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The RBP-Jκ Binding Sites within the RTA Promoter Regulate KSHV Latent Infection and Cell Proliferation

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PLoS Pathogens

Public Library of Science

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      Abstract

      Kaposi's sarcoma-associated herpesvirus (KSHV) is tightly linked to at least two lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). However, the development of KSHV-mediated lymphoproliferative disease is not fully understood. Here, we generated two recombinant KSHV viruses deleted for the first RBP-Jκ binding site (RTA1st) and all three RBP-Jκ binding sites (RTAall) within the RTA promoter. Our results showed that RTA1st and RTAall recombinant viruses possess increased viral latency and a decreased capability for lytic replication in HEK 293 cells, enhancing colony formation and proliferation of infected cells. Furthermore, recombinant RTA1st and RTAall viruses showed greater infectivity in human peripheral blood mononuclear cells (PBMCs) relative to wt KSHV. Interestingly, KSHV BAC36 wt, RTA1st and RTAall recombinant viruses infected both T and B cells and all three viruses efficiently infected T and B cells in a time-dependent manner early after infection. Also, the capability of both RTA1st and RTAall recombinant viruses to infect CD19+ B cells was significantly enhanced. Surprisingly, RTA1st and RTAall recombinant viruses showed greater infectivity for CD3+ T cells up to 7 days. Furthermore, studies in Telomerase-immortalized human umbilical vein endothelial (TIVE) cells infected with KSHV corroborated our data that RTA1st and RTAall recombinant viruses have enhanced ability to persist in latently infected cells with increased proliferation. These recombinant viruses now provide a model to explore early stages of primary infection in human PBMCs and development of KSHV-associated lymphoproliferative diseases.

      Author Summary

      Kaposi's sarcoma-associated herpesvirus (KSHV) is tightly linked to at least two lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). The life cycle of KSHV consists of latent and lytic phase. RTA is the master switch for viral lytic replication. In this study, we first show that recombinant viruses deleted for the RBP-Jκ sites within the RTA promoter have a decreased capability for lytic replication, and thus enhanced colony formation and proliferation of infected cells. Interestingly, the recombinant viruses show greater infectivity in human peripheral blood mononuclear cells (PBMCs). The recombinant viruses also infected CD19+ B cells and CD3+ T cells with increased efficiency in a time-dependent manner and now provide a model which can be used to explore the early stages of primary infection in human PBMCs, as well as the development of KSHV-associated lymphoproliferative diseases.

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      Most cited references 71

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      Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas.

      DNA fragments that appeared to belong to an unidentified human herpesvirus were recently found in more than 90 percent of Kaposi's sarcoma lesions associated with the acquired immunodeficiency syndrome (AIDS). These fragments were also found in 6 of 39 tissue samples without Kaposi's sarcoma, including 3 malignant lymphomas, from patients with AIDS, but not in samples from patients without AIDS. We examined the DNA of 193 lymphomas from 42 patients with AIDS and 151 patients who did not have AIDS. We searched the DNA for sequences of Kaposi's sarcoma-associated herpesvirus (KSHV) by Southern blot hybridization, the polymerase chain reaction (PCR), or both. The PCR products in the positive samples were sequences and compared with the KSHV sequences in Kaposi's sarcoma tissues from patients with AIDS. KSHV sequences were identified in eight lymphomas in patients infected with the human immunodeficiency virus. All eight, and only these eight, were body-cavity-based lymphomas--that is, they were characterized by pleural, pericardial, or peritoneal lymphomatous effusions. All eight lymphomas also contained the Epstein-Barr viral genome. KSHV sequences were not found in the other 185 lymphomas. KSHV sequences were 40 to 80 times more abundant in the body-cavity-based lymphomas than in the Kaposi's sarcoma lesions. A high degree of conservation of KSHV sequences in Kaposi's sarcoma and in the eight lymphomas suggests the presence of the same agent in both lesions. The recently discovered KSHV DNA sequences occur in an unusual subgroup of AIDS-related B-cell lymphomas, but not in any other lymphoid neoplasm studied thus far. Our finding strongly suggests that a novel herpesvirus has a pathogenic role in AIDS-related body-cavity-based lymphomas.
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        A highly efficient recombineering-based method for generating conditional knockout mutations.

        Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. Here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations. This method uses homologous recombination mediated by the lambda phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce loxP or FRT sites into the subcloned DNA. Unlike other methods that use short 45-55-bp regions of homology for recombineering, our method uses much longer regions of homology. We also make use of several new E. coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive lambda prophage, and the Cre or Flpe recombinases from an arabinose-inducible promoter. We also describe two new Neo selection cassettes that work well in both E. coli and mouse ES cells. Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in less than 2 wk. This method should also facilitate the generation of knock-in mutations and transgene constructs, as well as expedite the analysis of regulatory elements and functional domains in or near genes.
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          Kaposi's sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman's disease.

          Multicentric Castleman's disease (MCD) is an atypical lymphoproliferative disorder defined using clinical and pathologic criteria. A characteristic of the MCD is a close association with Kaposi's sarcoma (KS), which occurs during the clinical course of most human immunodeficiency virus (HIV)-associated MCD cases and also, but less frequently, in HIV-negative patients. Recently, sequences of a putative new Herpesvirus (KSHV) have been isolated and further detected in almost all the acquired immunodeficiency syndrome (AIDS) KS and in most of the non-AIDS KS samples. In this study, we searched for these Herpesvirus-like sequences in MCD samples of 31 patients. KSHV sequences were detected in 14 of 14 cases of HIV-associated MCD, including 5 cases without detectable KS. Moreover, KSHV was detected in 7 of 17 MCD cases in HIV-negative patients, including 1 case associated with a cutaneous KS. In 34 non-MCD reactive lymph nodes (follicular and/or interfollicular hyperplasia) in HIV-negative patients, KSHV was detected in only 1 case. In 1 HIV-negative case of MCD, KSHV was found in both the lymph node and peripheral blood samples. These data suggest that KSHV could play a role in the pathogenesis of MCD, especially in HIV-infected patients.
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            Author and article information

            Affiliations
            [1]Department of Microbiology and Tumor Virology Program of the Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
            [2]Department of Microbiology and Immunology, School of Medicine, University of Nevada, Reno, Nevada, United States of America
            University of North Carolina at Chapel Hill, United States of America
            Author notes

            Conceived and designed the experiments: JL SCV ESR. Performed the experiments: JL QC RKD. Analyzed the data: JL SCV. Contributed reagents/materials/analysis tools: AS. Wrote the paper: JL ESR.

            Contributors
            Role: Editor
            Journal
            PLoS Pathog
            plos
            plospath
            PLoS Pathogens
            Public Library of Science (San Francisco, USA)
            1553-7366
            1553-7374
            January 2012
            January 2012
            12 January 2012
            : 8
            : 1
            3257303
            22253595
            PPATHOGENS-D-11-01774
            10.1371/journal.ppat.1002479
            (Editor)
            Lu 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: 18
            Categories
            Research Article
            Biology
            Microbiology
            Virology

            Infectious disease & Microbiology

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