title: Factors affecting template usage in the development of K65R resistance in subtype C variants of HIV type-1.
abstract: BACKGROUND
  We have shown that the K65R resistance mutation in HIV type-1 (HIV-1) reverse transcriptase (RT) is selected more rapidly in subtype C than subtype B HIV-1 in biochemical, cell culture and clinical studies. Template-usage experiments demonstrated that subtype C nucleotide coding sequences caused RT to preferentially pause, leading to K65R acquisition. This new study now further establishes the basis for differential occurrence of both K65R and thymidine analogue mutations (TAMs) between subtypes.
  
  
  METHODS
  Gel-based nucleotide extension assays were used to study the homopolymeric sequence surrounding K65.
  
  
  RESULTS
  When positive double-stranded DNA synthesis was evaluated from a negative single-stranded DNA template, pausing at the 67 region, which is linked to occurrence of TAMs, was alleviated with both subtype B and C templates at high dCTP concentrations, but this alleviation was more pronounced with the subtype C template. By contrast, pausing at the 65 region on the subtype C but not subtype B template always occurred and was not alleviated at high levels of nucleotide triphosphates or by other means. Furthermore, templates containing repeats of the homopolymeric sequence spanning codons 64-66 of pol showed corresponding pausing repeats at the 65 region with the subtype C template only. Inverted RNA and DNA templates both displayed pausing at position K65 for the subtype C template and a ladder of pausing events culminating at codon 67 for the subtype B templates.
  
  
  CONCLUSIONS
  These results further establish a mechanistic basis for the exclusion of both K65R and TAMs on single templates as well as the preferential acquisition of K65R in subtype C viruses.
authors.affiliation: McGill University AIDS Center, Jewish General Hospital, Montréal, QC, Canada.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Coutsinos
authors.forename: Cédric F
authors.initials: CF
authors.lastname: Invernizzi
authors.forename: Hongtao
authors.initials: H
authors.lastname: Xu
authors.forename: Bluma G
authors.initials: BG
authors.lastname: Brenner
authors.forename: Mark A
authors.initials: MA
authors.lastname: Wainberg
mesh_terms: D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral; D004591:Electrophoresis, Polyacrylamide Gel; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D012367:RNA, Viral / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase