Molecular mechanism of HIV-1 resistance to 3′-azido-2′,3′-dideoxyguanosine

Abstract

We reported that 3’-azido-2’,3’-dideoxyguanosine (3’-azido-ddG) selected for the L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain of HIV-1 reverse transcriptase (RT). In this study, we have defined the molecular mechanisms of 3’-azido-ddG resistance by performing in-depth biochemical analyses of HIV-1 RT containing mutations L74V, F77L, V106I, L214F, R277K and K476N (SGS3). The SGS3 HIV-1 RT was from a single-genome-derived full-length RT sequence obtained from 3’-azido-ddG resistant HIV-1 selected in vitro. We also analyzed two additional constructs that either lacked the L74V mutation (SGS3-L74V) or the K476N mutation (SGS3-K476N). Pre-steady-state kinetic experiments revealed that the L74V mutation allows RT to effectively discriminate between the natural nucleotide (dGTP) and 3’-azido-ddG-triphosphate (3’-azido-ddGTP). 3’-azido-ddGTP discrimination was primarily driven by a decrease in 3’-azido-ddGTP binding affinity (Kd) and not by a decreased rate of incorporation (kpol). The L74V mutation was found to severely impair RT’s ability to excise the chain-terminating 3’-azido-ddG-monophosphate (3’-azido-ddGMP) moiety. However, the K476N mutation partially restored the enzyme’s ability to excise 3’-azido-ddGMP on an RNA/DNA, but not on a DNA/DNA, template/primer by selectively decreasing the frequency of secondary RNase H cleavage events. Collectively, these data provide strong additional evidence that the nucleoside base structure is major determinant of HIV-1 resistance to the 3’-azido-2’,3’-dideoxynucleosides.

Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART

“…In treated subject PID 002, we detected clonal plasma virus populations (orange arrows in Fig. 3B), as has been previously reported in ART-treated individuals (24,25). The identical plasma sequences did not match exactly any sequences detected in cell-associated HIV RNA or DNA by CARD-SGS (although several were only different by a few nucleotides), suggesting, again, that many of the transcriptionally active cells in blood do not produce readily detectable virions in plasma (this possibility is supported by the expression of hypermutants), and that the failure to detect frequent matches between cellular and plasma HIV RNA sequences is due to the small fraction of the diverse population of total infected cells sampled.…”

An integrated molecular dynamics, principal component analysis and residue interaction network approach reveals the impact of M184V mutation on HIV reverse transcriptase resistance to lamivudine

“…In order to act as a competitive inhibitor, NRTIs must be phosphorylated within the cell. 10 Rapid emergence of different mutant strains due to error proneness of HIV reverse transcriptase enzyme posses a great threat towards the treatment with NRTIs. 11,12 3TC or lamivudine is one of the crucial NRTIs, which presents in different HAART combinations such as Combivir (GSK), Trizivir (GSK), Epzicom (in USA, GSK)/Kivexa (in Europe, GSK).…”