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CD4؉ and CD8 ؉ memory T cells with stem cell-like properties (T SCM cells) have been identified in mice, humans, and nonhuman primates and are being investigated for antitumor and antiviral vaccines and immunotherapies. Whether CD4 ؉ T SCM cells are infected by human immunodeficiency virus (HIV) was investigated by using a combination HIV reporter virus system in vitro and by direct staining for HIV p24 antigen ex vivo. A proportion of T SCM cells were found to express the HIV coreceptors CCR5 and CXCR4 and were infected by HIV both in vitro and in vivo. Analysis of viral outcome following fusion using the combination reporter virus system revealed that T SCM cells can become productively or latently infected, although the vast majority of T SCM cells are abortively infected. Knockdown of the HIV restriction factor SAMHD1 using Vpx-containing simian immunodeficiency virus (SIV) virion-like particles enhanced the productive infection of T SCM cells, indicating that SAMHD1 contributes to abortive infection in these cells. These results demonstrate that CD4؉ T SCM cells are targets for HIV infection, that they become productively or latently infected at low levels, and that SAMHD1 expression promotes abortive infection of this important memory cell subset. IMPORTANCE Here we demonstrate the susceptibility of CD4 ؉ memory stem cells (T SCM cells) to infection by HIV in vitro and in vivo, provide an in-depth analysis of coreceptor expression, demonstrate the infection of naïve and memory CD4؉ T cell subsets with both CCR5-and CXCR4-tropic HIV, and also perform outcome analysis to calculate the percentage of cells that are productively, latently, or abortively infected. Through these outcome studies, we determined that the vast majority of T SCM cells are abortively infected by HIV, and we demonstrate that knockdown of SAMHD1 significantly increases the frequency of infection of this CD4 ؉ T cell subset, indicating that SAMHD1 is an active restriction factor in T SCM cells.

Latently infected cells remain a primary barrier to eradication of HIV-1. Over the past decade, a better understanding of the molecular mechanisms by which latency is established and maintained has led to the discovery of a number of compounds that selectively reactivate latent proviruses without inducing polyclonal T cell activation. Recently, the histone deacetylase (HDAC) inhibitor vorinostat has been demonstrated to induce HIV transcription from latently infected cells when administered to patients. While vorinostat will be given in the context of antiretroviral therapy (ART), infection of new cells by induced virus remains a clinical concern. Here, we demonstrate that vorinostat significantly increases the susceptibility of CD4؉ T cells to infection by HIV in a dose-and time-dependent manner that is independent of receptor and coreceptor usage. Vorinostat does not enhance viral fusion with cells but rather enhances the kinetics and efficiency of postentry viral events, including reverse transcription, nuclear import, and integration, and enhances viral production in a spreading-infection assay. Selective inhibition of the cytoplasmic class IIb HDAC6 with tubacin recapitulated the effect of vorinostat. These findings reveal a previously unknown cytoplasmic effect of HDAC inhibitors promoting productive infection of CD4 ؉ T cells that is distinct from their well-characterized effects on nuclear histone acetylation and long-terminal-repeat (LTR) transcription. Our results indicate that careful monitoring of patients and ART intensification are warranted during vorinostat treatment and indicate that HDAC inhibitors that selectively target nuclear class I HDACs could reactivate latent HIV without increasing the susceptibility of uninfected cells to HIV. IMPORTANCEHDAC inhibitors, particularly vorinostat, are currently being investigated clinically as part of a "shock-and-kill" strategy to purge latent reservoirs of HIV. We demonstrate here that vorinostat increases the susceptibility of uninfected CD4؉ T cells to infection with HIV, raising clinical concerns that vorinostat may reseed the viral reservoirs it is meant to purge, particularly under conditions of suboptimal drug exposure. We demonstrate that vorinostat acts following viral fusion and enhances the kinetics and efficiency of reverse transcription, nuclear import, and integration. The effect of vorinostat was recapitulated using the cytoplasmic histone deacetylase 6 (HDAC6) inhibitor tubacin, revealing a novel and previously unknown cytoplasmic mechanism of HDAC inhibitors on HIV replication that is distinct from their well-characterized effects of long-terminal-repeat (LTR)-driven gene expression. Moreover, our results suggest that treatment of patients with class I-specific HDAC inhibitors could induce latent viruses without increasing the susceptibility of uninfected cells to HIV.

Fusion between the viral membrane of human immunodeficiency virus (HIV) and the host cell marks the end of the HIV entry process and the beginning of a series of post-entry events including uncoating, reverse transcription, integration, and viral gene expression. The efficiency of post-entry events can be modulated by cellular factors including viral restriction factors and can lead to several distinct outcomes: productive, latent, or abortive infection. Understanding host and viral proteins impacting post-entry event efficiency and viral outcome is critical for strategies to reduce HIV infectivity and to optimize transduction of HIV-based gene therapy vectors. Here, we report a combination reporter virus system measuring both membrane fusion and viral promoter-driven gene expression. This system enables precise determination of unstimulated primary CD4+ T cell subsets targeted by HIV, the efficiency of post-entry viral events, and viral outcome and is compatible with high-throughput screening and cell-sorting methods.

BackgroundTemplate switching between two distinct HIV-1 RNA genomes during reverse transcription gives rise to recombinant viruses that greatly expand the genetic diversity of HIV-1 and have adverse implications for drug resistance, immune escape, and vaccine design. Virions with two distinct genomes are produced exclusively from cells infected with two or more viruses, or ‘doubly infected’ cells. Previous studies have revealed higher than expected frequencies of doubly infected cells compared to frequencies based on chance alone, suggesting non-random enhancement of double infection.MethodsWe investigated double infection of unstimulated primary CD4+ T cells using reporter viruses carrying genes for different fluorescent proteins, EGFP and mCherry, combined with sophisticated modeling techniques based on Poisson distribution. Additionally, through the use of multiparameter flow cytometry we examined the susceptibility of naïve and memory subsets of CD4+ T cells to double infection by HIV.ResultsUsing our double infection system, we confirm non-random enhancement of multiple infection events. Double infection of CD4+ T cells was not found to be a consequence of suboptimal provirus expression rescued by Tat in trans—as has been reported in cell lines—but rather due to a heterogeneous cell population in which only a fraction of primary peripheral blood CD4+ T cells are susceptible to HIV infection regardless of viral titer. Intriguingly, double infection of CD4+ T cells occurred preferentially in memory CD4+ T cells—particularly the central memory (TCM) subset—but was not a consequence of SAMHD1-mediated restriction of HIV infection in naïve cells.ConclusionsThese findings reveal that double infection in primary CD4+ T cells is primarily a consequences of cellular heterogeneity and not rescue of suboptimal provirus expression by Tat in trans. Additionally, we report a previously unappreciated phenomenon of enhanced double infection within primary TCM cells and suggest that these long-lived cells may serve as an archive that drive ongoing viral recombination events in vivo.