Skip to main content
eScholarship
Open Access Publications from the University of California

Nucleic acid template and the risk of a PCR-Induced HIV-1 drug resistance mutation.

  • Author(s): Varghese, Vici
  • Wang, Elijah
  • Babrzadeh, Farbod
  • Bachmann, Michael H
  • Shahriar, Rajin
  • Liu, Tommy
  • Mappala, Svetlana Jean M
  • Gharizadeh, Baback
  • Fessel, W Jeffrey
  • Katzenstein, David
  • Kassaye, Seble
  • Shafer, Robert W
  • et al.

Published Web Location

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0010992
No data is associated with this publication.
Abstract

The HIV-1 nucleoside RT inhibitor (NRTI)-resistance mutation, K65R confers intermediate to high-level resistance to the NRTIs abacavir, didanosine, emtricitabine, lamivudine, and tenofovir; and low-level resistance to stavudine. Several lines of evidence suggest that K65R is more common in HIV-1 subtype C than subtype B viruses.

We performed ultra-deep pyrosequencing (UDPS) and clonal dideoxynucleotide sequencing of plasma virus samples to assess the prevalence of minority K65R variants in subtype B and C viruses from untreated individuals. Although UDPS of plasma samples from 18 subtype C and 27 subtype B viruses showed that a higher proportion of subtype C viruses contain K65R (1.04% vs. 0.25%; p<0.001), limiting dilution clonal sequencing failed to corroborate its presence in two of the samples in which K65R was present in >1.5% of UDPS reads. We therefore performed UDPS on clones and site-directed mutants containing subtype B- and C-specific patterns of silent mutations in the conserved KKK motif encompassing RT codons 64 to 66 and found that subtype-specific nucleotide differences were responsible for increased PCR-induced K65R mutation in subtype C viruses.

This study shows that the RT KKK nucleotide template in subtype C viruses can lead to the spurious detection of K65R by highly sensitive PCR-dependent sequencing techniques. However, the study is also consistent with the subtype C nucleotide template being inherently responsible for increased polymerization-induced K65R mutations in vivo.

Item not freely available? Link broken?
Report a problem accessing this item