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Nuclear Retention of Multiply Spliced HIV-1 RNA in Resting CD4+ T Cells

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      Abstract

      HIV-1 latency in resting CD4 + T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART). We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4 + T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS) HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4 + T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4 + T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB) was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4 + T cells. Overexpression of PTB in resting CD4 + T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4 + T cells. Virus culture experiments showed that overexpression of PTB in resting CD4 + T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.

      Synopsis

      HIV-1 has the ability to establish a state of latent infection in resting memory CD4 + T cells. These latently infected cells represent a stable reservoir for the virus that is a major barrier to viral eradication. Understanding how this reservoir is established, maintained, and reactivated is essential for developing methods to target and eliminate these cells. Currently, many proposed mechanisms of HIV-1 latency involve a dramatic reduction in ongoing HIV-1 transcription. However, some HIV-1 mRNAs are made, and it has been unclear why the cells are unable to produce virus. This study describes the surprising observation that mRNAs encoding the viral regulatory proteins Tat and Rev are retained in the nucleus of infected resting CD4 + T cells. A cellular HIV-1 RNA-binding protein called polypyrimidine tract binding protein was shown to reverse latency when overexpressed in resting CD4 + T cells. This overexpression of polypyrimidine tract binding protein was sufficient to allow release of replication-competent HIV-1 from latently infected cells without inducing cellular stimulation. These experiments suggest that multiple factors contribute to the maintenance of HIV-1 latency in vivo; however, perturbation of the level of a specific cellular protein is sufficient to overcome these blocks and allow for virus production.

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      Most cited references 74

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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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        Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection.

        The capacity of HIV-1 to establish latent infection of CD4+ T cells may allow viral persistence despite immune responses and antiretroviral therapy. Measurements of infectious virus and viral RNA in plasma and of infectious virus, viral DNA and viral messenger RNA species in infected cells all suggest that HIV-1 replication continues throughout the course of infection. Uncertainty remains over what fraction of CD4+ T cells are infected and whether there are latent reservoirs for the virus. We show here that during the asymptomatic phase of infection there is an extremely low total body load of latently infected resting CD4+ T cells with replication-competent integrated provirus (<10(7) cells). The most prevalent form of HIV-1 DNA in resting and activated CD4+ T cells is a full-length, linear, unintegrated form that is not replication competent. The infection progresses even though at any given time in the lymphoid tissues integrated HIV-1 DNA is present in only a minute fraction of the susceptible populations, including resting and activated CD4+ T cells and macrophages.
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          HIV-1 integration in the human genome favors active genes and local hotspots.

          A defining feature of HIV replication is integration of the proviral cDNA into human DNA. The selection of chromosomal targets for integration is crucial for efficient viral replication, but the mechanism is poorly understood. Here we describe mapping of 524 sites of HIV cDNA integration on the human genome sequence. Genes were found to be strongly favored as integration acceptor sites. Global analysis of cellular transcription indicated that active genes were preferential integration targets, particularly genes that were activated in cells after infection by HIV-1. Regional hotspots for integration were also found, including a 2.4 kb region containing 1% of sites. These data document unexpectedly strong biases in integration site selection and suggest how selective targeting promotes aggressive HIV replication.
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            Author and article information

            Affiliations
            [1 ]Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
            [2 ]Howard Hughes Medical Institute, Baltimore, Maryland, United States of America
            King's College London, United Kingdom
            Author notes
            * To whom correspondence should be addressed. E-mail: rsiliciano@ 123456jhmi.edu
            Contributors
            Role: Editor
            Journal
            PLoS Pathog
            ppat
            PLoS Pathogens
            Public Library of Science (San Francisco, USA )
            1553-7366
            1553-7374
            July 2006
            7 July 2006
            : 2
            : 7
            1487174
            16839202
            10.1371/journal.ppat.0020068
            06-PLPA-RA-0048R3 plpa-02-07-03
            (Editor)
            Copyright: © 2006 Lassen 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
            HIV - AIDS
            Infectious Diseases
            Molecular Biology - Structural Biology
            Viruses
            Homo (Human)
            Custom metadata
            Lassen KG, Ramyar KX, Bailey JR, Zhou Y, Siliciano RF (2006) Nuclear retention of multiply spliced HIV-1 RNA in resting CD4 + T cells. PLoS Pathog 2(7): e68. DOI: 10.1371/journal.ppat.0020068

            Infectious disease & Microbiology

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