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Background:  The M184V, K65R, and L74V mutations in HIV-1 RT share several characteristics:  discrimination against incorporation of relevant nucleoside reverse transcriptase inhibitors (NRTI), diminished RT processivity and error rates in biochemical assays and reduced viral replicative capacity. In addition, both M184V and K65R mutations cause a reduction in the efficiency of excision of ZDV-terminated DNA. We wished to assess the effect of L74V in synthesis of viral DNA in real-time PCR assays and whether L74V might also compromise rescue of ZDV-terminated DNA synthesis.

Methods:  Viral replication capacity was determined by measuring copy numbers of (–)ssDNA and full length DNA by real-time PCR in a single round of infection. Recombinant wild type, L74V, M184V, and L74V/M184V-containing RT were purified and rescue of chain-terminated DNA synthesis was studied at a single template position. A DNA/DNA duplex 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 and DNA synthesis were monitored in time-course experiments.

Results:  Real-time PCR showed that L74V-containing viruses were compromised by more  than 50% in synthesis of both (–)ssDNA and full-length DNA. The simultaneous presence of the M184V mutation further impaired reverse transcription with differences being especially significant in regard to full-length viral DNA. In the presence of ATP, L74V-containing RT displayed a 50% reduction in the efficiency of excision of ZDV-MP from newly synthesized viral DNA. Wt enzyme was able to unblock 50% of the ZDV-terminated primer after ~ 40 minutes whereas L74V RT required ~ 80 minutes. M184V- and L74V-M184V-containing RT showed a dramatic impaired excision mechanism. In contrast, PPi-mediated excision was only impaired at very early time points of the rescue reaction.

Conclusions:  Diminished viral fitness of L74V may be due to reduced synthesis of (–)ssDNA as well as full-length DNA. Although ATP-mediated excision was compromised in RT containing L74V, PPi-mediated excision showed differences only at early time points, potentially highlighting the biological significance of ATP vs PPi in excision reactions. These findings support previous evidence that K65R, L74V and M184V should be considered as a group with regard to common mechanisms of resistance to NRTI and their effects on RT biochemistry.

Keywords: HIV; L74V; excision