Correlates of Virus Release in HIV-1

Abstract

Integration of HIV-1 within the host genome may result in latent infection, where a replication competent provirus remains in a state of transcriptional silence. The latent reservoir remains the major barrier to cure efforts as to achieve cure, the reservoir should be depleted of all replication competent proviruses. Various assays to measure the latent reservoir rely on virus release and vary widely in their estimations. Quantitative viral outgrowth assay (QVOA) which is the gold standard relies on reactivation of latent proviruses to release virions and thus falls short when proviruses are non-inducible. The correlates of virus release per cell were examined in this study. The TZ-5 zipcoded library which is a Jurkat cell line with one uniquely tagged HIV-1 integrant per cell was used in this study. The library consisted of infected cells expressing HIV-1 gag and pol with an inactivated env gene and vpr-. GFP is expressed as a Nef spliced product to indicate HIV-1 expression. Intracellular Gag staining coupled with the relative amounts of p24 detected in culture supernatant showed GFP expression is an accurate proxy for HIV-1 expression. High-throughput sequencing was carried out on PCR amplified zipcodes. The topmost 74 clones which represents ≥ 97% of the raw sequence reads was analysed in this study. Although cell proliferation is thought to drive HIV-1 persistence, clonal abundance was not found to correlate with high virus release. Rather a higher GFP+ expression as determined by the green proportion correlates with high virus release per cell. Spliced and unspliced mRNA products are needed to productively assemble virions in HIV-1 although Gag which is an unspliced product is the driving force. Clones with higher amounts of unspliced product to spliced had higher green proportions and subsequently overlapped with higher amounts of virus released. Taken together, higher green proportions and unspliced RNA may predict a higher amount of virus released per cell. As green proportion is a measure of overall HIV-1 expression by a clone, incorporating the extent of HIV-1 expression in QVOA measurements will calibrate HIV-1 expression among inducible and noninducible cells in the latent pool and ultimately the decay rate of such clones. The frequency of formation of host-virus read-through and read-in chimeras and packaging into virions was examined as these unusual species can be helpful in integration site determination and ultimately determine genetic correlates of chimeric RNA generation. RNase protection assays showed the presence of chimeric read-through and read-in RNAs both in cells and virus. The genomic context of integration may contribute to our observed findings as more read-in RNA was observed to be produced and packaged into virions than read-through RNA.