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      A novel quantitative approach for measuring the reservoir of latent HIV-1 proviruses

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          Abstract

          A stable latent reservoir for HIV-1 in resting CD4 + T-cells precludes cure 13 . Curative strategies targeting the reservoir are being tested 4, 5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays (QVOAs) for cells releasing infectious virus following one round of T-cell activation 1 . However, QVOAs and newer assays for cells producing viral RNA after activation 6 may underestimate reservoir size because one round of activation does not induce all proviruses 7 . Many studies rely on simple PCR-based assays to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority proviruses are defective 79 . We describe a novel approach that separately quantifies intact and defective proviruses and show that the dynamics of cells carrying intact and defective proviruses are different in vitro and in vivo, a finding with implications for targeting the intact proviruses that are a barrier to cure.

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          Most cited references16

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          Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy.

          The hypothesis that quiescent CD4+ T lymphocytes carrying proviral DNA provide a reservoir for human immunodeficiency virus-type 1 (HIV-1) in patients on highly active antiretroviral therapy (HAART) was examined. In a study of 22 patients successfully treated with HAART for up to 30 months, replication-competent virus was routinely recovered from resting CD4+ T lymphocytes. The frequency of resting CD4+ T cells harboring latent HIV-1 was low, 0.2 to 16.4 per 10(6) cells, and, in cross-sectional analysis, did not decrease with increasing time on therapy. The recovered viruses generally did not show mutations associated with resistance to the relevant antiretroviral drugs. This reservoir of nonevolving latent virus in resting CD4+ T cells should be considered in deciding whether to terminate treatment in patients who respond to HAART.
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            Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection.

            Treatment of infected patients with ABT-538, an inhibitor of the protease of human immunodeficiency virus type 1 (HIV-1), causes plasma HIV-1 levels to decrease exponentially (mean half-life, 2.1 +/- 0.4 days) and CD4 lymphocyte counts to rise substantially. Minimum estimates of HIV-1 production and clearance and of CD4 lymphocyte turnover indicate that replication of HIV-1 in vivo is continuous and highly productive, driving the rapid turnover of CD4 lymphocytes.
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              HIV reproducibly establishes a latent infection after acute infection of T cells in vitro.

              The presence of latent reservoirs has prevented the eradication of human immunodeficiency virus (HIV) from infected patients successfully treated with anti-retroviral therapy. The mechanism of postintegration latency is poorly understood, partly because of the lack of an in vitro model. We have used an HIV retroviral vector or a full-length HIV genome expressing green fluorescent protein to infect a T lymphocyte cell line in vitro and highly enrich for latently infected cells. HIV latency occurred reproducibly, albeit with low frequency, during an acute infection. Clonal cell lines derived from latent populations showed no detectable basal expression, but could be transcriptionally activated after treatment with phorbol esters or tumor necrosis factor alpha. Direct sequencing of integration sites demonstrated that latent clones frequently contain HIV integrated in or close to alphoid repeat elements in heterochromatin. This is in contrast to a productive infection where integration in or near heterochromatin is disfavored. These observations demonstrate that HIV can reproducibly establish a latent infection as a consequence of integration in or near heterochromatin.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                5 January 2019
                30 January 2019
                February 2019
                30 July 2019
                : 566
                : 7742
                : 120-125
                Affiliations
                [1 ]Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
                [2 ]Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
                [3 ]Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA.
                [4 ]Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA.
                [5 ]Department of Medicine, University of California San Francisco, San Francisco, CA 94110, USA.
                [6 ]Accelevir Diagnostics, Baltimore MD 21215.
                [7 ]Howard Hughes Medical Institute, Baltimore, MD 21205, USA.
                Author notes
                [†]

                Present address: Department of Biology, University of Texas, Austin, Texas, 78705, USA.

                [‡]

                Present address: Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT 06510, USA.

                Author Contributions KMB, ZW, GML and RFS designed the study. KMB, ZW, GML, FRS, AMB, KJK, SS, EJF, AARA, KMJ, LNB, JTK, and YCH performed experiments. KMB, ZW, FRS, AT, JDS, GML and RFS analyzed data. CN, JG and FDB provided integration site analysis. HZ performed cell sorting. SAB, AAC, JBM, JNB, and SGD provided patient samples. KMB and RFS wrote the manuscript.

                Correspondence and requests for materials should be addressed to rsiliciano@ 123456jhmi.edu .
                Article
                NIHMS1518047
                10.1038/s41586-019-0898-8
                6447073
                30700913
                752b1476-d59a-4ebe-999a-05c5f6ac9245

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