title: Resolution of Specific Nucleotide Mismatches by Wild-Type and AZT-Resistant Reverse Transcriptases during HIV-1 Replication.
abstract: 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 (AZT(R)) RT containing a thymidine analog mutation set-D67N, K70R, D215F, and K219Q-were compared. AZT(R) RT did not excise terminal nucleotides more frequently than WT, and for the majority of tested mismatches, both WT and AZT(R) 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 AZT(R) 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 AZT(R) 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.
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620, USA.
authors.forename: Siarhei
authors.initials: S
authors.lastname: Kharytonchyk
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620, USA.
authors.forename: Steven R
authors.initials: SR
authors.lastname: King
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620, USA.
authors.forename: Clement B
authors.initials: CB
authors.lastname: Ndongmo
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620, USA.
authors.forename: Krista L
authors.initials: KL
authors.lastname: Stilger
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620, USA.
authors.forename: Wenfeng
authors.initials: W
authors.lastname: An
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620, USA. Electronic address: ateles@umich.edu.
authors.forename: Alice
authors.initials: A
authors.lastname: Telesnitsky
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology; D053843:DNA Mismatch Repair / Q000187:drug effects / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D024882:Drug Resistance, Viral / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D009154:Mutation / Q000187:drug effects / Q000235:genetics; D009711:Nucleotides / Q000235:genetics*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D013698:Templates, Genetic; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics*
chemical_list: D019380:Anti-HIV Agents; D017931:DNA Primers; D004279:DNA, Viral; D009711:Nucleotides; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase
keywords: forced copy-choice recombination; retroviral error mechanisms