The role of Gag in HIV-1 DNA synthesis and sensitivity to reverse transcriptase inhibitor drugs

Résumé fourni par la source

We hypothesise that HIV-1 DNA synthesis occurs inside intact viral capsid (CA) cores. We propose that dNTPs are transported into the CA via an electrostatic channel, formed by six positively charged arginines in the centre of CA hexamers. Here, we consider whether reverse transcriptase inhibitor (RTI) sensitivity is altered when the nature of the channel is changed, either by Gag mutation or exchange with Gag from a non-pandemic HIV isolate with a different structure. There are two classes of RTIs: nucleoside/nucleotide based (NRTI) and non-nucleoside inhibitors (NNRTI). We hypothesised that negatively charged NRTIs would recruit to CA hexamers, to be transported into cores. However, NNRTIs are uncharged, yet potently inhibit DNA synthesis, suggesting that NNRTIs enter cores by diffusion or inhibit after uncoating. We tested HIV-1 vector sensitivity to RTIs, either bearing lab adapted M-group, transmitted founder, O-group or mutant Gag sequences. Viral inhibition was measured by comparing IC50 and IC90 values in a range of cell lines. Our data shows that some differences in Gag demonstrate a cell type-dependent effect on viral sensitivity to RTIs. We also tested the stage of RTI inhibition, measuring early and late–reverse transcription (RT) products of HIV-1 (M) and HIV-1 (O) virus in the presence of inhibitors. Our data show that both HIV-1 (M) and (O) vectors are inhibited after 2nd DNA strand transfer. We determined that a small number of vDNA strands are required to infect a U87 cell, which increases in the presence of RTIs or on R18G mutation. We conclude that differences in Gag have some small cell type-dependent effects on RTI sensitivity. We hypothesise this may be due to differences in the timing of CA uncoating between cell types, supported by our finding that all RTIs tested inhibit RT predominantly after 2nd strand transfer.

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