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

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

PloS one (2010-06-12)
Vici Varghese, Elijah Wang, Farbod Babrzadeh, Michael H Bachmann, Rajin Shahriar, Tommy Liu, Svetlana Jean M Mappala, Baback Gharizadeh, W Jeffrey Fessel, David Katzenstein, Seble Kassaye, Robert W Shafer
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.

MATERIALS
Product Number
Brand
Product Description

Roche
Expand High FidelityPLUS PCR System, dNTPack, sufficient for ≤125 reactions (04743725001), sufficient for ≤500 reactions (04743733001), suitable for PCR, dNTPs included