Article

Factors affecting template usage in the development of K65R resistance in subtype C variants of HIV type-1

McGill University AIDS Center, Jewish General Hospital, Montréal, QC, Canada.
Antiviral chemistry & chemotherapy 01/2010; 20(3):117-31. DOI: 10.3851/IMP1443
Source: PubMed

ABSTRACT 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.
Gel-based nucleotide extension assays were used to study the homopolymeric sequence surrounding K65.
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.
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.

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    • "Subtype C viruses apparently have an intrinsic difficulty in synthesizing stretches of adenine homopolymeric runs that leads to template pausing at codon 65, facilitating the acquisition of K65R under selective drug pressure [37] [38], whereas the subtype B template favors pausing at codon 67 that may facilitate the generation of D67N and TAMs rather than K65R [37] [38] [39]. In addition, the introduction of codons from positions 64 and 65 in the RT of subtype C into a subtype B backbone was sufficient to lead to selection of K65R by multiple NRTIs [37] [38] [39]. Figure 1 provides a pictorial representation of the preferential development of K65R in subtype C viruses. "
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    Dipartimento di Biologia, Dipartimento di Biologia, University of Rome Tor Vergata, University of Rome Tor Vergata, 01/2010, Degree: Phd, Supervisor: Prof. Carlo Federico Perno
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