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


RESULTS
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.


CONCLUSIONS
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.
Department of Medicine, Stanford University School of Medicine, Stanford, California, United States of America. viciv@stanford.edu
Vici
V
Varghese
Elijah
E
Wang
Farbod
F
Babrzadeh
Michael H
MH
Bachmann
Rajin
R
Shahriar
Tommy
T
Liu
Svetlana Jean M
SJ
Mappala
Baback
B
Gharizadeh
W Jeffrey
WJ
Fessel
David
D
Katzenstein
Seble
S
Kassaye
Robert W
RW
Shafer
D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic
D017931:DNA Primers; D004279:DNA, Viral