Latency reversing agents affect HIV RNA splicing
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2017
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Melbourne Authors
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Microbiology & Immunology
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PhD thesis
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Open Access
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© 2017 Dr. Talia Monique Mota
Abstract
Although antiretroviral therapy (ART) has changed HIV from a death sentence to a manageable chronic illness, there is still no cure and individuals living with HIV must endure lifelong therapy and continue to face stigma and discrimination associated with their infection. The largest barrier to an HIV cure is the existence of a viral reservoir in resting CD4+ T cells, in which an integrated copy of the viral genome persists within these cells for the lifespan of the individual. Latency reversing agents (LRAs) are epigenetic modifiers including histone deacetylase inhibitors (HDACi), and have been evaluated in clinical trials for HIV cure in an approach called “shock and kill” where the HDACi can induce viral transcription and hopefully enable death of the cell through viral mediated cytopathic effects or immune mediated clearance. Although HDACi have been investigated in vivo, there has been no significant decline in number of latently infected cells across the studies. The potency of HDACi as LRAs has largely been assessed by measuring increases in cell-associated unspliced (US) HIV RNA, but the effect of HDACi on viral splicing or on the function of viral Tat protein is not known. Additionally, the long term effects of HDACi on safety and virological aspects in vivo have never been assessed. In this thesis, we demonstrate through extended follow-up in individuals living with HIV who had participated in our clinical trial did not reveal any long-term toxicity or changes in markers of HIV persistence or transcription in participants on ART who had received 14 days of HDACi vorinostat. We additionally show that while HDACi increase the levels of US HIV RNA, they reduce the accumulation of cell-associated multiply spliced (MS) HIV RNA, and do this in the presence or absence of viral transactivator protein Tat. We show that this change in splicing is mediated through a reduction in HDAC1 and that romidepsin, a potent HDACi, also inhibits a number of key splicing factors, as well as polypyrimidine tract-binding protein (PTB), and CyclinT1. Additionally, Tat can be post-translationally modified and we show that various lysine and arginine mutations reduce the capacity of Tat to induce both transcription and splicing, and that modifications on lysine 28 and lysine 50 are required for synergistic function between Tat and HDACi, JQ1, or chaetocin in transcription. We also show that JQ1 is the only LRA that can induce splicing without Tat, or rescue splicing in the presence of mutations in Tat. Our data provide evidence that Tat expression is crucial in reactivation of latent HIV infection and that HDACi synergize with Tat to increase transcription, but JQ1 is more efficient in enabling splicing. Post translational modifications of different lysine residues of Tat are critical for synergism with LRAs. Overall, our data provide evidence of the limited potency of HDACi to reverse latency as a result of adverse effects on HIV splicing, a key step required for HIV protein and virion production in latently infected cells. This research will inform clinicians as they continue to pursue LRAs and other strategies for HIV cure.