We propose that a nucleotide template-based mechanism facilitates the acquisition of the K65R mutation in subtype C human immunodeficiency virus type 1 (HIV-1). Different patterns of DNA synthesis were observed using DNA templates from viruses of subtype B or C origin. When subtype C reverse transcriptase (RT) was employed to synthesize DNA from subtype C DNA templates, preferential pausing was seen at the nucleotide position responsible for the AAG-to-AGG K65R mutation. This did not occur when the subtype B RT and template were used. Template factors can therefore increase the probability of K65R development in subtype C HIV-1.

Resistance to antiviral therapy is the limiting factor in the successful management of HIV. In general, the K65R mutation is rarely selected (1.7-4%) with tenofovir disoproxil fumarate (TDF), abacavir (ABC), didanosine (ddI), and stavudine (d4T), as compared with the high incidence (>40%) of thymidine analog mutations associated with zidovudine and d4T. The high barrier to the development of K65R may reflect a combination of factors, including the high potency of K65R-selecting drugs, including recommended TDF/emtricitabine and ABC/lamivudine (ABC/3TC) combinations; the partial (low-intermediate level) profile of cross-resistance conferred by K65R to TDF, ABC and 3TC; the favorable viral fitness constraint imposed by K65R and the 3TC/emtricitabine-associated M184V mutations; the bidirectional antagonism between the K65R and thymidine analog mutation pathways; and unique RNA structural considerations in the region surrounding codon 65. Nevertheless, surprisingly high levels of treatment failures and K65R resistance may be associated with triple nucleoside analog regimens. The use of TDF + ABC, TDF + ddI and ABC + d4T in combination with 3TC or emtricitabine should be avoided. This selection of K65R may be reduced by the inclusion of zidovudine in two-four nucleoside reverse-transcriptase regimens. Clinical studies have demonstrated an increased frequency of K65R in association with suboptimal d4T and ddI regimens, as well as nevirapine and its resistance mutations Y181C and G190A. The potential for the development of the K65R mutation in subtype C is particularly problematic wherein a signature KKK nucleotide motif, at codons 64, 65 and 66 in reverse transcriptase, appear to lead to template pausing, facilitating the selection of K65R. Optimizing regimens may attenuate the emergence of K65R, leading to better long-term treatment management in different geographic settings. TDF-based regimens are the leading candidates for first-and second-line therapy, microbicides and chemoprophylaxis strategies.

Recently, we described a novel nucleotide template-based mechanism that may be the basis for the facilitated acquisition of the K65R resistance mutation in subtype C versus subtype B human immunodeficiency virus type 1 (HIV-1). In this article, we evaluated the effects of subtype C-specific silent polymorphisms in cell culture drug-selection experiments using nucleoside and nucleotide reverse-transcriptase inhibitors. The K65R pathway was selected more frequently in a subtype B virus that contained subtype C nucleotide polymorphisms at both positions 64 and 65 than in a wild-type NL4-3 subtype B virus. This is the first demonstration of the significance of silent nucleotide polymorphisms in the development of drug resistance.