Transcriptionally silent HIV proviruses form the major obstacle to eradicating HIV. Many studies of HIV latency have focused on the cellular mechanisms that maintain silencing of proviral DNA. Here we show that viral sequence variation affecting replicative ability leads to variable rates of silencing and ability to reactivate.
We studied naturally occurring and engineered polymorphisms in a recently identified exonic splice enhancer (ESEtat) that regulates tat mRNA splicing and constructed viruses with increased (M1), reduced (M2) or completely absent (ERK) binding of splicing factors essential for optimal production of tat mRNA resulting in a corresponding change in Tat activity. The mutations affected viral replication, with M1 having wild type kinetics, M2 exhibiting reduced kinetics and with replication completely abrogated in ERK. Using single round GFP expressing viruses to study proviral gene expression, we observed progressively greater rates of silencing relating to the degree of ESEtat disruption, with WT at 53%, M2 at 69% and ERK at 94%. By stimulating infected cells with each of the latency reversal agents PMA, panobinostat and JQ1, we observed that the dose required to achieve 50% of the maximum signal was lowest in WT, intermediate in M2 and highest in ERK, indicating a progressively higher threshold for reactivation.
These results suggest that the ability of silent proviruses to reactivate from latency is variable and that minor differences in the viral sequence can alter the proportion of silenced viruses as well as the threshold required to induce silenced viruses to reactivate and express.HPM is supported by the Medical Research Council, UK (MR/N02043X/1) and the Academy of Medical Sciences (UK). NJN is supported by the British Infection Association and the Medical Research Council (MR/M003515/1). This work was supported by the Evelyn Trust and the Cambridge Clinical Academic Reserve. Work in the laboratory is supported by the Cambridge NIHR Biomedical Research Centre and the MRC
