Resolution of Specific Nucleotide Mismatches by Wild-Type and AZT-Resistant Reverse Transcriptases during HIV-1 Replication

• The dissection of HIV-1 reverse transcriptase intracellular error mechanisms is challenging.
• A novel system to study mismatch resolution during HIV-1 replication was developed.
• Outcomes for wild-type and an AZT-resistant reverse transcriptase were quantified using three approaches.
• Most mismatches were extended to similar high extents by both enzymes.
• Striking mechanistic differences were revealed during dC–rC mispair resolution.

A key contributor to HIV-1 genetic variation is reverse transcriptase errors. Some mutations result because reverse transcriptase (RT) lacks 3′ to 5′ proofreading exonuclease and can extend mismatches. However, RT also excises terminal nucleotides to a limited extent, and this activity contributes to AZT resistance. Because HIV-1 mismatch resolution has been studied in vitro but only indirectly during replication, we developed a novel system to study mismatched base pair resolution during HIV-1 replication in cultured cells using vectors that force template switching at defined locations. These vectors generated mismatched reverse transcription intermediates, with proviral products diagnostic of mismatch resolution mechanisms. Outcomes for wild-type (WT) RT and an AZT-resistant (AZTR) RT containing a thymidine analog mutation set—D67N, K70R, D215F, and K219Q—were compared. AZTR RT did not excise terminal nucleotides more frequently than WT, and for the majority of tested mismatches, both WT and AZTR RTs extended mismatches in more than 90% of proviruses. However, striking enzyme-specific differences were observed for one mispair, with WT RT preferentially resolving dC–rC pairs either by excising the mismatched base or switching templates prematurely, while AZTR RT primarily misaligned the primer strand, causing deletions via dislocation mutagenesis. Overall, the results confirmed HIV-1 RT’s high capacity for mismatch extension during virus replication and revealed dramatic differences in aberrant intermediate resolution repertoires between WT and AZTR RTs on one mismatched replication intermediate. Correlating mismatch extension frequencies observed here with reported viral mutation rates suggests a complex interplay of nucleotide discrimination and mismatch extension drives HIV-1 mutagenesis.

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