Specific HIV integration sites are linked to clonal expansion and persistence of infected cells

164

219

HIV-1–infected individuals harbor a latent reservoir of infected CD4+ T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones.

Section: Resultssupporting

confidence: 54%

Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA

Lorenzi

Cohen

Cohn

et al. 2016

164

219

“…The in vivo presence of clonally expanded proviral populations has been reported by ourselves and others (14,(23)(24)(25)(26). However, there is minimal evidence for transcription of defective proviruses (14,27).…”

Section: Significancementioning

confidence: 92%

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

Imamichi

Dewar

Adelsberger

et al. 2016

295

313

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.

“…1C). We had previously estimated, based on integration site analysis, that AMBI-1 constituted a smaller fraction of the proviral DNA (at least 3.2%) (3). These data imply that a substantial fraction of the proviruses detected in the integration site analysis were defective and may have had large internal deletions, or hypermutations, which would have prevented their detection by PCR for pro-pol DNA.…”

Section: Significancementioning

confidence: 99%

“…A stringent filter was used to check quality of recovered integration sites. Sequences that have exactly the same integration site but different host DNA breakpoints came from different cells; this test identifies proviruses in clonally amplified cells (3).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Clonally expanded CD4 ⁺ T cells can produce infectious HIV-1 in vivo

Simonetti

Sobolewski

Fyne

et al. 2016

314

392

Reservoirs of infectious HIV-1 persist despite years of combination antiretroviral therapy and make curing HIV-1 infections a major challenge. Most of the proviral DNA resides in CD4+T cells. Some of these CD4+T cells are clonally expanded; most of the proviruses are defective. It is not known if any of the clonally expanded cells carry replication-competent proviruses. We report that a highly expanded CD4+ T-cell clone contains an intact provirus. The highly expanded clone produced infectious virus that was detected as persistent plasma viremia during cART in an HIV-1–infected patient who had squamous cell cancer. Cells containing the intact provirus were widely distributed and significantly enriched in cancer metastases. These results show that clonally expanded CD4+T cells can be a reservoir of infectious HIV-1.