Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing

Ocwieja, Karen E.; Sherrill-Mix, Scott; Mukherjee, Rithun; Custers-Allen, Rebecca; David, Patricia H; Brown, Michael S.; Wang, Susana; Link, Darren R.; Olson, Jeff; Travers, Kevin; Schadt, Eric; Bushman, Frederic D.

doi:10.1093/nar/gks753

Cited by 127 publications

(213 citation statements)

References 56 publications

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“…2B, the novel usHIV-RNA transcripts reported in this study did not contain any of the classic splice acceptor/donor sites in their genomes (28), nor did they contain cryptic splice sites that have been reported elsewhere (29,30). These data support the hypothesis that the "defective" proviruses seen in patients with HIV-1 infection lead to the production of novel HIV-RNA transcripts faithfully transcribed from these proviruses.…”

Section: Significancesupporting

confidence: 82%

Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy

Imamichi

Dewar

Adelsberger

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

295

313

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Despite years of plasma HIV-RNA levels <40 copies per milliliter during combination antiretroviral therapy (cART), the majority of HIV-infected patients exhibit persistent seropositivity to HIV-1 and evidence of immune activation. These patients also show persistence of proviruses of HIV-1 in circulating peripheral blood mononuclear cells. Many of these proviruses have been characterized as defective and thus thought to contribute little to HIV-1 pathogenesis. By combining 5′LTR-to-3′LTR single-genome amplification and direct amplicon sequencing, we have identified the presence of “defective” proviruses capable of transcribing novel unspliced HIV-RNA (usHIV-RNA) species in patients at all stages of HIV-1 infection. Although these novel usHIV-RNA transcripts had exon structures that were different from those of the known spliced HIV-RNA variants, they maintained translationally competent ORFs, involving elements of gag, pol, env, rev, and nef to encode a series of novel HIV-1 chimeric proteins. These novel usHIV-RNAs were detected in five of five patients, including four of four patients with prolonged viral suppression of HIV-RNA levels <40 copies per milliliter for more than 6 y. Our findings suggest that the persistent defective proviruses of HIV-1 are not “silent,” but rather may contribute to HIV-1 pathogenesis by stimulating host-defense pathways that target foreign nucleic acids and proteins.

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Section: Significancesupporting

confidence: 82%

Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy

Imamichi

Dewar

Adelsberger

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

295

313

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“…Real-time PCR was then performed by using the described primers and probe targeting the integrase region of pol (11). This described primer/probe pair also amplifies vif mRNA, which constitutes 0.21% of the 4.0-kb family of spliced mRNAs in T cells (32), and, thus, does not contribute significantly to quantification of unspliced HIV-1 RNA. The cellular gene IPO8 was quantified as an internal standard for cellular RNA recovery by using forward primer 5′-GCTCTGATAACTGTGCAG-3′, reverse primer 5′-CAGTGTGTACACCTCCTG-3′, and probe 5′-[6FAM]TGCTGTCCTCTGAT-CCTCGC[TAMRA]-3′.…”

Section: Methodsmentioning

confidence: 99%

Quantification of HIV-1 latency reversal in resting CD4 ⁺ T cells from patients on suppressive antiretroviral therapy

Cillo

Sobolewski

Bosch

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

212

218

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Reversal of proviral latency is being pursued as a curative strategy for HIV-1 infection. Recent clinical studies of in vivo administration of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat) show increases in unspliced cellular HIV-1 RNA levels in resting CD4 + T cells. A critical unknown, however, is the proportion of latent proviruses that can be transcriptionally reactivated by SAHA or T-cell activation. In this study, we quantified the fraction of HIV-1 proviruses in resting CD4 + T cells from patients on suppressive antiretroviral therapy that were reactivated ex vivo with SAHA or antibodies to CD3/CD28. At concentrations of SAHA achieved clinically, only 0.079% of proviruses in resting CD4 + T cells were reactivated to produce virions, compared with 1.5% of proviruses in cells treated with anti-CD3/CD28 antibodies after correcting for spontaneous virion production in the medium control. A significant positive correlation (ρ = 0.67, P < 0.001) was found between levels of virions in the supernatant and unspliced cellular HIV-1 RNA following anti-CD3/CD28 treatment, but not following SAHA treatment (ρ = 0.21, P = 0.99). These results reveal that the majority of HIV-1 proviruses are not reactivated by current therapeutic approaches and that more effective means of reversing proviral latency will likely be required to deplete HIV-1 reservoirs.HIV-1 persistence | HIV-1 eradication | HIV-1 cure | fractional provirus expression A ntiretroviral therapy (ART) for HIV-1 infection suppresses viral replication but is not curative. Assays of infectious virus recovery from quiescent CD4 + T cells isolated from patients on ART have revealed the existence of a reservoir of latent, replication competent HIV-1 with a half-life of 44 mo (1-4). In addition, low-level plasma viremia persists indefinitely on ART (5, 6), and the level of virus in plasma rebounds following cessation of ART (7,8). New therapeutic approaches are required to eliminate both persistent low-level viremia and the latent proviral reservoir. A "kick and kill" approach has been proposed in which latency reversing agents, administered in conjunction with ART, will "kick" proviruses out of latency, followed by a "kill" of the infected cells through viral cytopathic effects or immune-mediated cytotoxicity.Histone deacetylase inhibitors (HDACi) have been proposed as latency reversing agents, and single-dose or multidose administration of suberoylanilide hydroxamic acid (SAHA; vorinostat) in vivo was shown to increase expression of unspliced cellular HIV-1 RNA in resting CD4 + T (rCD4) cells in patients on suppressive ART (9, 10). Although three-to fivefold increases in cellular HIV-1 RNA were observed (9), the fraction of latent HIV-1 proviruses that were reactivated by SAHA was not quantified. It is possible that SAHA transcriptionally reactivated many latent proviruses, or alternatively reactivated only a minority of latent proviruses. These two alternatives have very different implications in terms of the impact SAHA coul...

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“…Transcription from the 5′ LTR generates a primary transcript, which is spliced into over 109 mRNAs to produce all viral proteins or is packaged into virions (7). Each LTR contains untranslated 3′ (U3), repeat (R), and untranslated 5′ (U5) regions.…”

Section: Historical Perspectivementioning

confidence: 99%

Molecular mechanisms of HIV latency

Cary¹,

Fujinaga²,

Peterlin³

2016

Journal of Clinical Investigation

123

133

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Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing

Cited by 127 publications

References 56 publications

Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy

Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy

Quantification of HIV-1 latency reversal in resting CD4 ⁺ T cells from patients on suppressive antiretroviral therapy

Molecular mechanisms of HIV latency

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Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing

Cited by 127 publications

References 56 publications

Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy

Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy

Quantification of HIV-1 latency reversal in resting CD4 + T cells from patients on suppressive antiretroviral therapy

Molecular mechanisms of HIV latency

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Quantification of HIV-1 latency reversal in resting CD4 ⁺ T cells from patients on suppressive antiretroviral therapy