Khan, Hataf; (2019) Investigating antagonism of innate immunity by HIV-1 accessory protein Vpr. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The role of the HIV-1 accessory protein Vpr has been obscure. Recent studies suggested that HIV-1 is sensitive to type-I Interferon stimulated by activation of cytoplasmic DNA sensor cGAS. Given that Vpr is packaged into HIV-1 particles and present during early stages of the viral lifecycle when its DNA is prone to detection by cGAS, it was hypothesised that Vpr may antagonise cGAS activation of innate immune responses. Consistent with this hypothesis, HIV-1 replication was Vpr dependent in macrophages activated with cGAMP, a product of activated cGAS. High dose infection of THP-1 cells by HIV-1 triggered a Vpr sensitive ISG response, which depended on cGAS but not MAVS. Vpr expression inhibited interferon stimulated genes (ISGs) mRNA and protein expression stimulated by cGAMP. Vpr mutants revealed that this activity required interaction with the DCAF1 E3 ubiquitin ligase complex and importin-α but is independent of Vpr cell cycle arrest function. DCAF1 requirement was further confirmed by DCAF1 depletion. Surprisingly, Vpr expression also inhibited LPS or Sendai virus activated ISG expression suggesting that Vpr targets a conserved step downstream of several innate immune sensors. Indeed, Vpr potently inhibited nuclear translocation of IRF3 without affecting IRF3 phosphorylation at serine386 which is necessary and sufficient for IRF3 activation. In addition to IRF3, Vpr also inhibited NF-ĸB nuclear translocation downstream of DNA sensing. Immunofluorescence analysis of Vpr correlated antagonism of immune signalling with localisation of Vpr to the nuclear envelope, suggesting that Vpr may target nuclear translocation of IRF3 and NF-ĸB at the nuclear pore. In parallel, investigation of Vpr in HEK293T cells revealed that Vpr inhibits mRNA expression from various promoters except the ubiquitin or EF1α promoter which lack NFĸB binding sites. This function correlated with Vpr localisation to the nuclear envelope and was independent of the cell cycle arrest function of Vpr. Interestingly, Vpr did not inhibit HIV-1 gene expression or infectivity. Moreover, nucleofection or integration of a reporter overcame the Vpr-mediated block to expression, suggesting that Vpr may inhibit nuclear import of co-transfected plasmids. In conclusion, I propose that during infection Vpr acts to suppress cGAS activation induced by inappropriately exposed HIV-1 DNA in infected cells and Vpr mediated block to expression from the co-transfected plasmids is a consequence of Vpr inhibition of IRF3 and NF-ĸB nuclear import

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