Intragenic proviral elements support transcription of defective HIV-1 proviruses

Abstract

HIV-1 establishes a persistent proviral reservoir by integrating into the genome of infected host cells. Current antiretroviral treatments do not target this persistent population of proviruses which include latently infected cells that upon treatment interruption can be reactivated to contribute to HIV-1 rebound. Deep sequencing of persistent HIV proviruses has revealed that greater than 90% of integrated HIV genomes are defective and unable to produce infectious virions. We hypothesized that intragenic elements in the HIV genome support transcription of aberrant HIV-1 RNAs from defective proviruses that lack long terminal repeats (LTRs). Using an intact provirus detection assay, we observed that resting CD4+ T cells and monocyte-derived macrophages (MDMs) are biased towards generating defective HIV-1 proviruses. Multiplex reverse transcription droplet digital PCR identified env and nef transcripts which lacked 5’ untranslated regions (UTR) in acutely infected CD4+ T cells and MDMs indicating transcripts are generated that do not utilize the promoter within the LTR. 5’UTR-deficient env transcripts were also identified in a cohort of people living with HIV (PLWH) on ART, suggesting that these aberrant RNAs are produced in vivo. Using 5’ rapid amplification of cDNA ends (RACE), we mapped the start site of these transcripts within the Env gene. This region bound several cellular transcription factors and functioned as a transcriptional regulatory element that could support transcription and translation of downstream HIV-1 RNAs. These studies provide mechanistic insights into how defective HIV-1 proviruses are persistently expressed to potentially drive inflammation in PLWH.

HIV silencing and cell survival signatures in infected T cell reservoirs

“…HIV genome coverage patterns of mapped reads were notable for isolated peaks interspersed with areas of no coverage (Fig. 4d ), suggesting atypical transcription start sites 52 , transcripts from proviruses with deletion mutations and/or chance sampling variations. Spliced transcripts were not detected even by manually inspecting and mapping individual mates of read pairs using BLAST.…”

Adaptation of a transmitted/founder simian-human immunodeficiency virus for enhanced replication in rhesus macaques

“…Genomic DNA (gDNA) was obtained by using the All-Prep DNA/RNA Mini kit (Qiagen # 80204). Digital droplet PCR was performed as previously described (26, 73–75). Briefly, 200-250 ng of DNA was added in each ddPCR reaction containing 11 μl of ddPCR 2x super mix (Bio Rad #1863024), 2 units of HindIII per reaction well (New England Biolabs # R0104S), 600 nM of each primer (Pol and ENV) and 200nM of each probe.…”

Defective HIV-1 genomes and their potential impact on HIV pathogenesis

“…In addition, sequences within the HIV-1 env gene have been identified as potential elements that control intragenic transcriptional activity and include transcription binding sites, the presence of methylated CpG islands, and increased DNAse I sensitivity which correlates with transcriptionally active elements [ 92 – 94 ]. We have extended these observations using 5ʹ RACE PCR to demonstrate that HIV transcripts are generated from an intragenic promoter within the envelope gene in in vitro infected primary cells [ 95 ]. Potential aberrant RNAs that contained env and nef but lacked 5ʹ LTR derived untranslated regions (UTRs) were detected in cDNAs generated from cell-associated RNA from PLWH on ART using multiplex reverse transcriptase droplet digital PCR [ 92 ].…”

“…Similarly, internal deletions and inversions within defective proviral genomes can generate novel open reading frames and translation of proteins [ 54 , 98 ]. HIV-1 proteins are also translated from transcripts generated from intragenic promoters and there have been reports of an antisense protein [ 80 , 82 , 95 , 99 ]. Translation from these aberrant or spurious RNAs would be consistent with the detection of HIV-1 proteins in PWLH on ART and in latently infected cells in the absence of viral replication.…”