Microbiology & Immunology - Theses

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Subject: HIV-1 × Start date: 2020 × End date: 2023 × Author: Stern, Jared ×

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    Understanding the drivers of natural variation of transcription in HIV-1 latency
    Stern, Jared ( 2022)
    An estimated 37.7 million people are currently living with HIV-1, with 27.5 million of those taking antiretroviral therapy (ART) that inhibits viral replication to prevent disease progression and onward transmission. Upon infection, HIV-1 establishes a latent state of infection characterised by (near) silent viral transcription and translation to avoid immune recognition and clearance, further ensuring the virus’s survival within its host. This, together with integration of the viral genome into CD4+ T-cells (and less frequently into myeloid-derived leukocytes), creates a persistent HIV-1 reservoir. These latently infected cells can quickly and spontaneously reactivate to cause viral rebound within weeks of ART cessation. Therefore, ART is required life-long which poses significant economic needs on countries as well as financial, physical and mental health burdens on people living with HIV (PLHIV). The main goal of cure strategies is to reduce the size of the reservoir, allowing for a greater chance of immune mediated control in the absence of ART. One such strategy, the “shock and kill” approach, aims to reactivate latent proviruses to induce viral gene and protein expression and allow for elimination of infected cells by immune-mediated clearance or even direct killing by viral cytopathic effects. Clinical trials of latency reversing agents (LRAs) in PLHIV have so far demonstrated viral reactivation, though this has not resulted in meaningful reductions of the reservoir or delays to viral rebound after cessation of ART – highlighting a need for new, more potent LRAs to achieve a cure. In a prospective clinical trial of PLHIV on ART, we observed that cell-associated HIV-1 RNA - a measure of HIV-1 transcription - varied significantly on three separate occasions prior to any intervention. The variation in HIV-1 RNA was independently associated with time and visit, with higher HIV-1 RNA being measured earlier in the day. Circadian rhythms dictate numerous physiologic and behavioural changes over the 24-hour day and are entrained at a systemic level, as well as by cell-autonomous circadian molecular clocks. Such cell-autonomous circadian cycles consist of classical feedback loops driven by the major transcription factors, CLOCK and BMAL1, together with their repressors, Period and Cryptochrome. Indeed, the immune system’s activity displays circadian rhythmicity and various pathogens are known to modulate – or be modulated by – the host’s circadian cycles. In this research project, we investigated the temporal variation in HIV-1 in vivo, its source and whether this variation can be exploited to reverse latency. In a prospective observational study of virally suppressed male PLHIV on ART, we identified a circadian rhythmicity in cell-associated unspliced HIV-1 RNA and the HIV RNA-to-DNA ratio in vivo. HIV-1 DNA itself, however, remained stable over time – signifying that the circadian rhythms of HIV-1 RNA were due to variation in viral transcription or RNA clearance, rather than cell trafficking. Expression of core circadian genes, Clock, Bmal1, Period1-3, and Cryptochrome1-2 cycled in a circadian manner, indicating that PLHIV maintain intact cell-autonomous circadian cycles within peripheral CD4+ T-cells, despite chronic infection. Furthermore, there was a relationship between oestradiol’s circadian cycle and that of the HIV RNA-to-DNA ratio. We have therefore shown that cell-associated unspliced HIV-1 RNA has a circadian rhythmicity in vivo, contributing to the new paradigm that HIV-1 is not always completely latent. These observations could be leveraged for new interventions. Using the HIV-1-reporter cell line, J-Lat Tat-IRES-GFP clone A2, we next established a high-throughput assay to screen the latency-reversing potential of circadian-modulating compounds. We identified several compounds with acceptable toxicities that activated the HIV-1 long terminal repeat (LTR) promoter, including; the organic selenium compounds, methaneseleninic acid (MSA) and methylselenocysteine; the SIRT1 activator, resveratrol; and the nuclear import inhibitor, ivermectin. Of these, MSA exhibited the greatest increase in LTR activation with tolerable toxicities and was therefore characterised further. In the latently infected cell lines, J-Lat 10.6 and ACH2, MSA potently induced HIV-1 RNA and protein expression, as well as cell-associated unspliced HIV-1 RNA in primary CD4+ T-cells from PLHIV on ART ex vivo. Expression of the major circadian activator, Bmal1, was also increased by MSA, demonstrating that latency reversal was associated with perturbations to cell-autonomous circadian cycles. Additionally, MSA did not induce sustained cellular activation or proliferation. Together, this research identified a novel LRA that induced both viral and circadian gene transcription, in the absence of cellular activation or proliferation. Finally, using molecular techniques, we addressed the source of circadian variation in HIV-1 RNA and the association between circadian disruption and latency reversal. Co-expression of both Clock and Bmal1 transcription factors activated the HIV-1 LTR in vitro. By generating various LTR mutants, we identified that this activation was entirely dependent on a single E-box motif within the LTR recognised by CLOCK:BMAL1 heterodimers, indicating that cell-autonomous circadian cycles may directly interact with the integrated HIV-1 provirus to initiate transcription and contribute to the circadian rhythmicity of HIV-1 transcription observed in vivo. Collectively, this research has demonstrated circadian rhythmicity in HIV-1 transcription in vivo despite suppressive ART and the cell-autonomous circadian cycles of latently infected cells may directly activate their harboured provirus. These data highlight the dynamic nature of viral activity throughout the 24-hour day, and necessitate consideration when designing future clinical trials. Moreover, we demonstrated that cell-autonomous circadian clocks offer a novel, druggable pathway to target as part of the shock and kill approach to a cure for HIV-1.