Transcriptional regulation of the HTLV-1 provirus

Abstract

The deltaretrovirus Human T cell Leukaemia Virus type 1 (HTLV-1) causes infections which persist for the lifetime of its host, following its integration into the host genome. Infection can result in an aggressive leukemia, ATL, or a debilitating neuropathy known as HAM/TSP, which together affect approximately 10% of HTLV-1 carriers. Proviral gene expression maintains a stable proviral load through its conferral of pro-survival and pro-proliferative effects on infected cells. The expression of the proviral sense strand, which encodes most HTLV-1 proteins, is heterogeneous even within clonal infected cell populations, and live-cell imaging has revealed that it is intermittently expressed. The dynamics and underlying molecular mechanisms of this discontinuous gene expression are unknown. Here, I used multiple methods to investigate potential mechanisms regulating proviral expression in naturally infected primary, and clonal, HTLV-infected T cells. I used bulk RNA-seq to examine the transcriptomes of HTLV-1-infected cells undergoing reactivation ex vivo, and observed a conserved progression of changes to gene expression in infected cells. Histone ubiquitination was highlighted as a potential mechanism for limiting proviral expression following reactivation, with several components involved in the deposition and removal of H2AK119ub1 significantly affected by proviral expression. Using single-molecule RNA FISH, I quantified sense-strand transcription over six days, finding that an existing three promoter state transcriptional model is insufficient to explain the observed transcript trajectories, with changes to parameter values mid-run necessary to recreate measured RNA counts, suggesting additional mechanisms are required to explain the observed data. Finally, I examined whether transcriptional interference between the proviral sense and antisense strands impacts proviral expression. Following a block of proviral transcription using CRISPRi to reduce oncoming RNA polymerase traffic, I found no evidence of transcriptional interference between the two proviral strands, suggesting transcription can occur from both strands without adverse effects on output. The findings presented in this thesis are relevant to our understanding of the regulation of HTLV-1 proviral transcription.