Resting CD4 T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals carry integrated HIV-1 genomes within actively transcribed host genes

Department of Medicine, Ross 1049, School of Medicine, Johns Hopkins University, 720 Rutland Ave., Baltimore, MD 21205, USA.
Journal of Virology (Impact Factor: 4.44). 07/2004; 78(12):6122-33. DOI: 10.1128/JVI.78.12.6122-6133.2004
Source: PubMed


Resting CD4+ T-cell populations from human immunodeficiency virus type 1 (HIV-1)-infected individuals include cells with integrated HIV-1
DNA. In individuals showing suppression of viremia during highly active antiretroviral therapy (HAART), resting CD4+ T-cell populations do not produce virus without cellular activation. To determine whether the nonproductive nature of the
infection in resting CD4+ T cells is due to retroviral integration into chromosomal regions that are repressive for transcription, we used inverse
PCR to characterize the HIV-1 integration sites in vivo in resting CD4+ T cells from patients on HAART. Of 74 integration sites from 16 patients, 93% resided within transcription units, usually
within introns. Integration was random with respect to transcriptional orientation relative to the host gene and with respect
to position within the host gene. Of integration sites within well-characterized genes, 91% (51 of 56) were in genes that
were actively expressed in resting CD4+ T cells, as directly demonstrated by reverse transcriptase PCR (RT-PCR). These results predict that HIV-1 sequences may be
included in the primary transcripts of host genes as part of rapidly degraded introns. RT-PCR experiments confirmed the presence
of HIV-1 sequences within transcripts initiating upstream of the HIV-1 transcription start site. Taken together, these results
demonstrate that HIV-1 genomes reside within actively transcribed host genes in resting CD4+ T cells in vivo.

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Available from: Kara Lassen, May 22, 2014
    • "We analyzed the genomic location of the integration sites obtained from viremic controllers and untreated and treated progressors and compared our results to published data obtained from HIV-1-infected individuals (Han et al., 2004; Ho et al., 2013; Ikeda et al., 2007; Schrö der et al., 2002). In agreement with the work of others, the majority of integration sites in each group are genic (Figure 1C and Figure S1A). "
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    ABSTRACT: The barrier to curing HIV-1 is thought to reside primarily in CD4(+) T cells containing silent proviruses. To characterize these latently infected cells, we studied the integration profile of HIV-1 in viremic progressors, individuals receiving antiretroviral therapy, and viremic controllers. Clonally expanded T cells represented the majority of all integrations and increased during therapy. However, none of the 75 expanded T cell clones assayed contained intact virus. In contrast, the cells bearing single integration events decreased in frequency over time on therapy, and the surviving cells were enriched for HIV-1 integration in silent regions of the genome. Finally, there was a strong preference for integration into, or in close proximity to, Alu repeats, which were also enriched in local hotspots for integration. The data indicate that dividing clonally expanded T cells contain defective proviruses and that the replication-competent reservoir is primarily found in CD4(+) T cells that remain relatively quiescent. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Jan 2015 · Cell
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    • "The site of integration is partly responsible for this transcriptional suppression. In latently infected cells, the provirus tends to reside either in compacted heterochromatic regions or in very highly expressed genes that cause transcriptional interference (Han et al., 2004; Lenasi et al., 2008; Lewinski et al., 2005). Low transcriptional levels during latency can also result from decreased availability or activity of transcriptional factors that are dependent on T cell activation. "
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    ABSTRACT: Long-lived pools of latently infected cells are a significant barrier to the development of a cure for HIV-1 infection. A better understanding of the mechanisms of reactivation from latency is needed to facilitate the development of novel therapies that address this problem. Here we show that chemical inhibitors of the sulfonation pathway prevent virus reactivation, both in latently infected J-Lat and U1 cell lines and in a primary human CD4+ T cell model of latency. In each of these models, sulfonation inhibitors decreased transcription initiation from the HIV-1 promoter. These inhibitors block transcription initiation at a step that lies downstream of nucleosome remodeling and affects RNA polymerase II recruitment to the viral promoter. These results suggest that the sulfonation pathway acts by a novel mechanism to regulate efficient virus transcription initiation during reactivation from latency, and further that augmentation of this pathway could be therapeutically useful.
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    • "However, high-throughput analysis of HIV-1 integration sites indicates that integrations into such regions are highly disfavored [9,10]. Instead, most proviruses are located within actively transcribed genes that are enriched for histone marks associated with active chromatin (H3K4me3, lysine acetylation), and depleted for marks associated with repressive chromatin (H3K9me3, H3K27me3) [9,10,12,38,39]. We speculate that latency models using cellular activation and long-term culturing to identify and establish latency could select for the most strongly repressed latent proviruses, thereby resulting in an over-representation of such disfavored integration locations. "
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    ABSTRACT: Molecular latency allows HIV-1 to persist in resting memory CD4+ T-cells as transcriptionally silent provirus integrated into host chromosomal DNA. Multiple transcriptional regulatory mechanisms for HIV-1 latency have been described in the context of progressive epigenetic silencing and maintenance. However, our understanding of the determinants critical for the establishment of latency in newly infected cells is limited. In this study, we used a recently described, doubly fluorescent HIV-1 latency model to dissect the role of proviral integration sites and cellular activation state on direct non-productive infections at the single cell level. Proviral integration site mapping of infected Jurkat T-cells revealed that productively and non-productively infected cells are indistinguishable in terms of genomic landmarks, surrounding epigenetic landscapes, and proviral orientation relative to host genes. However, direct non-productive infections were inversely correlated with both cellular activation state and NFkappaB activity. Furthermore, modulating NFkappaB with either small molecules or by conditional overexpression of NFkappaB subunits was sufficient to alter the propensity of HIV-1 to directly enter a non-productive latent state in newly infected cells. Importantly, this modulatory effect was limited to a short time window post-infection. Taken together, our data suggest that cellular activation state and NFkappaB activity during the time of infection, but not the site of proviral integration, are important regulators of direct HIV-1 non-productive infections.
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