Replication competent virus as an important source of bias in HIV latency models utilizing single round viral constructs

Bonczkowski, Pawel; Spiegelaere, Ward De; Bosque, Alberto; White, Cory H.; Nuffel, Anouk Van; Malatinková, Eva; Kiselinova, Maja; Trypsteen, Wim; Witkowski, Wojciech; Vermeíre, Jolien; Verhasselt, Bruno; Martins, Laura J.; Woelk, Christopher H.; Planelles, Vicente; Vandekerckhove, Linos

doi:10.1186/preaccept-1619001546133883

Cited by 3 publications

(3 citation statements)

References 7 publications

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“…Compared to αCD3/αCD28-stimulated cells at 6 days postinfection, untreated resting CD4 + T cells showed significantly lower levels of HIV infection but, nonetheless, permitted both productive and latent infection (Fig 1C). Importantly, productive and latent infection of resting CD4 + T cells over the 6-day time-course was not the result of replication-competent virus being present in our HIV Duo-Fluo I viral stocks (S2 Fig) [48]. …”

Section: Resultsmentioning

confidence: 99%

HIV Latency Is Established Directly and Early in Both Resting and Activated Primary CD4 T Cells

2015

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Highly active antiretroviral therapy (HAART) suppresses human immunodeficiency virus (HIV) replication to undetectable levels but cannot fully eradicate the virus because a small reservoir of CD4+ T cells remains latently infected. Since HIV efficiently infects only activated CD4+ T cells and since latent HIV primarily resides in resting CD4+ T cells, it is generally assumed that latency is established when a productively infected cell recycles to a resting state, trapping the virus in a latent state. In this study, we use a dual reporter virus—HIV Duo-Fluo I, which identifies latently infected cells immediately after infection—to investigate how T cell activation affects the estab-lishment of HIV latency. We show that HIV latency can arise from the direct infection of both resting and activated CD4+ T cells. Importantly, returning productively infected cells to a resting state is not associated with a significant silencing of the integrated HIV. We further show that resting CD4+ T cells from human lymphoid tissue (tonsil, spleen) show increased latency after infection when compared to peripheral blood. Our findings raise significant questions regarding the most commonly accepted model for the establishment of latent HIV and suggest that infection of both resting and activated primary CD4+ T cells produce latency.

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Section: Resultsmentioning

confidence: 99%

HIV Latency Is Established Directly and Early in Both Resting and Activated Primary CD4 T Cells

2015

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“…A recent study indicated that HIV-1 constructs rendered incapable of replicating by mutations or deletions of the env gene can revert to wild type through recombination with envelope expression plasmids following co-transfection of producer cells [ 51 ]. To test if replication competent viruses might be contributing to our results, we treated infected resting CD4 T cells with the protease inhibitor indinavir on the day of infection and on day 5 post infection with the non-nucleoside reverse transcriptase inhibitor efavirenz in order to block the spread of any reverted viruses.…”

Section: Response Of Latent Unintegrated Vs Integrated Hiv-1 To a Sementioning

confidence: 99%

HIV-1 latency and virus production from unintegrated genomes following direct infection of resting CD4 T cells

et al. 2016

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BackgroundHIV-1 integration is prone to a high rate of failure, resulting in the accumulation of unintegrated viral genomes (uDNA) in vivo and in vitro. uDNA can be transcriptionally active, and circularized uDNA genomes are biochemically stable in non-proliferating cells. Resting, non-proliferating CD4 T cells are prime targets of HIV-1 infection and latently infected resting CD4 T cells are the major barrier to HIV cure. Our prior studies demonstrated that uDNA generates infectious virions when T cell activation follows rather than precedes infection.ResultsHere, we characterize in primary resting CD4 T cells the dynamics of integrated and unintegrated virus expression, genome persistence and sensitivity to latency reversing agents. Unintegrated HIV-1 was abundant in directly infected resting CD4 T cells. Maximal gene expression from uDNA was delayed compared with integrated HIV-1 and was less toxic, resulting in uDNA enrichment over time relative to integrated proviruses. Inhibiting integration with raltegravir shunted the generation of durable latency from integrated to unintegrated genomes. Latent uDNA was activated to de novo virus production by latency reversing agents that also activated latent integrated proviruses, including PKC activators, histone deacetylase inhibitors and P-TEFb agonists. However, uDNA responses displayed a wider dynamic range, indicating differential regulation of expression relative to integrated proviruses. Similar to what has recently been demonstrated for latent integrated proviruses, one or two applications of latency reversing agents failed to activate all latent unintegrated genomes. Unlike integrated proviruses, uDNA gene expression did not down modulate expression of HLA Class I on resting CD4 T cells. uDNA did, however, efficiently prime infected cells for killing by HIV-1-specific cytotoxic T cells.ConclusionsThese studies demonstrate that contributions by unintegrated genomes to HIV-1 gene expression, virus production, latency and immune responses are inherent properties of the direct infection of resting CD4 T cells. Experimental models of HIV-1 latency employing directly infected resting CD4 T cells should calibrate the contribution of unintegrated HIV-1.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0234-9) contains supplementary material, which is available to authorized users.

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“…When samples from a single donor were profiled over time, a large number of genes were identified as dysregulated during the latent phase (N = 227) and were associated with chemokine receptors, cytokine signaling, and general immune responses. We previously used RNA-Seq to profile latently infected and uninfected samples from 4 donors [17] from the first iteration of a cultured primary central memory CD4 T cell (T CM ) model [18,19]. This study demonstrated that the defective vectors used to seed the latent reservoir were recombining to reconstitute actively replicating HIV-1.…”

Section: Introductionmentioning

confidence: 99%

Transcriptomic Analysis Implicates the p53 Signaling Pathway in the Establishment of HIV-1 Latency in Central Memory CD4 T Cells in an In Vitro Model

et al. 2016

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The search for an HIV-1 cure has been greatly hindered by the presence of a viral reservoir that persists despite antiretroviral therapy (ART). Studies of HIV-1 latency in vivo are also complicated by the low proportion of latently infected cells in HIV-1 infected individuals. A number of models of HIV-1 latency have been developed to examine the signaling pathways and viral determinants of latency and reactivation. A primary cell model of HIV-1 latency, which incorporates the generation of primary central memory CD4 T cells (TCM), full-length virus infection (HIVNL4-3) and ART to suppress virus replication, was used to investigate the establishment of HIV latency using RNA-Seq. Initially, an investigation of host and viral gene expression in the resting and activated states of this model indicated that the resting condition was reflective of a latent state. Then, a comparison of the host transcriptome between the uninfected and latently infected conditions of this model identified 826 differentially expressed genes, many of which were related to p53 signaling. Inhibition of the transcriptional activity of p53 by pifithrin-α during HIV-1 infection reduced the ability of HIV-1 to be reactivated from its latent state by an unknown mechanism. In conclusion, this model may be used to screen latency reversing agents utilized in shock and kill approaches to cure HIV, to search for cellular markers of latency, and to understand the mechanisms by which HIV-1 establishes latency.

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Replication competent virus as an important source of bias in HIV latency models utilizing single round viral constructs

Cited by 3 publications

References 7 publications

HIV Latency Is Established Directly and Early in Both Resting and Activated Primary CD4 T Cells

HIV Latency Is Established Directly and Early in Both Resting and Activated Primary CD4 T Cells

HIV-1 latency and virus production from unintegrated genomes following direct infection of resting CD4 T cells

Transcriptomic Analysis Implicates the p53 Signaling Pathway in the Establishment of HIV-1 Latency in Central Memory CD4 T Cells in an In Vitro Model

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