possession of a plethora of "accessory" genes and encoded proteins in addition to the core, conserved herpesvirus and gammaherpesvirus genes that are necessary for basic biological functions of these viruses. The HHV-8 accessory proteins specify not only activities deducible from their cellular protein homologies but also novel, unsuspected activities that have revealed new mechanisms of virus-host interaction that serve virus replication or latency and may contribute to the development and progression of virus-associated neoplasia. These proteins include viral interleukin-6 (vIL-6), viral chemokines (vCCLs), viral G protein-coupled receptor (vGPCR), viral interferon regulatory factors (vIRFs), and viral antiapoptotic proteins homologous to FLICE (FADD-like IL-1β converting enzyme)-inhibitory protein (FLIP) and survivin. Other HHV-8 proteins, such as signaling membrane receptors encoded by open reading frames K1 and K15, also interact with host mechanisms in unique ways and have been implicated in viral pathogenesis. Additionally, a set of micro-RNAs encoded by HHV-8 appear to modulate expression of multiple host proteins to provide conditions conducive to virus persistence within the host and could also contribute to HHV-8-induced neoplasia. Here, we review the molecular biology underlying these novel virus-host interactions and their potential roles in both virus biology and virus-associated disease.

  15. Comprehensive analysis of LANA interacting proteins essential for viral genome tethering and persistence.

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    Subhash C Verma

    Full Text Available Kaposi's sarcoma associated herpesvirus is tightly linked to multiple human malignancies including Kaposi's sarcoma (KS, Primary Effusion Lymphoma (PEL and Multicentric Castleman's Disease (MCD. KSHV like other herpesviruses establishes life-long latency in the infected host by persisting as chromatin and tethering to host chromatin through the virally encoded protein Latency Associated Nuclear Antigen (LANA. LANA, a multifunctional protein, is capable of binding to a large number of cellular proteins responsible for transcriptional regulation of various cellular and viral pathways involved in blocking cell death and promoting cell proliferation. This leads to enhanced cell division and replication of the viral genome, which segregates faithfully in the dividing tumor cells. The mechanism of genome segregation is well known and the binding of LANA to nucleosomal proteins, throughout the cell cycle, suggests that these interactions play an important role in efficient segregation. Various biochemical methods have identified a large number of LANA binding proteins, including histone H2A/H2B, histone H1, MeCP2, DEK, CENP-F, NuMA, Bub1, HP-1, and Brd4. These nucleosomal proteins may have various functions in tethering of the viral genome during specific phases of the viral life cycle. Therefore, we performed a comprehensive analysis of their interaction with LANA using a number of different assays. We show that LANA binds to core nucleosomal histones and also associates with other host chromatin proteins including histone H1 and high mobility group proteins (HMGs. We used various biochemical assays including co-immunoprecipitation and in-vivo localization by split GFP and fluorescence resonance energy transfer (FRET to demonstrate their association.

  16. Deep sequencing reveals direct targets of gammaherpesvirus-induced mRNA decay and suggests that multiple mechanisms govern cellular transcript escape.

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    Karen Clyde

    Full Text Available One characteristic of lytic infection with gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV, Epstein-Barr virus (EBV and murine herpesvirus 68 (MHV68, is the dramatic suppression of cellular gene expression in a process known as host shutoff. The alkaline exonuclease proteins (KSHV SOX, MHV-68 muSOX and EBV BGLF5 have been shown to induce shutoff by destabilizing cellular mRNAs. Here we extend previous analyses of cellular mRNA abundance during lytic infection to characterize the effects of SOX and muSOX, in the absence of other viral genes, utilizing deep sequencing technology (RNA-seq. Consistent with previous observations during lytic infection, the majority of transcripts are downregulated in cells expressing either SOX or muSOX, with muSOX acting as a more potent shutoff factor than SOX. Moreover, most cellular messages fall into the same expression class in both SOX- and muSOX-expressing cells, indicating that both factors target similar pools of mRNAs. More abundant mRNAs are more efficiently downregulated, suggesting a concentration effect in transcript targeting. However, even among highly expressed genes there are mRNAs that escape host shutoff. Further characterization of select escapees reveals multiple mechanisms by which cellular genes can evade downregulation. While some mRNAs are directly refractory to SOX, the steady state levels of others remain unchanged, presumably as a consequence of downstream effects on mRNA biogenesis. Collectively, these studies lay the framework for dissecting the mechanisms underlying the susceptibility of mRNA to destruction during lytic gammaherpesvirus infection.

  17. SUMO and KSHV Replication

    Directory of Open Access Journals (Sweden)

    Pei-Ching Chang

    2014-09-01

    Full Text Available Small Ubiquitin-related MOdifier (SUMO modification was initially identified as a reversible post-translational modification that affects the regulation of diverse cellular processes, including signal transduction, protein trafficking, chromosome segregation, and DNA repair. Increasing evidence suggests that the SUMO system also plays an important role in regulating chromatin organization and transcription. It is thus not surprising that double-stranded DNA viruses, such as Kaposi’s sarcoma-associated herpesvirus (KSHV, have exploited SUMO modification as a means of modulating viral chromatin remodeling during the latent-lytic switch. In addition, SUMO regulation allows the disassembly and assembly of promyelocytic leukemia protein-nuclear bodies (PML-NBs, an intrinsic antiviral host defense, during the viral replication cycle. Overcoming PML-NB-mediated cellular intrinsic immunity is essential to allow the initial transcription and replication of the herpesvirus genome after de novo infection. As a consequence, KSHV has evolved a way as to produce multiple SUMO regulatory viral proteins to modulate the cellular SUMO environment in a dynamic way during its life cycle. Remarkably, KSHV encodes one gene product (K-bZIP with SUMO-ligase activities and one gene product (K-Rta that exhibits SUMO-targeting ubiquitin ligase (STUbL activity. In addition, at least two viral products are sumoylated that have functional importance. Furthermore, sumoylation can be modulated by other viral gene products, such as the viral protein kinase Orf36. Interference with the sumoylation of specific viral targets represents a potential therapeutic strategy when treating KSHV, as well as other oncogenic herpesviruses. Here, we summarize the different ways KSHV exploits and manipulates the cellular SUMO system and explore the multi-faceted functions of SUMO during KSHV’s life cycle and pathogenesis.

  18. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.

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    Denis Avey

    2015-07-01

    Full Text Available Kaposi's Sarcoma-Associated Herpesvirus (KSHV is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK mitogen-activated protein kinase (MAPK pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5' UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45

  19. Unbiased mutagenesis of MHV68 LANA reveals a DNA-binding domain required for LANA function in vitro and in vivo.

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    Clinton R Paden

    2012-09-01

    Full Text Available The Latency-Associated Nuclear Antigen (LANA, encoded by ORF73, is a conserved gene among the γ2-herpesviruses (rhadinoviruses. The Kaposi's Sarcoma-Associated Herpesvirus (KSHV LANA is consistently expressed in KSHV-associated malignancies. In the case of the rodent γ2-herpesvirus, murine gammaherpesvirus 68 (MHV68, the LANA homolog (mLANA is required for efficient virus replication, reactivation from latency and immortalization of murine fetal liver-derived B cells. To gain insights into mLANA function(s, knowing that KSHV LANA binds DNA and can modulate transcription of a variety of promoters, we sought out and identified a mLANA-responsive promoter which maps to the terminal repeat (TR of MHV68. Notably, mLANA strongly repressed activity from this promoter. We extended these analyses to demonstrate direct, sequence-specific binding of recombinant mLANA to TR DNA by DNase I footprinting. To assess whether the DNA-binding and/or transcription modulating function is important in the known mLANA phenotypes, we generated an unbiased library of mLANA point mutants using error-prone PCR, and screened a large panel of mutants for repression of the mLANA-responsive promoter to identify loss of function mutants. Notably, among the mutant mLANA proteins recovered, many of the mutations are in a predicted EBNA-1-like DNA-binding domain. Consistent with this prediction, those tested displayed loss of DNA binding activity. We engineered six of these mLANA mutants into the MHV68 genome and tested the resulting mutant viruses for: (i replication fitness; (ii efficiency of latency establishment; and (iii reactivation from latency. Interestingly, each of these mLANA-mutant viruses exhibited phenotypes similar to the mLANA-null mutant virus, indicating that DNA-binding is critical for mLANA function.

  20. KSHV LANA and EBV LMP1 induce the expression of UCH-L1 following viral transformation

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    Bentz, Gretchen L.; Bheda-Malge, Anjali; Wang, Ling [Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill (United States); Shackelford, Julia [Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill (United States); Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill (United States); Damania, Blossom [Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill (United States); Departments of Medicine and of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC (United States); Pagano, Joseph S., E-mail: joseph_pagano@med.unc.edu [Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill (United States); Departments of Medicine and of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC (United States)

    2014-01-05

    Ubiquitin C-terminal Hydrolase L1 (UCH-L1) has oncogenic properties and is highly expressed during malignancies. We recently documented that Epstein-Barr virus (EBV) infection induces uch-l1 expression. Here we show that Kaposi's Sarcoma-associated herpesvirus (KSHV) infection induced UCH-L1 expression, via cooperation of KSHV Latency-Associated Nuclear Antigen (LANA) and RBP-Jκ and activation of the uch-l1 promoter. UCH-L1 expression was also increased in Primary Effusion Lymphoma (PEL) cells co-infected with KSHV and EBV compared with PEL cells infected only with KSHV, suggesting EBV augments the effect of LANA on uch-l1. EBV latent membrane protein 1 (LMP1) is one of the few EBV products expressed in PEL cells. Results showed that LMP1 was sufficient to induce uch-l1 expression, and co-expression of LMP1 and LANA had an additive effect on uch-l1 expression. These results indicate that viral latency products of both human γ-herpesviruses contribute to uch-l1 expression, which may contribute to the progression of lymphoid malignancies. - Highlights: • Infection of endothelial cells with KSHV induced UCH-L1 expression. • KSHV LANA is sufficient for the induction of uch-l1. • Co-infection with KSHV and EBV (observed in some PELs) results in the additive induction of uch-l1. • EBV LMP1 also induced UCH-L1 expression. • LANA- and LMP1-mediated activation of the uch-l1 promoter is in part through RBP-Jκ.

  1. An Oncogenic Virus Promotes Cell Survival and Cellular Transformation by Suppressing Glycolysis.

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    Ying Zhu

    2016-05-01

    Full Text Available Aerobic glycolysis is essential for supporting the fast growth of a variety of cancers. However, its role in the survival of cancer cells under stress conditions is unclear. We have previously reported an efficient model of gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV-induced cellular transformation of rat primary mesenchymal stem cells. KSHV-transformed cells efficiently induce tumors in nude mice with pathological features reminiscent of Kaposi's sarcoma tumors. Here, we report that KSHV promotes cell survival and cellular transformation by suppressing aerobic glycolysis and oxidative phosphorylation under nutrient stress. Specifically, KSHV microRNAs and vFLIP suppress glycolysis by activating the NF-κB pathway to downregulate glucose transporters GLUT1 and GLUT3. While overexpression of the transporters rescues the glycolytic activity, it induces apoptosis and reduces colony formation efficiency in softagar under glucose deprivation. Mechanistically, GLUT1 and GLUT3 inhibit constitutive activation of the AKT and NF-κB pro-survival pathways. Strikingly, GLUT1 and GLUT3 are significantly downregulated in KSHV-infected cells in human KS tumors. Furthermore, we have detected reduced levels of aerobic glycolysis in several KSHV-infected primary effusion lymphoma cell lines compared to a Burkitt's lymphoma cell line BJAB, and KSHV infection of BJAB cells reduced aerobic glycolysis. These results reveal a novel mechanism by which an oncogenic virus regulates a key metabolic pathway to adapt to stress in tumor microenvironment, and illustrate the importance of fine-tuning the metabolic pathways for sustaining the proliferation and survival of cancer cells, particularly under stress conditions.

  2. The Viral KSHV Chemokine vMIP-II Inhibits the Migration of Naive and Activated Human NK Cells by Antagonizing Two Distinct Chemokine Receptors

    Science.gov (United States)

    Yamin, Rachel; Kaynan, Noa S.; Glasner, Ariella; Vitenshtein, Alon; Tsukerman, Pinchas; Bauman, Yoav; Ophir, Yael; Elias, Shlomo; Bar-On, Yotam; Gur, Chamutal; Mandelboim, Ofer

    2013-01-01

    Natural killer (NK) cells are innate immune cells able to rapidly kill virus-infected and tumor cells. Two NK cell populations are found in the blood; the majority (90%) expresses the CD16 receptor and also express the CD56 protein in intermediate levels (CD56Dim CD16Pos) while the remaining 10% are CD16 negative and express CD56 in high levels (CD56Bright CD16Neg). NK cells also reside in some tissues and traffic to various infected organs through the usage of different chemokines and chemokine receptors. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human virus that has developed numerous sophisticated and versatile strategies to escape the attack of immune cells such as NK cells. Here, we investigate whether the KSHV derived cytokine (vIL-6) and chemokines (vMIP-I, vMIP-II, vMIP-III) affect NK cell activity. Using transwell migration assays, KSHV infected cells, as well as fusion and recombinant proteins, we show that out of the four cytokine/chemokines encoded by KSHV, vMIP-II is the only one that binds to the majority of NK cells, affecting their migration. We demonstrate that vMIP-II binds to two different receptors, CX3CR1 and CCR5, expressed by naïve CD56Dim CD16Pos NK cells and activated NK cells, respectively. Furthermore, we show that the binding of vMIP-II to CX3CR1 and CCR5 blocks the binding of the natural ligands of these receptors, Fractalkine (Fck) and RANTES, respectively. Finally, we show that vMIP-II inhibits the migration of naïve and activated NK cells towards Fck and RANTES. Thus, we present here a novel mechanism in which KSHV uses a unique protein that antagonizes the activity of two distinct chemokine receptors to inhibit the migration of naïve and activated NK cells. PMID:23966863

  3. The RING-CH ligase K5 antagonizes restriction of KSHV and HIV-1 particle release by mediating ubiquitin-dependent endosomal degradation of tetherin.

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    Claire Pardieu

    2010-04-01

    Full Text Available Tetherin (CD317/BST2 is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV is sensitive to tetherin restriction and its activity is counteracted by the KSHV encoded RING-CH E3 ubiquitin ligase K5. Tetherin expression in KSHV-infected cells inhibits viral particle release, as does depletion of K5 protein using RNA interference. K5 induces a species-specific downregulation of human tetherin from the cell surface followed by its endosomal degradation. We show that K5 targets a single lysine (K18 in the cytoplasmic tail of tetherin for ubiquitination, leading to relocalization of tetherin to CD63-positive endosomal compartments. Tetherin degradation is dependent on ESCRT-mediated endosomal sorting, but does not require a tyrosine-based sorting signal in the tetherin cytoplasmic tail. Importantly, we also show that the ability of K5 to substitute for Vpu in HIV-1 release is entirely dependent on K18 and the RING-CH domain of K5. By contrast, while Vpu induces ubiquitination of tetherin cytoplasmic tail lysine residues, mutation of these positions has no effect on its antagonism of tetherin function, and residual tetherin is associated with the trans-Golgi network (TGN in Vpu-expressing cells. Taken together our results demonstrate that K5 is a mechanistically distinct viral countermeasure to tetherin-mediated restriction, and that herpesvirus particle release is sensitive to this mode of antiviral inhibition.

  4. Herpes and polyoma family viruses in thyroid cancer.

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    Stamatiou, Dimitris P; Derdas, Stavros P; Zoras, Odysseas L; Spandidos, Demetrios A

    2016-03-01

    Thyroid cancer is considered the most common malignancy that affects the endocrine system. Generally, thyroid cancer derives from follicular epithelial cells, and thyroid cancer is divided into well-differentiated papillary (80% of cases) and follicular (15% of cases) carcinoma. Follicular thyroid cancer is further divided into the conventional and oncocytic (Hürthle cell) type, poorly differentiated carcinoma and anaplastic carcinoma. Both poorly differentiated and anaplastic carcinoma can arise either de novo , or secondarily from papillary and follicular thyroid cancer. The incidence of thyroid cancer has significantly increased for both males and females of all ages, particularly for females between 55-64 years of age, from 1999 through 2008. The increased rates refer to tumors of all stages, though they were mostly noted in localized disease. Recently, viruses have been implicated in the direct regulation of epithelial-mesenchymal transition (EMT) and the development of metastases. More specifically, Epstein-Barr virus (EBV) proteins may potentially lead to the development of metastasis through the regulation of the metastasis suppressor, Nm23, and the control of Twist expression. The significant enhancement of the metastatic potential, through the induction of angiogenesis and changes to the tumor microenvironment, subsequent to viral infection, has been documented, while EMT also contributes to cancer cell permissiveness to viruses. A number of viruses have been identified to be associated with carcinogenesis, and these include lymphotropic herpesviruses, namely EBV and Kaposi's sarcoma-associated herpesvirus [KSHV, also known as human herpesvirus type 8 (HHV8)]; two hepatitis viruses, hepatitis B virus (HBV) and hepatitis C virus (HCV); human papillomaviruses (HPVs); human T cell lymphoma virus (HTLV); and a new polyomavirus, Merkel cell polyomavirus identified in 2008. In this review, we examined the association between thyroid cancer and two oncogenic

  5. Tumor viruses and cancer biology: Modulating signaling pathways for therapeutic intervention.

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    Saha, Abhik; Kaul, Rajeev; Murakami, Masanao; Robertson, Erle S

    2010-11-15

    Tumor viruses have provided relatively simple genetic systems, which can be manipulated for understanding the molecular mechanisms of the cellular transformation process. A growing body of information in the tumor virology field provides several prospects for rationally targeted therapies. However, further research is needed to better understand the multiple mechanisms utilized by these viruses in cancer progression in order to develop therapeutic strategies. Initially viruses were believed to be associated with cancers as causative agents only in animals. It was almost half a century before the first human tumor virus, Epstein-Barr virus (EBV), was identified in 1964. Subsequently, several human tumor viruses have been identified including Kaposi sarcoma associated herpesvirus (KSHV), human Papillomaviruses (HPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human T lymphotropic virus (HTLV-1) and recently identified Merkel cell Polyomavirus (MCPyV). Tumor viruses are sub-categorized as either DNA viruses, which include EBV, KSHV, HPV, HBV, and MCPyV, or RNA viruses such as HCV and HTLV-1. Tumor-viruses induce oncogenesis through manipulating an array of different cellular pathways. These viruses initiate a series of cellular events, which lead to immortalization and proliferation of the infected cells by disrupting the mitotic checkpoint upon infection of the host cell. This is often accomplished by functional inhibition or proteasomal degradation of many tumor suppressor proteins by virally encoded gene products. The virally infected cells can either be eliminated via cell-mediated apoptosis or persist in a state of chronic infection. Importantly, the chronic persistence of infection by tumor viruses can lead to oncogenesis. This review discusses the major human tumor associated viruses and their ability to modulate numerous cell signaling pathways, which can be targeted for potential therapeutic approaches.

  6. Plasma Viral miRNAs Indicate a High Prevalence of Occult Viral Infections

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    Enrique Fuentes-Mattei

    2017-06-01

    Full Text Available Prevalence of Kaposi sarcoma-associated herpesvirus (KSHV/HHV-8 varies greatly in different populations. We hypothesized that the actual prevalence of KSHV/HHV8 infection in humans is underestimated by the currently available serological tests. We analyzed four independent patient cohorts with post-surgical or post-chemotherapy sepsis, chronic lymphocytic leukemia and post-surgical patients with abdominal surgical interventions. Levels of specific KSHV-encoded miRNAs were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR, and KSHV/HHV-8 IgG were measured by immunoassay. We also measured specific miRNAs from Epstein Barr Virus (EBV, a virus closely related to KSHV/HHV-8, and determined the EBV serological status by ELISA for Epstein-Barr nuclear antigen 1 (EBNA-1 IgG. Finally, we identified the viral miRNAs by in situ hybridization (ISH in bone marrow cells. In training/validation settings using independent multi-institutional cohorts of 300 plasma samples, we identified in 78.50% of the samples detectable expression of at least one of the three tested KSHV-miRNAs by RT-qPCR, while only 27.57% of samples were found to be seropositive for KSHV/HHV-8 IgG (P < 0.001. The prevalence of KSHV infection based on miRNAs qPCR is significantly higher than the prevalence determined by seropositivity, and this is more obvious for immuno-depressed patients. Plasma viral miRNAs quantification proved that EBV infection is ubiquitous. Measurement of viral miRNAs by qPCR has the potential to become the “gold” standard method to detect certain viral infections in clinical practice.

  7. Plasma Viral miRNAs Indicate a High Prevalence of Occult Viral Infections.

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    Fuentes-Mattei, Enrique; Giza, Dana Elena; Shimizu, Masayoshi; Ivan, Cristina; Manning, John T; Tudor, Stefan; Ciccone, Maria; Kargin, Osman Aykan; Zhang, Xinna; Mur, Pilar; do Amaral, Nayra Soares; Chen, Meng; Tarrand, Jeffrey J; Lupu, Florea; Ferrajoli, Alessandra; Keating, Michael J; Vasilescu, Catalin; Yeung, Sai-Ching Jim; Calin, George A

    2017-06-01

    Prevalence of Kaposi sarcoma-associated herpesvirus (KSHV/HHV-8) varies greatly in different populations. We hypothesized that the actual prevalence of KSHV/HHV8 infection in humans is underestimated by the currently available serological tests. We analyzed four independent patient cohorts with post-surgical or post-chemotherapy sepsis, chronic lymphocytic leukemia and post-surgical patients with abdominal surgical interventions. Levels of specific KSHV-encoded miRNAs were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and KSHV/HHV-8 IgG were measured by immunoassay. We also measured specific miRNAs from Epstein Barr Virus (EBV), a virus closely related to KSHV/HHV-8, and determined the EBV serological status by ELISA for Epstein-Barr nuclear antigen 1 (EBNA-1) IgG. Finally, we identified the viral miRNAs by in situ hybridization (ISH) in bone marrow cells. In training/validation settings using independent multi-institutional cohorts of 300 plasma samples, we identified in 78.50% of the samples detectable expression of at least one of the three tested KSHV-miRNAs by RT-qPCR, while only 27.57% of samples were found to be seropositive for KSHV/HHV-8 IgG (P<0.001). The prevalence of KSHV infection based on miRNAs qPCR is significantly higher than the prevalence determined by seropositivity, and this is more obvious for immuno-depressed patients. Plasma viral miRNAs quantification proved that EBV infection is ubiquitous. Measurement of viral miRNAs by qPCR has the potential to become the "gold" standard method to detect certain viral infections in clinical practice. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Sphingosine kinase-2 maintains viral latency and survival for KSHV-infected endothelial cells.

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    Lu Dai

    Full Text Available Phosphorylation of sphingosine by sphingosine kinases (SphK1 and SphK2 generates sphingosine-1-phosphate (S1P, a bioactive sphingolipid which promotes cancer cell survival and tumor progression in vivo. We have recently reported that targeting SphK2 induces apoptosis for human primary effusion lymphoma (PEL cell lines infected by the Kaposi's sarcoma-associated herpesvirus (KSHV, and this occurs in part through inhibition of canonical NF-κB activation. In contrast, pharmacologic inhibition of SphK2 has minimal impact for uninfected B-cell lines or circulating human B cells from healthy donors. Therefore, we designed additional studies employing primary human endothelial cells to explore mechanisms responsible for the selective death observed for KSHV-infected cells during SphK2 targeting. Using RNA interference and a clinically relevant pharmacologic approach, we have found that targeting SphK2 induces apoptosis selectively for KSHV-infected endothelial cells through induction of viral lytic gene expression. Moreover, this effect occurs through repression of KSHV-microRNAs regulating viral latency and signal transduction, including miR-K12-1 which targets IκBα to facilitate activation of NF-κB, and ectopic expression of miR-K12-1 restores NF-κB activation and viability for KSHV-infected endothelial cells during SphK2 inhibition. These data illuminate a novel survival mechanism and potential therapeutic target for KSHV-infected endothelial cells: SphK2-associated maintenance of viral latency.

  9. Mutational analysis of Kaposica reveals that bridging of MG2 and CUB domains of target protein is crucial for the cofactor activity of RCA proteins.

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    Gautam, Avneesh Kumar; Panse, Yogesh; Ghosh, Payel; Reza, Malik Johid; Mullick, Jayati; Sahu, Arvind

    2015-10-13

    The complement system has evolved to annul pathogens, but its improper regulation is linked with diseases. Efficient regulation of the system is primarily provided by a family of proteins termed regulators of complement activation (RCA). The knowledge of precise structural determinants of RCA proteins critical for imparting the regulatory activities and the molecular events underlying the regulatory processes, nonetheless, is still limited. Here, we have dissected the structural requirements of RCA proteins that are crucial for one of their two regulatory activities, the cofactor activity (CFA), by using the Kaposi's sarcoma-associated herpesvirus RCA homolog Kaposica as a model protein. We have scanned the entire Kaposica molecule by sequential mutagenesis using swapping and site-directed mutagenesis, which identified residues critical for its interaction with C3b and factor I. Mapping of these residues onto the modeled structure of C3b-Kaposica-factor I complex supported the mutagenesis data. Furthermore, the model suggested that the C3b-interacting residues bridge the CUB (complement C1r-C1s, Uegf, Bmp1) and MG2 (macroglobulin-2) domains of C3b. Thus, it seems that stabilization of the CUB domain with respect to the core of the C3b molecule is central for its CFA. Identification of CFA-critical regions in Kaposica guided experiments in which the equivalent regions of membrane cofactor protein were swapped into decay-accelerating factor. This strategy allowed CFA to be introduced into decay-accelerating factor, suggesting that viral and human regulators use a common mechanism for CFA.

  10. KSHV-Mediated Angiogenesis in Tumor Progression

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    Pravinkumar Purushothaman

    2016-07-01

    Full Text Available Human herpesvirus 8 (HHV-8, also known as Kaposi’s sarcoma-associated herpesvirus (KSHV, is a malignant human oncovirus belonging to the gamma herpesvirus family. HHV-8 is closely linked to the pathogenesis of Kaposi’s sarcoma (KS and two other B-cell lymphoproliferative diseases: primary effusion lymphoma (PEL and a plasmablastic variant of multicentric Castleman’s disease (MCD. KS is an invasive tumor of endothelial cells most commonly found in untreated HIV-AIDS or immuno-compromised individuals. KS tumors are highly vascularized and have abnormal, excessive neo-angiogenesis, inflammation, and proliferation of infected endothelial cells. KSHV directly induces angiogenesis in an autocrine and paracrine fashion through a complex interplay of various viral and cellular pro-angiogenic and inflammatory factors. KS is believed to originate due to a combination of KSHV’s efficient strategies for evading host immune systems and several pro-angiogenic and pro-inflammatory stimuli. In addition, KSHV infection of endothelial cells produces a wide array of viral oncoproteins with transforming capabilities that regulate multiple host-signaling pathways involved in the activation of angiogenesis. It is likely that the cellular-signaling pathways of angiogenesis and lymph-angiogenesis modulate the rate of tumorigenesis induction by KSHV. This review summarizes the current knowledge on regulating KSHV-mediated angiogenesis by integrating the findings reported thus far on the roles of host and viral genes in oncogenesis, recent developments in cell-culture/animal-model systems, and various anti-angiogenic therapies for treating KSHV-related lymphoproliferative disorders.

  11. KSHV inhibits stress granule formation by viral ORF57 blocking PKR activation.

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    Nishi R Sharma

    2017-10-01

    Full Text Available TIA-1 positive stress granules (SG represent the storage sites of stalled mRNAs and are often associated with the cellular antiviral response. In this report, we provide evidence that Kaposi's sarcoma-associated herpesvirus (KSHV overcomes the host antiviral response by inhibition of SG formation via a viral lytic protein ORF57. By immunofluorescence analysis, we found that B lymphocytes with KSHV lytic infection are refractory to SG induction. KSHV ORF57, an essential post-transcriptional regulator of viral gene expression and the production of new viral progeny, inhibits SG formation induced experimentally by arsenite and poly I:C, but not by heat stress. KSHV ORF37 (vSOX bearing intrinsic endoribonuclease activity also inhibits arsenite-induced SG formation, but KSHV RTA, vIRF-2, ORF45, ORF59 and LANA exert no such function. ORF57 binds both PKR-activating protein (PACT and protein kinase R (PKR through their RNA-binding motifs and prevents PACT-PKR interaction in the PKR pathway which inhibits KSHV production. Consistently, knocking down PKR expression significantly promotes KSHV virion production. ORF57 interacts with PKR to inhibit PKR binding dsRNA and its autophosphorylation, leading to inhibition of eIF2α phosphorylation and SG formation. Homologous protein HSV-1 ICP27, but not EBV EB2, resembles KSHV ORF57 in the ability to block the PKR/eIF2α/SG pathway. In addition, KSHV ORF57 inhibits poly I:C-induced TLR3 phosphorylation. Altogether, our data provide the first evidence that KSHV ORF57 plays a role in modulating PKR/eIF2α/SG axis and enhances virus production during virus lytic infection.

  12. A population-based study of how children are exposed to saliva in KwaZulu-Natal Province, South Africa: implications for the spread of saliva-borne pathogens to children.

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    Butler, L M; Neilands, T B; Mosam, A; Mzolo, S; Martin, J N

    2010-04-01

    In sub-Saharan Africa, many viral infections, including Epstein-Barr virus, cytomegalovirus, Kaposi's sarcoma-associated herpesvirus and hepatitis B are acquired in childhood. While saliva is an important transmission conduit for these viruses, little is known about how saliva is passed to African children. We endeavoured to identify the range and determinants of acts by which African children are exposed to saliva. To identify the range of acts by which African children are exposed to saliva, we conducted focus groups, semi-structured interviews and participant observations in an urban and a rural community in South Africa. To measure the prevalence and determinants of the identified acts, we administered a questionnaire to a population-based sample of caregivers. We identified 12 caregiving practices that expose a child's oral-respiratory mucosa, cutaneous surfaces or anal-rectal mucosa to saliva. Several acts were heretofore not described in the contemporary literature (e.g., caregiver inserting finger lubricated with saliva into child's rectum to relieve constipation). Among 896 participants in the population-based survey, many of the acts were commonly practised by all respondent types (mothers, fathers, grandmothers and siblings). The most common were premastication of food, sharing sweets and premastication of medicinal plants that are spit onto a child's body. African children are exposed to saliva through a variety of acts, practised by a variety of caregivers, with no single predominant practice. This diversity poses challenges for epidemiologic work seeking to identify specific saliva-passing practices that transmit viruses. Most acts could be replaced by other actions and are theoretically preventable.

  13. The first endogenous herpesvirus, identified in the tarsier genome, and novel sequences from primate rhadinoviruses and lymphocryptoviruses.

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    Amr Aswad

    2014-06-01

    Full Text Available Herpesviridae is a diverse family of large and complex pathogens whose genomes are extremely difficult to sequence. This is particularly true for clinical samples, and if the virus, host, or both genomes are being sequenced for the first time. Although herpesviruses are known to occasionally integrate in host genomes, and can also be inherited in a Mendelian fashion, they are notably absent from the genomic fossil record comprised of endogenous viral elements (EVEs. Here, we combine paleovirological and metagenomic approaches to both explore the constituent viral diversity of mammalian genomes and search for endogenous herpesviruses. We describe the first endogenous herpesvirus from the genome of the Philippine tarsier, belonging to the Roseolovirus genus, and characterize its highly defective genome that is integrated and flanked by unambiguous host DNA. From a draft assembly of the aye-aye genome, we use bioinformatic tools to reveal over 100,000 bp of a novel rhadinovirus that is the first lemur gammaherpesvirus, closely related to Kaposi's sarcoma-associated virus. We also identify 58 genes of Pan paniscus lymphocryptovirus 1, the bonobo equivalent of human Epstein-Barr virus. For each of the viruses, we postulate gene function via comparative analysis to known viral relatives. Most notably, the evidence from gene content and phylogenetics suggests that the aye-aye sequences represent the most basal known rhadinovirus, and indicates that tumorigenic herpesviruses have been infecting primates since their emergence in the late Cretaceous. Overall, these data show that a genomic fossil record of herpesviruses exists despite their extremely large genomes, and expands the known diversity of Herpesviridae, which will aid the characterization of pathogenesis. Our analytical approach illustrates the benefit of intersecting evolutionary approaches with metagenomics, genetics and paleovirology.

  14. Deep sequencing reveals direct targets of gammaherpesvirus-induced mRNA decay and suggests that multiple mechanisms govern cellular transcript escape.

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    Clyde, Karen; Glaunsinger, Britt A

    2011-05-09

    One characteristic of lytic infection with gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and murine herpesvirus 68 (MHV68), is the dramatic suppression of cellular gene expression in a process known as host shutoff. The alkaline exonuclease proteins (KSHV SOX, MHV-68 muSOX and EBV BGLF5) have been shown to induce shutoff by destabilizing cellular mRNAs. Here we extend previous analyses of cellular mRNA abundance during lytic infection to characterize the effects of SOX and muSOX, in the absence of other viral genes, utilizing deep sequencing technology (RNA-seq). Consistent with previous observations during lytic infection, the majority of transcripts are downregulated in cells expressing either SOX or muSOX, with muSOX acting as a more potent shutoff factor than SOX. Moreover, most cellular messages fall into the same expression class in both SOX- and muSOX-expressing cells, indicating that both factors target similar pools of mRNAs. More abundant mRNAs are more efficiently downregulated, suggesting a concentration effect in transcript targeting. However, even among highly expressed genes there are mRNAs that escape host shutoff. Further characterization of select escapees reveals multiple mechanisms by which cellular genes can evade downregulation. While some mRNAs are directly refractory to SOX, the steady state levels of others remain unchanged, presumably as a consequence of downstream effects on mRNA biogenesis. Collectively, these studies lay the framework for dissecting the mechanisms underlying the susceptibility of mRNA to destruction during lytic gammaherpesvirus infection.

  15. Delta-9 tetrahydrocannabinol (THC inhibits lytic replication of gamma oncogenic herpesviruses in vitro

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    Friedman Herman

    2004-09-01

    Full Text Available Abstract Background The major psychoactive cannabinoid compound of marijuana, delta-9 tetrahydrocannabinol (THC, has been shown to modulate immune responses and lymphocyte function. After primary infection the viral DNA genome of gamma herpesviruses persists in lymphoid cell nuclei in a latent episomal circular form. In response to extracellular signals, the latent virus can be activated, which leads to production of infectious virus progeny. Therefore, we evaluated the potential effects of THC on gamma herpesvirus replication. Methods Tissue cultures infected with various gamma herpesviruses were cultured in the presence of increasing concentrations of THC and the amount of viral DNA or infectious virus yield was compared to those of control cultures. The effect of THC on Kaposi's Sarcoma Associated Herpesvirus (KSHV and Epstein-Barr virus (EBV replication was measured by the Gardella method and replication of herpesvirus saimiri (HVS of monkeys, murine gamma herpesvirus 68 (MHV 68, and herpes simplex type 1 (HSV-1 was measured by yield reduction assays. Inhibition of the immediate early ORF 50 gene promoter activity was measured by the dual luciferase method. Results Micromolar concentrations of THC inhibit KSHV and EBV reactivation in virus infected/immortalized B cells. THC also strongly inhibits lytic replication of MHV 68 and HVS in vitro. Importantly, concentrations of THC that inhibit virus replication of gamma herpesviruses have no effect on cell growth or HSV-1 replication, indicating selectivity. THC was shown to selectively inhibit the immediate early ORF 50 gene promoter of KSHV and MHV 68. Conclusions THC specifically targets viral and/or cellular mechanisms required for replication and possibly shared by these gamma herpesviruses, and the endocannabinoid system is possibly involved in regulating gamma herpesvirus latency and lytic replication. The immediate early gene ORF 50 promoter activity was specifically inhibited by THC

  16. Structural basis for the conserved binding mechanism of MDM2-inhibiting peptides and anti-apoptotic Bcl-2 family proteins.

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    Lee, Min-Sung; Ha, Ji-Hyang; Yoon, Ho Sup; Lee, Chong-Kil; Chi, Seung-Wook

    2014-02-28

    The interaction between tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins serves a critical role in the transcription-independent apoptosis mechanism of p53. Our previous studies showed that an MDM2-inhibiting motif (residues 15-29) in the p53 transactivation domain (p53TAD) mediates the interaction with anti-apoptotic Bcl-2 family proteins. In this study, we provided structural models of the complexes between the MDM2-inhibiting p53TAD peptide and Mcl-1, Bcl-w, and Kaposi sarcoma-associated herpes virus (KSHV) Bcl-2 using NMR chemical shift perturbation data. The binding mode of the MDM2-inhibiting p53TAD peptide is highly conserved among the anti-apoptotic Bcl-2 family proteins despite their distinct specificities for pro-apoptotic Bcl-2 family proteins. We also identified the binding of a phage-display-derived MDM2-inhibiting peptide 12-1 to anti-apoptotic Bcl-XL protein by using NMR spectroscopy. The structural model of the Bcl-XL/12-1 peptide complex revealed that the conserved residues Phe4, Trp8, and Leu11 in the MDM2-inhibiting peptide fit into a hydrophobic cleft of Bcl-XL in a manner similar to that of pro-apoptotic Bcl-2 homology 3 (BH3) peptides. Our results shed light on the mechanism underlying dual-targeting of the FxxxWxxL-based α-helical motif to MDM2 and anti-apoptotic Bcl-2 family proteins for anticancer therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Sangivamycin induces apoptosis by suppressing Erk signaling in primary effusion lymphoma cells

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    Wakao, Kazufumi [Department of Biotechnology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Kofu-shi 400-8511 (Japan); Watanabe, Tadashi [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan); Takadama, Tadatoshi; Ui, Sadaharu [Department of Biotechnology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Kofu-shi 400-8511 (Japan); Shigemi, Zenpei; Kagawa, Hiroki [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan); Higashi, Chizuka; Ohga, Rie; Taira, Takahiro [Department of Molecular Cell Biology, Faculty of Medicine, University of Yamanashi, Chuoh-shi 409-3898 (Japan); Fujimuro, Masahiro, E-mail: fuji2@mb.kyoto-phu.ac.jp [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan)

    2014-02-07

    Highlights: • Sangivamycin induces the apoptosis of B cell lymphoma PEL cells. • Sangivamycin suppresses Erk signaling by inhibiting Erk phosphorylation in PEL cells. • The activation of Erk signaling is essential for PEL cell survival. • Sangivamycin induces the apoptosis of PEL cells without production of progeny virus. • Sangivamycin may serve as a novel drug for the treatment of PEL. - Abstract: Sangivamycin, a structural analog of adenosine and antibiotic exhibiting antitumor and antivirus activities, inhibits protein kinase C and the synthesis of both DNA and RNA. Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi’s sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients and HIV-infected homosexual males. PEL cells are derived from post-germinal center B cells, and are infected with KSHV. Herein, we asked if sangivamycin might be useful to treat PEL. We found that sangivamycin killed PEL cells, and we explored the underlying mechanism. Sangivamycin treatment drastically decreased the viability of PEL cell lines compared to KSHV-uninfected B lymphoma cell lines. Sangivamycin induced the apoptosis of PEL cells by activating caspase-7 and -9. Further, sangivamycin suppressed the phosphorylation of Erk1/2 and Akt, thus inhibiting activation of the proteins. Inhibitors of Akt and MEK suppressed the proliferation of PEL cells compared to KSHV-uninfected cells. It is known that activation of Erk and Akt signaling inhibits apoptosis and promotes proliferation in PEL cells. Our data therefore suggest that sangivamycin induces apoptosis by inhibiting Erk and Akt signaling in such cells. We next investigated whether sangivamycin, in combination with an HSP90 inhibitor geldanamycin (GA) or valproate (valproic acid), potentiated the cytotoxic effects of the latter drugs on PEL cells. Compared to treatment with GA or valproate alone, the addition of sangivamycin enhanced cytotoxic activity. Our data thus indicate that

  18. A Tiny RNA that Packs a Big Punch: The Critical Role of a Viral miR-155 Ortholog in Lymphomagenesis in Marek’s Disease

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    Guoqing Zhuang

    2017-06-01

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs that have been identified in animals, plants, and viruses. These small RNAs play important roles in post-transcriptional regulation of various cellular processes, including development, differentiation, and all aspects of cancer biology. Rapid-onset T-cell lymphoma of chickens, namely Marek’s disease (MD, induced by Gallid alphaherpesvirus 2 (GaHV2, could provide an ideal natural animal model for herpesvirus-related cancer research. GaHV2 encodes 26 mature miRNAs derived from 14 precursors assembled in three distinct gene clusters in the viral genome. One of the most highly expressed GaHV2 miRNAs, miR-M4-5p, shows high sequence similarity to the cellular miR-155 and the miR-K12-11 encoded by Kaposi’s sarcoma-associated herpesvirus, particularly in the miRNA “seed region.” As with miR-K12-11, miR-M4-5p shares a common set of host and viral target genes with miR-155, suggesting that they may target the same regulatory cellular networks; however, differences in regulatory function between miR-155 and miR-M4-5p may distinguish non-viral and viral mediated tumorigenesis. In this review, we focus on the functions of miR-M4-5p as the viral ortholog of miR-155 to explore how the virus mimics a host pathway to benefit the viral life cycle and trigger virus-induced tumorigenesis.

  19. Arctigenin induces the apoptosis of primary effusion lymphoma cells under conditions of glucose deprivation.

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    Baba, Yusuke; Shigemi, Zenpei; Hara, Naoko; Moriguchi, Misato; Ikeda, Marina; Watanabe, Tadashi; Fujimuro, Masahiro

    2017-12-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of primary effusion lymphoma (PEL) and Kaposi's sarcoma. PEL is a type of non-Hodgkin's B-cell lymphoma, affecting immunosuppressed individuals, such as post-transplant or AIDS patients. However, since PEL is resistant to chemotherapeutic regimens, new effective treatment strategies are required. Arctigenin, a natural lignan compound found in the plant Arctium lappa, has been widely investigated as a potential anticancer agent in the clinical setting. In the present study, we examined the cytotoxic effects of arctigenin by cell viability assay and found that arctigenin markedly inhibited the proliferation of PEL cells compared with KSHV-uninfected B-lymphoma cells under conditions of glucose deprivation. Arctigenin decreased cellular ATP levels, disrupted mitochondrial membrane potential and triggered caspase-9-mediated apoptosis in the glucose-deprived PEL cells. In addition, western blot analysis using phospho-specific antibodies were used to evaluate activity changes in the signaling pathways of interest. As a result, arctigenin suppressed the activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways by inhibiting ERK and p38 MAPK phosphorylation in the glucose-deprived PEL cells. We confirmed that an inhibitor of ERK (U0126) or p38 MAPK (SB202190 and SB203580) suppressed the proliferation of the BC3 PEL cells compared with the KSHV-negative DG75 cells. Moreover, RT-PCR and luciferase reporter assay revealed that arctigenin and p38 MAPK inhibition by SB202190 or SB203580 downregulated the transcriptional expression of unfolded protein response (UPR)‑related molecules, including GRP78 and ATF6α under conditions of glucose deprivation. Finally, we confirmed that arctigenin did not affect KSHV replication in PEL cells, suggesting that arctigenin treatment for PEL does not contribute to the risk of de novo KSHV

  20. KSHV PAN RNA Associates with Demethylases UTX and JMJD3 to Activate Lytic Replication through a Physical Interaction with the Virus Genome

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    Rossetto, Cyprian C.; Pari, Gregory

    2012-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the cause of Kaposi's sarcoma and body cavity lymphomas. KSHV lytic infection produces PAN RNA, a highly abundant noncoding polyadenylated transcript that is retained in the nucleus. We recently demonstrated that PAN RNA interacts with several viral and cellular factors and can disregulate the expression of genes that modulate immune response. In an effort to define the role of PAN RNA in the context of the virus genome we generated a recombinant BACmid that deleted the PAN RNA locus. Because of the apparent duplication of the PAN RNA locus in BAC36, we generated BAC36CR, a recombinant BACmid that removes the duplicated region. BAC36CR was used as a template to delete most of the PAN RNA locus to generate BAC36CRΔPAN. BAC36CRΔPAN failed to produce supernatant virus and displayed a general decrease in mRNA accumulation of representative immediate early, early and late genes. Most strikingly, K-Rta expression was decreased in lytically induced BAC36CRΔPAN-containing cell lines at early and late time points post induction. Expression of PAN RNA in trans in BAC36CRΔPAN containing cells resulted in an increase in K-Rta expression, however K-Rta over expression failed to rescue BAC36CRΔPAN, suggesting that PAN RNA plays a wider role in virus replication. To investigate the role of PAN RNA in the activation of K-Rta expression, we demonstrate that PAN RNA physically interacts with the ORF50 promoter. RNA chromatin immunoprecipitation assays show that PAN RNA interacts with demethylases JMJD3 and UTX, and the histone methyltransferase MLL2. Consistent with the interaction with demethylases, expression of PAN RNA results in a decrease of the repressive H3K27me3 mark at the ORF50 promoter. These data support a model where PAN RNA is a multifunctional regulatory transcript that controls KSHV gene expression by mediating the modification of chromatin by targeting the KSHV repressed genome. PMID:22589717

  1. Tick-Borne Transmission of Murine Gammaherpesvirus 68

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    Valeria Hajnická

    2017-10-01

    Full Text Available Herpesviruses are a large group of DNA viruses infecting mainly vertebrates. Murine gammaherpesvirus 68 (MHV68 is often used as a model in studies of the pathogenesis of clinically important human gammaherpesviruses such as Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. This rodent virus appears to be geographically widespread; however, its natural transmission cycle is unknown. Following detection of MHV68 in field-collected ticks, including isolation of the virus from tick salivary glands and ovaries, we investigated whether MHV68 is a tick-borne virus. Uninfected Ixodes ricinus ticks were shown to acquire the virus by feeding on experimentally infected laboratory mice. The virus survived tick molting, and the molted ticks transmitted the virus to uninfected laboratory mice on which they subsequently fed. MHV68 was isolated from the tick salivary glands, consistent with transmission via tick saliva. The virus survived in ticks without loss of infectivity for at least 120 days, and subsequently was transmitted vertically from one tick generation to the next, surviving more than 500 days. Furthermore, the F1 generation (derived from F0 infected females transmitted MHV68 to uninfected mice on which they fed, with MHV68 M3 gene transcripts detected in blood, lung, and spleen tissue of mice on which F1 nymphs and F1 adults engorged. These experimental data fulfill the transmission criteria that define an arthropod-borne virus (arbovirus, the largest biological group of viruses. Currently, African swine fever virus (ASFV is the only DNA virus recognized as an arbovirus. Like ASFV, MHV68 showed evidence of pathogenesis in ticks. Previous studies have reported MHV68 in free-living ticks and in mammals commonly infested with I. ricinus, and neutralizing antibodies to MHV68 have been detected in large mammals (e.g., deer including humans. Further studies are needed to determine if these reports are the result of tick-borne transmission

  2. Pyrrolidinium fullerene induces apoptosis by activation of procaspase-9 via suppression of Akt in primary effusion lymphoma

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    Watanabe, Tadashi [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan); Nakamura, Shigeo [Department of Chemistry, Nippon Medical School, 1-7-1 Kyonan-cho, Musashino, Tokyo 180-0023 (Japan); Ono, Toshiya; Ui, Sadaharu [Department of Biotechnology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Kofu 400-8511 (Japan); Yagi, Syota; Kagawa, Hiroki [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan); Watanabe, Hisami [Center of Molecular Biosciences, Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213 (Japan); Ohe, Tomoyuki; Mashino, Tadahiko [Department of Pharmaceutical Sciences, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512 (Japan); Fujimuro, Masahiro, E-mail: fuji2@mb.kyoto-phu.ac.jp [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan)

    2014-08-15

    Highlights: • Seven fullerenes were evaluated in terms of their cytotoxic effects on B-lymphomas. • Pyrrolidinium fullerene induced apoptosis of KSHV-infected B-lymphoma PEL cells. • The activation of Akt is essential for PEL cell survival. • Pyrrolidinium fullerene activated caspase-9 by inactivating Akt in PEL cells. • Pyrrolidinium fullerene have potential as novel drugs for the treatment of PEL. - Abstract: Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin’s B-cell lymphoma and is an aggressive neoplasm caused by Kaposi’s sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. In general, PEL cells are derived from post-germinal center B-cells and are infected with KSHV. To evaluate potential novel anti-tumor compounds against KSHV-associated PEL, seven water-soluble fullerene derivatives were evaluated as potential drug candidates for the treatment of PEL. Herein, we discovered a pyrrolidinium fullerene derivative, 1,1,1′,1′-tetramethyl [60]fullerenodipyrrolidinium diiodide, which induced apoptosis of PEL cells via a novel mechanism, the caspase-9 activation by suppressing the caspase-9 phosphorylation, causing caspase-9 inactivation. Pyrrolidinium fullerene treatment reduced significantly the viability of PEL cells compared with KSHV-uninfected lymphoma cells, and induced the apoptosis of PEL cells by activating caspase-9 via procaspase-9 cleavage. Pyrrolidinium fullerene additionally reduced the Ser473 phosphorylation of Akt and Ser196 of procaspase-9. Ser473-phosphorylated Akt (i.e., activated Akt) phosphorylates Ser196 in procaspase-9, causing inactivation of procaspase-9. We also demonstrated that Akt inhibitors suppressed the proliferation of PEL cells compared with KSHV-uninfected cells. Our data therefore suggest that Akt activation is essential for cell survival in PEL and a pyrrolidinium fullerene derivative induced apoptosis by activating caspase-9 via suppression of Akt in PEL cells. In addition, we evaluated

  3. Abortive lytic reactivation of KSHV in CBF1/CSL deficient human B cell lines.

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    Barbara A Scholz

    Full Text Available Since Kaposi's sarcoma associated herpesvirus (KSHV establishes a persistent infection in human B cells, B cells are a critical compartment for viral pathogenesis. RTA, the replication and transcription activator of KSHV, can either directly bind to DNA or use cellular DNA binding factors including CBF1/CSL as DNA adaptors. In addition, the viral factors LANA1 and vIRF4 are known to bind to CBF1/CSL and modulate RTA activity. To analyze the contribution of CBF1/CSL to reactivation in human B cells, we have successfully infected DG75 and DG75 CBF1/CSL knock-out cell lines with recombinant KSHV.219 and selected for viral maintenance by selective medium. Both lines maintained the virus irrespective of their CBF1/CSL status. Viral reactivation could be initiated in both B cell lines but viral genome replication was attenuated in CBF1/CSL deficient lines, which also failed to produce detectable levels of infectious virus. Induction of immediate early, early and late viral genes was impaired in CBF1/CSL deficient cells at multiple stages of the reactivation process but could be restored to wild-type levels by reintroduction of CBF1/CSL. To identify additional viral RTA target genes, which are directly controlled by CBF1/CSL, we analyzed promoters of a selected subset of viral genes. We show that the induction of the late viral genes ORF29a and ORF65 by RTA is strongly enhanced by CBF1/CSL. Orthologs of ORF29a in other herpesviruses are part of the terminase complex required for viral packaging. ORF65 encodes the small capsid protein essential for capsid shell assembly. Our study demonstrates for the first time that in human B cells viral replication can be initiated in the absence of CBF1/CSL but the reactivation process is severely attenuated at all stages and does not lead to virion production. Thus, CBF1/CSL acts as a global hub which is used by the virus to coordinate the lytic cascade.

  4. Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of MiRNA-mRNA Target Pairs in KSHV-Infected Cells.

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    Coralie Viollet

    Full Text Available Kaposi's sarcoma associated herpesvirus (KSHV causes several tumors, including primary effusion lymphoma (PEL and Kaposi's sarcoma (KS. Cellular and viral microRNAs (miRNAs have been shown to play important roles in regulating gene expression. A better knowledge of the miRNA-mediated pathways affected by KSHV infection is therefore important for understanding viral infection and tumor pathogenesis. In this study, we used deep sequencing to analyze miRNA and cellular mRNA expression in a cell line with latent KSHV infection (SLKK as compared to the uninfected SLK line. This approach revealed 153 differentially expressed human miRNAs, eight of which were independently confirmed by qRT-PCR. KSHV infection led to the dysregulation of ~15% of the human miRNA pool and most of these cellular miRNAs were down-regulated, including nearly all members of the 14q32 miRNA cluster, a genomic locus linked to cancer and that is deleted in a number of PEL cell lines. Furthermore, we identified 48 miRNAs that were associated with a total of 1,117 predicted or experimentally validated target mRNAs; of these mRNAs, a majority (73% were inversely correlated to expression changes of their respective miRNAs, suggesting miRNA-mediated silencing mechanisms were involved in a number of these alterations. Several dysregulated miRNA-mRNA pairs may facilitate KSHV infection or tumor formation, such as up-regulated miR-708-5p, associated with a decrease in pro-apoptotic caspase-2 and leukemia inhibitory factor LIF, or down-regulated miR-409-5p, associated with an increase in the p53-inhibitor MDM2. Transfection of miRNA mimics provided further evidence that changes in miRNAs are driving some observed mRNA changes. Using filtered datasets, we also identified several canonical pathways that were significantly enriched in differentially expressed miRNA-mRNA pairs, such as the epithelial-to-mesenchymal transition and the interleukin-8 signaling pathways. Overall, our data

  5. The ORF59 DNA polymerase processivity factor homologs of Old World primate RV2 rhadinoviruses are highly conserved nuclear antigens expressed in differentiated epithelium in infected macaques

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    Burnside Kellie L

    2009-11-01

    Full Text Available Abstract Background ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV, is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication. Results We cloned, sequenced and expressed the genes encoding related ORF59 proteins from the RV1 rhadinovirus homologs of KSHV from chimpanzee (PtrRV1 and three species of macaques (RFHVMm, RFHVMn and RFHVMf, and have compared them with ORF59 proteins obtained from members of the more distantly-related RV2 rhadinovirus lineage infecting the same non-human primate species (PtrRV2, RRV, MneRV2, and MfaRV2, respectively. We found that ORF59 homologs of the RV1 and RV2 Old World primate rhadinoviruses are highly conserved with distinct phylogenetic clustering of the two rhadinovirus lineages. RV1 and RV2 ORF59 C-terminal domains exhibit a strong lineage-specific conservation. Rabbit antiserum was developed against a C-terminal polypeptide that is highly conserved between the macaque RV2 ORF59 sequences. This anti-serum showed strong reactivity towards ORF59 encoded by the macaque RV2 rhadinoviruses, RRV (rhesus and MneRV2 (pig-tail, with no cross reaction to human or macaque RV1 ORF59 proteins. Using this antiserum and RT-qPCR, we determined that RRV ORF59 is expressed early after permissive infection of both rhesus primary fetal fibroblasts and African green monkey kidney epithelial cells (Vero in vitro. RRV- and MneRV2-infected foci showed strong nuclear expression of ORF59 that correlated with production of infectious progeny virus. Immunohistochemical studies of an MneRV2-infected macaque revealed strong nuclear expression of ORF59 in infected cells within the differentiating layer of epidermis corroborating previous observations that differentiated epithelial cells are permissive for replication of KSHV-like rhadinoviruses

  6. Epstein-Barr Virus BKRF4 Gene Product Is Required for Efficient Progeny Production.

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    Masud, H M Abdullah Al; Watanabe, Takahiro; Yoshida, Masahiro; Sato, Yoshitaka; Goshima, Fumi; Kimura, Hiroshi; Murata, Takayuki

    2017-09-13

    Epstein-Barr virus (EBV), a member of human gammaherpesvirus, infects mainly B cells. EBV has two alternative life cycles, latent and lytic, and is reactivated occasionally from the latent stage to the lytic cycle. To combat EBV-associated disorders, understanding the molecular mechanisms of the EBV lytic replication cycle is also important. Here, we focused on an EBV lytic gene, BKRF4. Using our anti-BKRF4 antibody, we revealed that the BKRF4 gene product is expressed during the lytic cycle with late kinetics. To characterize the role of BKRF4, we constructed BKRF4-knockout mutants using the bacterial artificial chromosome (BAC) and CRISPR/Cas9 systems. While disruption of the BKRF4 gene had almost no effect on viral protein expression and DNA synthesis, it significantly decreased progeny virion levels in HEK293 and Akata cells. Furthermore, we show that BKRF4 is involved not only in production of progeny virions but also in increasing the infectivity of the virus particles. Immunoprecipitation assays revealed that BKRF4 interacted with a virion protein, BGLF2. We showed that the C-terminal region of BKRF4 was critical for this interaction and for efficient progeny production. Immunofluorescence analysis revealed that BKRF4 partially colocalized with BGLF2 in the nucleus and perinuclear region. Finally, we showed that BKRF4 is a phosphorylated, possible tegument protein and that the EBV protein kinase BGLF4 may be important for this phosphorylation. Taken together, our data suggest that BKRF4 is involved in the production of infectious virions.IMPORTANCE While the latent genes of EBV have been studied extensively, the lytic genes are less well characterized. This study focused on one such lytic gene, BKRF4, which is conserved only among gammaherpesviruses (ORF45 of Kaposi's sarcoma-associated herpesvirus or murine herpesvirus-68). After preparing the BKRF4 knockout virus using B95-8 EBV-BAC, we demonstrated that the BKRF4 gene was involved in infectious progeny

  7. Murine gammaherpesvirus M2 protein induction of IRF4 via the NFAT pathway leads to IL-10 expression in B cells.

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    Udaya S Rangaswamy

    2014-01-01

    Full Text Available Reactivation of the gammaherpesviruses Epstein-Barr virus (EBV, Kaposi's sarcoma-associated herpesvirus (KSHV and murine gammaherpesvirus 68 (MHV68 from latently infected B cells has been linked to plasma cell differentiation. We have previously shown that the MHV68 M2 protein is important for virus reactivation from B cells and, when expressed alone in primary murine B cells, can drive B cell differentiation towards a pre-plasma cell phenotype. In addition, expression of M2 in primary murine B cells leads to secretion of high levels of IL-10 along with enhanced proliferation and survival. Furthermore, the absence of M2 in vivo leads to a defect in the appearance of MHV68 infected plasma cells in the spleen at the peak of MHV68 latency. Here, employing an inducible B cell expression system, we have determined that M2 activates the NFAT pathway in a Src kinase-dependent manner--leading to induction of the plasma cell-associated transcription factor, Interferon Regulatory Factor-4 (IRF4. Furthermore, we show that expression of IRF4 alone in a B cell line up-regulates IL-10 expression in culture supernatants, revealing a novel role for IRF4 in B cell induced IL-10. Consistent with the latter observation, we show that IRF4 can regulate the IL-10 promoter in B cells. In primary murine B cells, addition of cyclosporine (CsA resulted in a significant decrease in M2-induced IL-10 levels as well as IRF4 expression, emphasizing the importance of the NFAT pathway in M2- -mediated induction of IL-10. Together, these studies argue in favor of a model wherein M2 activation of the NFAT pathway initiates events leading to increased levels of IRF4--a key player in plasma cell differentiation--which in turn triggers IL-10 expression. In the context of previous findings, the data presented here provides insights into how M2 facilitates plasma cell differentiation and subsequent virus reactivation.

  8. Risk factors for high anti-HHV-8 antibody titers (≥1:51,200 in black, HIV-1 negative South African cancer patients: a case control study

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    Ruff Paul

    2003-09-01

    Full Text Available Abstract Background Infection with human herpesvirus 8 (HHV-8, also known as Kaposi's sarcoma-associated herpesvirus (KSHV, is the necessary causal agent in the development of Kaposi's sarcoma (KS. Infection with HIV-1, male gender and older age all increase risk for KS. However, the geographic distribution of HHV-8 and KS both prior to the HIV/AIDS epidemic and with HIV/AIDS suggest the presence of an additional co-factor in the development of KS. Methods Between January 1994 and October 1997, we interviewed 2576 black in-patients with cancer in Johannesburg and Soweto, South Africa. Blood was tested for antibodies against HIV-1 and HHV-8 and the study was restricted to 2191 HIV-1 negative patients. Antibodies against the latent nuclear antigen of HHV-8 encoded by orf73 were detected with an indirect immunofluorescence assay. We examined the relationship between high anti-HHV-8 antibody titers (≥1:51,200 and sociodemographic and behavioral factors using unconditional logistic regression models. Variables that were significant at p = 0.10 were included in multivariate analysis. Results Of the 2191 HIV-1 negative patients who did not have Kaposi's sarcoma, 854 (39.0% were positive for antibodies against HHV-8 according to the immunofluorescent assay. Among those seropositive for HHV-8, 530 (62.1% had low titers (1:200, 227 (26.6% had medium titers (1:51,200 and 97 (11.4% had highest titers (1:204,800. Among the 2191 HIV-1 negative patients, the prevalence of high anti-HHV-8 antibody titers (≥1:51,200 was independently associated with increasing age (ptrend = 0.04, having a marital status of separated or divorced (p = 0.003, using wood, coal or charcoal as fuel for cooking 20 years ago instead of electricity (p = 0.02 and consuming traditional maize beer more than one time a week (p = 0.02; p-trend for increasing consumption = 0.05 although this may be due to chance given the large number of predictors considered in this analysis. Conclusions

  9. Development of a real-time QPCR assay for the detection of RV2 lineage-specific rhadinoviruses in macaques and baboons

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    Thouless Margaret E

    2005-01-01

    Full Text Available Abstract Background Two distinct lineages of rhadinoviruses related to Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8 have been identified in macaques and other Old World non-human primates. We have developed a real-time quantitative PCR (QPCR assay using a TaqMan probe to differentially detect and quantitate members of the rhadinovirus-2 (RV2 lineage. PCR primers were derived from sequences within ORF 60 and the adjacent ORF 59/60 intergenic region which were highly conserved between the macaque RV2 rhadinoviruses, rhesus rhadinovirus (RRV and Macaca nemestrina rhadinovirus-2 (MneRV2. These primers showed little similarity to the corresponding sequences of the macaque RV1 rhadinoviruses, retroperitoneal fibromatosis herpesvirus Macaca nemestrina (RFHVMn and Macaca mulatta (RFHVMm. To determine viral loads per cell, an additional TaqMan QPCR assay was developed to detect the single copy cellular oncostatin M gene. Results We show that the RV2 QPCR assay is linear from less than 2 to more than 300,000 copies using MneRV2 DNA, and is non-reactive with RFHVMn DNA up to 1 billion DNA templates per reaction. RV2 loads ranging from 6 to 2,300 viral genome equivalent copies per 106 cells were detected in PBMC from randomly sampled macaques from the Washington National Primate Research Center. Screening tissue from other primate species, including another macaque, Macaca fascicularis, and a baboon, Papio cynocephalus, revealed the presence of novel rhadinoviruses, MfaRV2 and PcyRV2, respectively. Sequence comparison and phylogenetic analysis confirmed their inclusion within the RV2 lineage of KSHV-like rhadinoviruses. Conclusions We describe a QPCR assay which provides a quick and sensitive method for quantitating rhadinoviruses belonging to the RV2 lineage of KSHV-like rhadinoviruses found in a variety of macaque species commonly used for biomedical research. While this assay broadly detects different RV2 rhadinovirus species, it is unreactive with

  10. A Novel γ2-Herpesvirus of the Rhadinovirus 2 Lineage in Chimpanzees

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    Lacoste, Vincent; Mauclère, Philippe; Dubreuil, Guy; Lewis, John; Georges-Courbot, Marie-Claude; Gessain, Antoine

    2001-01-01

    Old World monkeys and, recently, African great apes have been shown, by serology and polymerase chain reaction (PCR), to harbor different γ2-herpesviruses closely related to Kaposi's sarcoma-associated Herpesvirus (KSHV). Although the presence of two distinct lineages of KSHV-like rhadinoviruses, RV1 and RV2, has been revealed in Old World primates (including African green monkeys, macaques, and, recently, mandrills), viruses belonging to the RV2 genogroup have not yet been identified from great apes. Indeed, the three yet known γ2-herpesviruses in chimpanzees (PanRHV1a/PtRV1, PanRHV1b) and gorillas (GorRHV1) belong to the RV1 group. To investigate the putative existence of a new RV2 Rhadinovirus in chimpanzees and gorillas we have used the degenerate consensus primer PCR strategy for the Herpesviral DNA polymerase gene on 40 wild-caught animals. This study led to the discovery, in common chimpanzees, of a novel γ2-herpesvirus belonging to the RV2 genogroup, termed Pan Rhadino-herpesvirus 2 (PanRHV2). Use of specific primers and internal oligonucleotide probes demonstrated the presence of this novel γ2-herpesvirus in three wild-caught animals. Comparison of a 1092-bp fragment of the DNA polymerase obtained from these three animals of the Pan troglodytes troglodytes subspecies, one from Gabon and the two others from Cameroon, revealed <1% of nucleotide divergence. The geographic colocalization as well as the phylogenetic “relationship” of the human and simian γ2-herpesviruses support the model according to which herpesviruses have diversified from a common ancestor in a manner mediating cospeciation of herpesviruses with their host species. By demonstrating the existence of two distinct Rhadinovirus lineages in common chimpanzees, our finding indicates the possible existence of a novel human γ2-herpesvirus belonging to the RV2 genogroup. [The Herpesviral DNA polymerase sequence data determined herein have been deposited at the GenBank database under

  11. Biphasic euchromatin-to-heterochromatin transition on the KSHV genome following de novo infection.

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    Zsolt Toth

    Full Text Available The establishment of latency is an essential step for the life-long persistent infection and pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV. While the KSHV genome is chromatin-free in the virions, the viral DNA in latently infected cells has a chromatin structure with activating and repressive histone modifications that promote latent gene expression but suppress lytic gene expression. Here, we report a comprehensive epigenetic study of the recruitment of chromatin regulatory factors onto the KSHV genome during the pre-latency phase of KSHV infection. This demonstrates that the KSHV genome undergoes a biphasic chromatinization following de novo infection. Initially, a transcriptionally active chromatin (euchromatin, characterized by high levels of the H3K4me3 and acetylated H3K27 (H3K27ac activating histone marks, was deposited on the viral episome and accompanied by the transient induction of a limited number of lytic genes. Interestingly, temporary expression of the RTA protein facilitated the increase of H3K4me3 and H3K27ac occupancy on the KSHV episome during de novo infection. Between 24-72 hours post-infection, as the levels of these activating histone marks declined on the KSHV genome, the levels of the repressive H3K27me3 and H2AK119ub histone marks increased concomitantly with the decline of lytic gene expression. Importantly, this transition to heterochromatin was dependent on both Polycomb Repressive Complex 1 and 2. In contrast, upon infection of human gingiva-derived epithelial cells, the KSHV genome underwent a transcription-active euchromatinization, resulting in efficient lytic gene expression. Our data demonstrate that the KSHV genome undergoes a temporally-ordered biphasic euchromatin-to-heterochromatin transition in endothelial cells, leading to latent infection, whereas KSHV preferentially adopts a transcriptionally active euchromatin in oral epithelial cells, resulting in lytic gene expression. Our results

  12. KSHV PAN RNA associates with demethylases UTX and JMJD3 to activate lytic replication through a physical interaction with the virus genome.

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    Rossetto, Cyprian C; Pari, Gregory

    2012-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the cause of Kaposi's sarcoma and body cavity lymphomas. KSHV lytic infection produces PAN RNA, a highly abundant noncoding polyadenylated transcript that is retained in the nucleus. We recently demonstrated that PAN RNA interacts with several viral and cellular factors and can disregulate the expression of genes that modulate immune response. In an effort to define the role of PAN RNA in the context of the virus genome we generated a recombinant BACmid that deleted the PAN RNA locus. Because of the apparent duplication of the PAN RNA locus in BAC36, we generated BAC36CR, a recombinant BACmid that removes the duplicated region. BAC36CR was used as a template to delete most of the PAN RNA locus to generate BAC36CRΔPAN. BAC36CRΔPAN failed to produce supernatant virus and displayed a general decrease in mRNA accumulation of representative immediate early, early and late genes. Most strikingly, K-Rta expression was decreased in lytically induced BAC36CRΔPAN-containing cell lines at early and late time points post induction. Expression of PAN RNA in trans in BAC36CRΔPAN containing cells resulted in an increase in K-Rta expression, however K-Rta over expression failed to rescue BAC36CRΔPAN, suggesting that PAN RNA plays a wider role in virus replication. To investigate the role of PAN RNA in the activation of K-Rta expression, we demonstrate that PAN RNA physically interacts with the ORF50 promoter. RNA chromatin immunoprecipitation assays show that PAN RNA interacts with demethylases JMJD3 and UTX, and the histone methyltransferase MLL2. Consistent with the interaction with demethylases, expression of PAN RNA results in a decrease of the repressive H3K27me3 mark at the ORF50 promoter. These data support a model where PAN RNA is a multifunctional regulatory transcript that controls KSHV gene expression by mediating the modification of chromatin by targeting the KSHV repressed genome.

  13. Latent KSHV Infected Endothelial Cells Are Glutamine Addicted and Require Glutaminolysis for Survival.

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    Erica L Sanchez

    2015-07-01

    Full Text Available Kaposi's Sarcoma-associated Herpesvirus (KSHV is the etiologic agent of Kaposi's Sarcoma (KS. KSHV establishes a predominantly latent infection in the main KS tumor cell type, the spindle cell, which is of endothelial cell origin. KSHV requires the induction of multiple metabolic pathways, including glycolysis and fatty acid synthesis, for the survival of latently infected endothelial cells. Here we demonstrate that latent KSHV infection leads to increased levels of intracellular glutamine and enhanced glutamine uptake. Depletion of glutamine from the culture media leads to a significant increase in apoptotic cell death in latently infected endothelial cells, but not in their mock-infected counterparts. In cancer cells, glutamine is often required for glutaminolysis to provide intermediates for the tri-carboxylic acid (TCA cycle and support for the production of biosynthetic and bioenergetic precursors. In the absence of glutamine, the TCA cycle intermediates alpha-ketoglutarate (αKG and pyruvate prevent the death of latently infected cells. Targeted drug inhibition of glutaminolysis also induces increased cell death in latently infected cells. KSHV infection of endothelial cells induces protein expression of the glutamine transporter, SLC1A5. Chemical inhibition of SLC1A5, or knockdown by siRNA, leads to similar cell death rates as glutamine deprivation and, similarly, can be rescued by αKG. KSHV also induces expression of the heterodimeric transcription factors c-Myc-Max and related heterodimer MondoA-Mlx. Knockdown of MondoA inhibits expression of both Mlx and SLC1A5 and induces a significant increase in cell death of only cells latently infected with KSHV, again, fully rescued by the supplementation of αKG. Therefore, during latent infection of endothelial cells, KSHV activates and requires the Myc/MondoA-network to upregulate the glutamine transporter, SLC1A5, leading to increased glutamine uptake for glutaminolysis. These findings

  14. KSHV Entry and Trafficking in Target Cells—Hijacking of Cell Signal Pathways, Actin and Membrane Dynamics

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    Binod Kumar

    2016-11-01

    Full Text Available Kaposi’s sarcoma associated herpesvirus (KSHV is etiologically associated with human endothelial cell hyperplastic Kaposi’s sarcoma and B-cell primary effusion lymphoma. KSHV infection of adherent endothelial and fibroblast cells are used as in vitro models for infection and KSHV enters these cells by host membrane bleb and actin mediated macropinocytosis or clathrin endocytosis pathways, respectively. Infection in endothelial and fibroblast cells is initiated by the interactions between multiple viral envelope glycoproteins and cell surface associated heparan sulfate (HS, integrins (α3β1, αVβ3 and αVβ5, and EphA2 receptor tyrosine kinase (EphA2R. This review summarizes the accumulated studies demonstrating that KSHV manipulates the host signal pathways to enter and traffic in the cytoplasm of the target cells, to deliver the viral genome into the nucleus, and initiate viral gene expression. KSHV interactions with the cell surface receptors is the key platform for the manipulations of host signal pathways which results in the simultaneous induction of FAK, Src, PI3-K, Rho-GTPase, ROS, Dia-2, PKC ζ, c-Cbl, CIB1, Crk, p130Cas and GEF-C3G signal and adaptor molecules that play critical roles in the modulation of membrane and actin dynamics, and in the various steps of the early stages of infection such as entry and trafficking towards the nucleus. The Endosomal Sorting Complexes Required for Transport (ESCRT proteins are also recruited to assist in viral entry and trafficking. In addition, KSHV interactions with the cell surface receptors also induces the host transcription factors NF-κB, ERK1/2, and Nrf2 early during infection to initiate and modulate viral and host gene expression. Nuclear delivery of the viral dsDNA genome is immediately followed by the host innate responses such as the DNA damage response (DDR, inflammasome and interferon responses. Overall, these studies form the initial framework for further studies of

  15. A systems biology approach identified different regulatory networks targeted by KSHV miR-K12-11 in B cells and endothelial cells.

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    Yang, Yajie; Boss, Isaac W; McIntyre, Lauren M; Renne, Rolf

    2014-08-08

    Kaposi's sarcoma associated herpes virus (KSHV) is associated with tumors of endothelial and lymphoid origin. During latent infection, KSHV expresses miR-K12-11, an ortholog of the human tumor gene hsa-miR-155. Both gene products are microRNAs (miRNAs), which are important post-transcriptional regulators that contribute to tissue specific gene expression. Advances in target identification technologies and molecular interaction databases have allowed a systems biology approach to unravel the gene regulatory networks (GRNs) triggered by miR-K12-11 in endothelial and lymphoid cells. Understanding the tissue specific function of miR-K12-11 will help to elucidate underlying mechanisms of KSHV pathogenesis. Ectopic expression of miR-K12-11 differentially affected gene expression in BJAB cells of lymphoid origin and TIVE cells of endothelial origin. Direct miRNA targeting accounted for a small fraction of the observed transcriptome changes: only 29 genes were identified as putative direct targets of miR-K12-11 in both cell types. However, a number of commonly affected biological pathways, such as carbohydrate metabolism and interferon response related signaling, were revealed by gene ontology analysis. Integration of transcriptome profiling, bioinformatic algorithms, and databases of protein-protein interactome from the ENCODE project identified different nodes of GRNs utilized by miR-K12-11 in a tissue-specific fashion. These effector genes, including cancer associated transcription factors and signaling proteins, amplified the regulatory potential of a single miRNA, from a small set of putative direct targets to a larger set of genes. This is the first comparative analysis of miRNA-K12-11's effects in endothelial and B cells, from tissues infected with KSHV in vivo. MiR-K12-11 was able to broadly modulate gene expression in both cell types. Using a systems biology approach, we inferred that miR-K12-11 establishes its GRN by both repressing master TFs and influencing

  16. Functional Analysis of Genes Comprising the Locus of Heat Resistance in Escherichia coli.

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    Mercer, Ryan; Nguyen, Oanh; Ou, Qixing; McMullen, Lynn; Gänzle, Michael G

    2017-10-15

    The locus of heat resistance (LHR) is a 15- to 19-kb genomic island conferring exceptional heat resistance to organisms in the family Enterobacteriaceae , including pathogenic strains of Salmonella enterica and Escherichia coli The complement of LHR-comprising genes that is necessary for heat resistance and the stress-induced or growth-phase-induced expression of LHR-comprising genes are unknown. This study determined the contribution of the seven LHR-comprising genes yfdX1 GI , yfdX2 , hdeD GI , orf11 , trx GI , kefB , and psiE GI by comparing the heat resistances of E. coli strains harboring plasmid-encoded derivatives of the different LHRs in these genes. (Genes carry a subscript "GI" [genomic island] if an ortholog of the same gene is present in genomes of E. coli ) LHR-encoded heat shock proteins sHSP20, ClpK GI , and sHSP GI are not sufficient for the heat resistance phenotype; YfdX1, YfdX2, and HdeD are necessary to complement the LHR heat shock proteins and to impart a high level of resistance. Deletion of trx GI , kefB , and psiE GI from plasmid-encoded copies of the LHR did not significantly affect heat resistance. The effect of the growth phase and the NaCl concentration on expression from the putative LHR promoter p2 was determined by quantitative reverse transcription-PCR and by a plasmid-encoded p2:GFP promoter fusion. The expression levels of exponential- and stationary-phase E. coli cells were not significantly different, but the addition of 1% NaCl significantly increased LHR expression. Remarkably, LHR expression in E. coli was dependent on a chromosomal copy of evgA In conclusion, this study improved our understanding of the genes required for exceptional heat resistance in E. coli and factors that increase their expression in food. IMPORTANCE The locus of heat resistance (LHR) is a genomic island conferring exceptional heat resistance to several foodborne pathogens. The exceptional level of heat resistance provided by the LHR questions the

  17. Rutamarin, an Active Constituent from Ruta angustifolia Pers., Induced Apoptotic Cell Death in the HT29 Colon Adenocarcinoma Cell Line.

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    Suhaimi, Shafinah Ahmad; Hong, Sok Lai; Abdul Malek, Sri Nurestri

    2017-07-01

    : ACN: Acetonitrile, ANOVA: One-way analysis of variance, BrdU: Bromodeoxyuridine, 13C-NMR: Carbon-13 Nuclear magnetic resonance, CAD: Caspase-activated endonuclease, CCD-18Co: Human colon normal, DLD1: Human Duke's type C colorectal adenocarcinoma, DMRT: Duncan's multiple range test, DMSO: Dimethyl sulfoxide, DNA: Deoxyribonucleic acid, DR4/5: Death receptor 4/5 protein, EMEM: Eagle's minimum essential media, FBS: Fetal bovine serum, FITC Annexin V: Annexin V conjugated with fluorescein isothiocyanate, FITC-DEVD-FMK: Fluorescein isothiocyanate conjugate of caspase inhibitor Asp-Glu-Val-Asp-fluoromethyl ketone, FITC-IETD-FMK: Fluorescein isothiocyanate conjugate of caspase inhibitor Ile-Glu-Thr-Asp-fluoromethyl ketone, FITC-LEHD-FMK: Fluorescein isothiocyanate conjugate of caspase inhibitor Leu-Glu-His-Asp-fluoromethyl ketone, G0: Quiescent phase of cell cycle, G1: Gap 1 phase of cell cycle, G2: Gap 2 phase of cell cycle, GC-MS: Gas chromatography-mass spectrometry, HeLa: Human cervical adenocarcinoma, HPLC: High performance liquid chromatography, HT29: Human colon adenocarcinoma, Huh7.5: Human hepatocellular carcinoma, IC50: Half maximal inhibitory concentration, KSHV: Kaposi's sarcoma-associated herpesvirus, M phase: Mitotic phase of cell cycle, MCF7: Human breast adenocarcinoma, NMR: Nuclear magnetic resonance, PBS: Phosphate-buffered saline, PI: Propidium iodide, RNase: Ribonuclease, rt: Retention time, S phase: Synthesis phase of cell cycle, SD: Standard deviation, SRB: Sulforhodamine B, TCA: Trichloroacetic acid, TLC: Thin layer chromatography, TNF-R1: Tumor necrosis factor receptor 1 protein, TUNEL: Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling, UV: Ultraviolet.