CAGE-Seq Reveals that HIV-1 Latent Infection Does Not Trigger Unique Cellular Responses in a Jurkat T Cell Model

The cure for HIV-1 is currently stalled by our inability to specifically identify and target latently infected cells. HIV-1 viral RNA/DNA or viral proteins are recognized by cellular mechanisms and induce interferon responses in virus-producing cells, but changes in latently infected cells remain unknown. HIVGKO contains a green fluorescent protein (GFP) reporter under the HIV-1 promoter and a monomeric Kusabira orange 2 (mKO2) reporter under the internal elongation factor alpha (EF1α) promoter. This viral construct enables direct identification of both productively and latently HIV-1-infected cells. In this study, we aim to identify specific cellular transcriptional responses triggered by HIV-1 entry and integration using cap analysis of gene expression (CAGE). We deep sequenced CAGE tags in non-infected and latently and productively infected cells and compared their differentially expressed transcription start site (TSS) profiles. Virus-producing cells had differentially expressed TSSs related to T-cell activation and apoptosis compared to those of non-infected cells or latently infected cells. Surprisingly, latently infected cells had only 33 differentially expressed TSSs compared to those of non-infected cells. Among these, SPP1 and APOE were downregulated in latently infected cells. SPP1 or APOE knockdown in Jurkat T cells increased susceptibility to HIVGKO infection, suggesting that they have antiviral properties. Components of the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway, MLST8, 4EBP, and RPS6, were significant TSSs in productively infected cells, and S6 kinase (S6K) phosphorylation was increased compared to that in latently infected cells, suggesting that mTOR pathway activity plays a role in establishing the latent reservoir. These findings indicate that HIV-1 entry and integration do not trigger unique transcriptional responses when infection becomes latent

Understanding HIV Latency: The Road to an HIV Cure

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Mutations that delay completion of reverse transcription do not increase viral sensitivity to hTRIM5α.

<p>Untransduced CRFK cells, and those transduced with lentiviral vectors resulting in the overexpression of β-galactosidase (CRFK-LacZ), N-terminal hemagglutinin-tagged hTRIM5α (CRFK-HA-TRIM5α) or hTRIM5α were infected with serial two-fold dilutions of the indicated VSV-pseudotyped viruses, which express <i>Renilla</i> luciferase in the place of Nef, and RLU was measured 40 h after infection. Infectivity was expressed as the slope of the RLU vs ng p24 curves, determined by linear regression. In the top panels (A), results are the mean ± SEM for three independent experiments expressed relative to infectivity observed in untransduced CRFK cells. ** indicates p<0.01 compared to results for NL4-3 in the same cell line. In the bottom panels (B), results in untransduced CRFK cells for each virus (n = 3) are expressed relative to infectivity observed for NL4-3 (left bottom panel) or Bru (right bottom panel).</p

Delaying Reverse Transcription Does Not Increase Sensitivity of HIV-1 to Human TRIM5α

<div><h3>Background</h3><p>Because uncoating of the capsid is linked to reverse transcription, modifications that delay this process lead to the persistence in the cytoplasm of capsids susceptible to recognition by the human restriction factor TRIM5α (hTRIM5α). It is unknown, however, if increasing the time available for capsid-hTRIM5α interactions would actually render viruses more sensitive to hTRIM5α.</p> <h3>Results</h3><p>Viral sensitivity to hTRIM5α was evaluated by comparing their replication in human U373-X4 cells in which hTRIM5α activity had or had not been inhibited by overexpression of human TRIM5γ. No differences were observed comparing wild-type HIV-1 and variants carrying mutations in reverse transcriptase or the central polypurine tract that delayed the completion of reverse transcription. In addition, the effect of delaying the onset of reverse transcription for several hours by treating target cells with nevirapine was evaluated using viral isolates with different sensitivities to hTRIM5α. Delaying reverse transcription led to a time-dependent loss in viral infectivity that was increased by inhibiting capsid-cyclophilin A interactions, but did not result in increased viral sensitivity to hTRIM5α, regardless of their intrinsic sensitivity to this restriction factor.</p> <h3>Conclusions</h3><p>Consistent with prior studies, the HIV-1 capsid can be targeted for destruction by hTRIM5α, but different strains display considerable variability in their sensitivity to this restriction factor. Capsids can also be lost more slowly through a TRIM5α-independent process that is accelerated when capsid-cyclophilin A interactions are inhibited, an effect that may reflect changes in the intrinsic stability of the capsid. Blocking the onset or delaying reverse transcription does not, however, increase viral sensitivity to hTRIM5α, indicating that the recognition of the capsids by hTRIM5α is completed rapidly following entry into the cytoplasm, as previously observed for the simian restriction factors TRIM-Cyp and rhesus TRIM5α.</p> </div

Delaying the onset of reverse transcription under conditions where CypA-CA interactions are inhibited does not increase viral sensitivity to hTRIM5α.

<p>Experiments were performed and results are expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052434#pone-0052434-g002" target="_blank">Figure 2</a>, legend, except that all cultures were maintained in the continuous presence of 1 µg/ml Debio-025. Shown are the mean ± SEM for three independent experiments performed using fresh viral stocks. * p<0.05, ** p<0.01 compared to U373-X4-LacZ cells.</p

Mutagenesis Primers.

*<p>Reverse primers were the reverse-complement of the indicated sequence. <b>T</b> = mutation introduced.</p

Delaying the onset of reverse transcription does not increase viral sensitivity to hTRIM5α.

<p>Untransduced U373-X4 cells and U373-X4 cells transduced with lentiviral vectors resulting in the overexpression of β-galactosidase (LacZ) or human TRIM5γ (TRIM5γ) were cultured overnight in the presence of 100 U/ml IFNα, and infected with 3 ng p24/well of the indicated recombinant VSV-pseudotyped viruses, which express <i>Renilla</i> luciferase in the place of Nef, and luciferase activity was measured 40 h after infection. Parallel cultures were maintained in the presence of 250 ng/ml NVP for the indicated times prior to washing the cells to remove NVP. Cultures not receiving NVP were washed 1 h after infection. In the left panels, results for cells not treated with NVP are expressed relative to RLU measured in untransduced U373-X4 cells. In the right panels, results for each cell line are expressed relative to RLU measured in cultures not treated with NVP. Shown are the mean ± SEM for three independent experiments performed using fresh viral stocks. ** indicates p<0.01 compared to U373-X4-LacZ cells.</p

Strain-Specific Differences in the Impact of Human TRIM5α, Different TRIM5α Alleles, and the Inhibition of Capsid-Cyclophilin A Interactions on the Infectivity of HIV-1 ▿ †

HIV-1 infectivity is strongly restricted by TRIM5α from certain primate species but has been described as being only marginally susceptible to human TRIM5α. In this study, we evaluated the effects of the modulation of human TRIM5α activity (pretreatment of target cells with alpha interferon, expression of a pre-miRNA targeting TRIM5α, and/or overexpression of TRIM5γ), the inhibition of cyclophilin A (CypA)-CA interactions, and the expression of different allelic variants of human TRIM5α on the infectivity of a series of recombinant viruses carrying different patient-derived Gag-protease sequences. We show that HIV-1 displays virus-specific differences in its sensitivity to human TRIM5α and in its sensitivity to different TRIM5α alleles. The effect of inhibiting CypA-CA interactions is also strain specific, and blocking these interactions can either inhibit or improve viral infectivity, depending on the isolate studied. The inhibition of CypA-CA interactions also modulates viral sensitivity to human TRIM5α. In the absence of CypA-CA interactions, most viruses displayed increased sensitivity to the inhibitory effects of TRIM5α on viral replication, but one isolate showed a paradoxical decrease in sensitivity to TRIM5α. Taken together, these findings support a model in which three interlinked factors—capsid sequence, CypA levels, and TRIM5α—interact to determine capsid stability and therefore viral infectivity