Investigating early events in HIV-1 replication: the role of envelope signalling and PAF1 restriction factor
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To enter cells, HIV utilises the envelope (Env) protein to engage the CD4 receptor and a co-receptor, CXCR4 or CCR5. In addition to its role in HIV entry, the Env protein also triggers intracellular signalling events. Expression of the HIV-1 and HIV-2 Env gp41 cytoplasmic tails triggered a significant decrease in microtubule acetylation at the single cell level. Since microtubule stability is controlled by Rac1, the role of Rac1 in HIV-1 infection was investigated. Treatment of TZM-bl cells with a Rac1 inhibitor that blocks its upstream activators, Tiam1 and TrioN, inhibited HIV-1 NL4.3 (X4-tropic) but not HIV-1 BaL (R5-tropic) suggesting a role for these activators which is possibly, co-receptor dependent. Subsequently, an siRNA screen including 145 upstream regulators of Rac1, cdc42 and RhoA were investigated in their role in HIV-1 NL4.3 and HIV-1 BaL infection. As a result, 39 upstream regulators of Rho-GTPases were identified of which 5 proteins had effects on both HIV-1 BaL and HIV-1 NL4.3 infection which suggests a novel role of Rho-GTPase upstream regulators in HIV-1 infection. Early in infection, HIV-1 also encounters restriction factors such as RNA polymerase II-Associated Factor 1 (PAF1). The possible cytoplasmic or nuclear role of PAF1 in HIV-1 infection was investigated. Confocal imaging and cellular fractionation suggested that endogenous PAF1 is localised mostly in the nuclei but also in the cytoplasm. Overexpression of a murine PAF1 cytoplasm-localised mutant decreased HIV-1 infection similarly to wild-type overexpression. On the other hand, overexpression of a nuclear PAF1 construct decreased infection better than the wild-type. This suggests that PAF1 restricts HIV-1 via a nuclear function. In addition, infection time-course experiments revealed that HIV-1 counteracts the effect of PAF1 within 30 minutes of infection. Overall, the evidence presented, suggests that PAF1 has a nuclear mechanism and that its downregulation could take place in the cytoplasm.
AuthorsGuerrero Alonso, Ana
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