Correlates of Virus Release in HIV-1
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Date
2019-03
Authors
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Journal ISSN
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Publisher
University of Ghana
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.
Description
MPhil. Molecular Cell Biology of Infectious Diseases
Keywords
HIV-1, Virus release, Ghana, Gag, RNA, Quantitative viral outgrowth assay, QVOA