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Abstract
The L74V mutation in HIV-1 RT impairs unblocking of ZDV-terminated DNA primers and reduces synthesis of viral DNA in real-time PCR
Frankel F.1, Marchand B.1, Götte M.1, Wainberg M.A.1
1McGill University AIDS Centre, Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada
Introduction: The M184V, K65R and L74V mutations in HIV-1 RT share numerous characteristics: diminished viral replication capacity, discrimination against relevant NRTIs and reduced RT processivity in biochemical assays. Moreover, both M184V and K65R mutations cause reductions in the efficiency of excision of ZDV-terminated DNA. We wished to assess whether L74V might compromise unblocking of ZDV-terminated DNA primers and synthesis of viral DNA in real-time PCR assays.
Methods: Recombinant wt, L74V, M184V and L74V/M184V-containing RTs were purified and unblocking of chain-terminated DNA primers was studied at a single template position. A DNA/DNA template/primer was incubated with either wt or mutant RT in a buffer containing 10 mM dCTP and 10 mM ZDV-TP. ATP or PPi-dependent excision of the ZDV-terminated primer was monitored in time-course experiments. Viral replication capacity was determined by measuring concentrations of (–)ssDNA and gag DNA by real-time PCR in a single round of infection.
Results: In the presence of ATP, L74V-containing RTs displayed a 50% reduction in the efficiency of excision of ZDV-MP from newly synthesized viral DNA. Wt enzyme unblocked 50% of the ZDV-terminated primer after 59 min whereas L74V RT required more than 90 min. M184V and L74V-M184V-containing RTs showed a significant impaired excision mechanism. In contrast, PPi-mediated excision was only impaired at early time points of the rescue reaction. Real-time PCR showed that L74V-containing viruses were compromised by more than 50% in synthesis of both (-)ssDNA and gag DNA. Addition of the M184V mutation further impaired reverse transcription with differences being especially significant in regard to gag viral DNA.
Conclusions: Although ATP-mediated excision was compromised in L74V RT, PPi-mediated excision showed differences only at early time points, potentially highlighting the biological significance of ATP vs PPi in excision reactions. Diminished viral replication capacity of L74V may be due to reduced synthesis of (-)ssDNA as well as full-length DNA.
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