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Murine Gamma-herpesvirus Immortalization of Fetal Liver-Derived B Cells Requires both the Viral Cyclin D Homolog and Latency-Associated Nuclear Antigen

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      Abstract

      Human gammaherpesviruses are associated with the development of lymphoproliferative diseases and B cell lymphomas, particularly in immunosuppressed hosts. Understanding the molecular mechanisms by which human gammaherpesviruses cause disease is hampered by the lack of convenient small animal models to study them. However, infection of laboratory strains of mice with the rodent virus murine gammaherpesvirus 68 (MHV68) has been useful in gaining insights into how gammaherpesviruses contribute to the genesis and progression of lymphoproliferative lesions. In this report we make the novel observation that MHV68 infection of murine day 15 fetal liver cells results in their immortalization and differentiation into B plasmablasts that can be propagated indefinitely in vitro, and can establish metastasizing lymphomas in mice lacking normal immune competence. The phenotype of the MHV68 immortalized B cell lines is similar to that observed in lymphomas caused by KSHV and resembles the favored phenotype observed during MHV68 infection in vivo. All established cell lines maintained the MHV68 genome, with limited viral gene expression and little or no detectable virus production - although virus reactivation could be induced upon crosslinking surface Ig. Notably, transcription of the genes encoding the MHV68 viral cyclin D homolog (v-cyclin) and the homolog of the KSHV latency-associated nuclear antigen (LANA), both of which are conserved among characterized γ2-herpesviruses, could consistently be detected in the established B cell lines. Furthermore, we show that the v-cyclin and LANA homologs are required for MHV68 immortalization of murine B cells. In contrast the M2 gene, which is unique to MHV68 and plays a role in latency and virus reactivation in vivo, was dispensable for B cell immortalization. This new model of gammaherpesvirus-driven B cell immortalization and differentiation in a small animal model establishes an experimental system for detailed investigation of the role of gammaherpesvirus gene products and host responses in the genesis and progression of gammaherpesvirus-associated lymphomas, and presents a convenient system to evaluate therapeutic modalities.

      Author Summary

      Herpesviruses are ubiquitous viruses, members of which infect all known mammalian species. A notable feature of all herpesvirus infections is that these infections cannot be cleared and persist for the lifetime of the host. In most cases these infections are benign and often without notable symptoms. However, for a subgroup of herpesviruses – the gammaherpesviruses – some infected individuals develop lymphomas, as well as several other types of cancer. There are two known gammaherpesviruses that infected humans, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), both of which have been the subject of intensive investigation. However, a major shortcoming of research on these viruses is the absence of an appropriate small animal model since these viruses only infect humans. To circumvent this limitation, infection of mice with a rodent gammaherpesvirus, murine gammaherpesvirus 68 (MHV68), is being characterized. Like EBV and KSHV, MHV68 infection of mice is also associated with the development of lymphoma under some experimental conditions. Here we show for the first time that a hallmark of EBV infection of human B lymphocytes – growth transformation of infected B cells in tissue culture – can be recapitulated by MHV68 infection of murine fetal liver-derived B cells. Furthermore, we identify two MHV68 genes that are required for B cell growth transformation. Finally, we show that MHV68 growth transformed B cell lines cause aggressive lymphomas in mice lacking an intact immune system, but not in immune competent mice. The latter result opens the door for studies on the role of viral genes in driving B cell growth, as well as host immune responses that control outgrowth of MHV68 infected B cells.

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      Most cited references 58

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      Epstein-Barr virus: 40 years on.

      Epstein-Barr virus (EBV) was discovered 40 years ago from examining electron micrographs of cells cultured from Burkitt's lymphoma, a childhood tumour that is common in sub-Saharan Africa, where its unusual geographical distribution - which matches that of holoendemic malaria -indicated a viral aetiology. However, far from showing a restricted distribution, EBV - a gamma-herpesvirus - was found to be widespread in all human populations and to persist in the vast majority of individuals as a lifelong, asymptomatic infection of the B-lymphocyte pool. Despite such ubiquity, the link between EBV and 'endemic' Burkitt's lymphoma proved consistent and became the first of an unexpectedly wide range of associations discovered between this virus and tumours.
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        Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.

        Representational difference analysis was used to isolate unique sequences present in more than 90 percent of Kaposi's sarcoma (KS) tissues obtained from patients with acquired immunodeficiency syndrome (AIDS). These sequences were not present in tissue DNA from non-AIDS patients, but were present in 15 percent of non-KS tissue DNA samples from AIDS patients. The sequences are homologous to, but distinct from, capsid and tegument protein genes of the Gammaherpesvirinae, herpesvirus saimiri and Epstein-Barr virus. These KS-associated herpesvirus-like (KSHV) sequences appear to define a new human herpesvirus.
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          Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme.

          Induced overexpression of AID in CH12F3-2 B lymphoma cells augmented class switching from IgM to IgA without cytokine stimulation. AID deficiency caused a complete defect in class switching and showed a hyper-IgM phenotype with enlarged germinal centers containing strongly activated B cells before or after immunization. AID-/- spleen cells stimulated in vitro with LPS and cytokines failed to undergo class switch recombination although they expressed germline transcripts. Immunization of AID-/- chimera with 4-hydroxy-3-nitrophenylacetyl (NP) chicken gamma-globulin induced neither accumulation of mutations in the NP-specific variable region gene nor class switching. These results suggest that AID may be involved in regulation or catalysis of the DNA modification step of both class switching and somatic hypermutation.
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            Author and article information

            Affiliations
            [1 ]Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia, United States of America
            [2 ]Department of Microbiology & Immunology, Emory University School of Medicine, Atlanta, Georgia, United States of America
            [3 ]Immunology and Molecular Pathogenesis Graduate Program, Emory University School of Medicine, Atlanta, Georgia, United States of America
            [4 ]Microbiology and Molecular Genetics Graduate Program, Emory University School of Medicine, Atlanta, Georgia, United States of America
            University of North Carolina at Chapel Hill, United States of America
            Author notes

            Conceived and designed the experiments: XL CRP JJ RPP SHS. Performed the experiments: XL CRP FMM RPP. Analyzed the data: XL CRP RPP JJ SHS. Contributed reagents/materials/analysis tools: FMM CRP RPP JJ. Wrote the paper: XL CRP JJ SHS.

            Contributors
            Role: Editor
            Journal
            PLoS Pathog
            plos
            plospath
            PLoS Pathogens
            Public Library of Science (San Francisco, USA )
            1553-7366
            1553-7374
            September 2011
            September 2011
            8 September 2011
            : 7
            : 9
            3169539
            21931547
            PPATHOGENS-D-11-00810
            10.1371/journal.ppat.1002220
            (Editor)
            Liang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
            Counts
            Pages: 12
            Categories
            Research Article
            Biology
            Microbiology
            Virology
            Model Organisms
            Animal Models
            Mouse

            Infectious disease & Microbiology

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