Background
Elucidating mechanisms governing proviral integration and transcription may aid in identifying strategies to limit HIV-1 reservoirs. Chimeric host-viral transcripts are a mechanism by which HIV-1 modulates host gene expression to promote reservoir persistence, though they are poor characterized in vivo. We analyzed SIV RNA+ CD4 T cells for viral, host, and chimeric transcripts during acute infection by scRNAseq, identifying integration sites (IS) and their expression.
Methods
Memory CD4 T-cells from lymph node and peripheral blood collected 2-3 weeks post-SIV infection of two rhesus macaques were single-cell RNA sequenced using SMART-Seq libraries and Illumina NovaSeq. Spliced viral RNA (svRNA)+ cells (n=150) were analyzed for chimeric mRNA and IS by mapping to rhesus and SIV genomes. Separate cell aliquots from the same specimens were DNA sequenced in bulk using linker-mediated PCR to both 5’ and 3’ SIV LTR and mapping to RheMac10 (UCSC).
Results
Single-cell RNAseq identified 72,757 chimeric SIV-rhesus transcripts within replication-active svRNA+ cells, representing ~0.1% of SIV reads and present in 75% of cells. Chimeric mRNA formed predominantly through transcription initiated at the SIV promoter, with 3’ LTR readthrough and splicing from virus donor to host acceptor sites comprising 76% of chimeras. 125 IS were mapped in 84 cells, one third of which had multiple proviruses. Consistent with prior analyses, protein coding genes and intronic loci predominated and average expression of targeted genes exceeded that of non-targeted genes (6-fold). Strikingly, insertional activation was observed for 7% of IS genes, while 9% lacked evidence of abundant transcription. DNA sequencing corroborated chimeric mRNA-identified IS with six shared clones between the analyses. Of the >200,000 IS identified by DNA sequencing, strong biases and non-randomness were observed (Chi-squared with Monte Carlo simulation, p<0.0005), with 50% of integrations occurring in <1000 genes.
Conclusions
Host-virus mRNA chimeras were found in most replication-active infected cells with transcription initiated predominantly at the viral promoter. Elevated average expression of IS genes identified by chimeric mRNA is consistent with a preference for actively transcribed genes, though IS gene expression is not required for proviral transcription. Frequent T cell superinfection may diversify proviral sequences during acute infection.