Chen, N; (2016) HIV-1 capsid is required for post-nuclear entry steps. Doctoral thesis , UCL (University College London).

Abstract

Since the start of global HIV-1 pandemic about 30 years ago, great research resources and efforts devoted to this field have changed the picture of AIDS from a deadly disease to a manageable chronic illness sharing similar life span expectation as the normal population. However, despite achieving great clinical improvements, HIV infection is still far from a cure, therefore still more research is needed into the fundamental mechanisms of viral replication and to bring new treatment options to light. During my PhD project, I used a recently identified antiretroviral compound, Coumermycin A1 (CA1), as a tool to study early events in HIV-1 infection. I found that C-A1 inhibits HIV- 1 integration in a capsid-dependent way without affecting reverse transcription or nuclear import. Using molecular docking, we have identified an extended pocket in the N-terminal domain of capsid where C-A1 is predicted to bind. Isothermal titration calorimetry (ITC) confirmed that C-A1 binds to capsid. Time-of–uncoating assays in Jurkat CD4+ T cells expressing engineered human TRIM5-CypA showed that C-A1 causes faster and greater escape from TRIMCyp restriction of wild type but not A105S or N74D capsid mutant viruses. This suggests that C-A1 induces faster uncoating, yet it did not affect reverse transcription. Sub-cellular fractionation showed that small amounts of capsid accumulated in the nuclei of infected cells and C-A1 reduced the nuclear capsid. A105S and N74D mutant viruses did not accumulate capsid in the nucleus, irrespective of C-A1 treatment. Furthermore, depletion of Nup153, a nucleoporin located at the nuclear side of the nuclear pore that binds to HIV-1 capsid, made the virus less susceptible to TRIMCyp restriction, suggesting that Nup153 may help maintaining some integrity of the viral core. The results suggest that capsid may be required for a post-nuclear entry event that affects integration. I describe a novel step of the HIV-1 life cycle that can be targeted by a small molecule. I also tried to adapt the cyclosporin washout assay to a high throughput system to screen for CA targeting compounds from two small chemical libraries. Although I did not identify any specific hit, the assay will be useful for future larger screening.

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