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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.

We wished to investigate the effects of various mutations in HIV-1 reverse transcriptase (RT) on biochemical inhibition by the active form of a novel nucleoside termed apricitabine. Accordingly, we studied the efficiency of chain-termination mediated by apricitabine triphosphate (TP) in cell-free assays that used either recombinant wild-type or mutated RTs. We also performed steady-state-kinetics and primer-unblocking assays. Subtype C RTs were also analysed. The results showed that the K65R mutation in RT caused reductions in the efficiency of chain-termination of apricitabine-TP by increasing its Ki. However, K65R did not affect rates of primer unblocking for apricitabine-TP. No significant differences were found between subtype C and subtype B RTs with regard to any of the parameters studied. Other mutations such as M184V, L74V and K103N had no effect on the efficiency of chain termination by apricitabine-TP. Thus, the mechanism of reduced susceptibility to apricitabine of viruses containing K65R in RT seems to be mediated exclusively through a reduction in binding or incorporation of apricitabine-TP. Unlike some other nucleoside analogues, increased excision of incorporated apricitabine does not seem to be a cause of resistance to apricitabine.

Objectives: We recently reported the preferential selection of the K65R resistance mutation in subtype C HIV-1 compared with subtype B and showed the underlying mechanism to be dependent on subtype C-specific silent nucleotide polymorphisms, i.e. genomic mutations that change the genotype but not the phenotype. The number of clinical reports demonstrating elevated numbers of K65R nevertheless suggests the existence of factors limiting the increased incidence of K65R mutations. Thus, we investigated the contributions of subtype C-specific silent nucleotide polymorphisms at thymidine analogue mutation (TAM) sites 70, 210 and/or 219 that might reduce the previously described preferential selection of K65R in subtype C HIV-1 associated with subtype C-specific nucleotide polymorphisms at sites 64/65. Methods: Cell culture drug selections were performed with various drugs in MT2 cells. Results: The use of nucleoside/nucleotide reverse transcriptase inhibitors [N(t)RTIs] as single drugs or in combination confirmed the more frequent selection of K65R by multiple N(t)RTIs in a subtype B virus that contained the 64/ 65 nucleotide polymorphisms of subtype C than in a wild-type subtype B virus. This effect was attenuated in the presence of several silent TAM nucleotide polymorphisms, except when stavudine was employed in the selection protocol. Conclusions: These results further demonstrate that stavudine can preferentially select for K65R in subtype C virus and also provide a basis for understanding the importance of silent nucleotide polymorphisms in regard to altered HIV drug resistance profiles.

Background: The host protein APOBEC3G (A3G) can limit HIV-1 replication. Its protective effect is overcome by the HIV-1 ‘viral infectivity factor' (Vif), which targets A3G for proteosomal degradation. Although Vif is considered to be essential for HIV-1 replication, the effect of Vif variability among commonly used HIV-1 molecular clones of different genetic backgrounds on viral infectiousness and pathogenesis has not been fully determined. Methods: We cloned the intact Vif coding regions of available molecular clones of different subtypes into expression vectors. Δvif full-length HIV-1 clonal variants were generated from corresponding subtype-specific full-length molecular clones. Replication-competent viruses were produced in 293T cells in the presence or absence of A3G, with Vif being supplied by the full-length HIV-1 clone or in trans. The extent of A3G-mediated restriction was then determined in a viral replication assay using a reporter cell line. Results and Conclusions: In the absence of A3G, Vif subtype origin did not impact viral replication. In the presence of A3G the subtype origin of Vif had a differential effect on viral replication. Vif derived from a subtype C molecular clone was less effective at overcoming A3G-mediated inhibition than Vif derived from either subtype B or CRF02_AG molecular clones.

Numerous studies have suggested that the K65R reverse transcriptase (RT) mutation develops more readily in subtype C than subtype B HIV-1. We recently showed that this discrepancy lies partly in the subtype C template coding sequence that predisposes RT to pause at the site of K65R mutagenesis. However, the mechanism underlying this observation and the elevated rates of K65R development remained unknown. Here, we report that DNA synthesis performed with subtype C templates consistently produced more K65R-containing transcripts than subtype B templates, regardless of the subtype-origin of the RT enzymes employed. These findings confirm that the mechanism involved is template-specific and RT-independent. In addition, a pattern of DNA synthesis characteristic of site-specific primer/template slippage and dislocation was only observed with the subtype C sequence. Analysis of RNA secondary structure suggested that the latter was unlikely to impact on K65R development between subtypes and that Streisinger strand slippage during DNA synthesis at the homopolymeric nucleotide stretch of the subtype C K65 region might occur, resulting in misalignment of the primer and template. Consequently, slippage would lead to a deletion of the middle adenine of codon K65 and the production of a -1 frameshift mutation, which upon dislocation and realignment of the primer and template, would lead to development of the K65R mutation. These findings provide additional mechanistic evidence for the facilitated development of the K65R mutation in subtype C HIV-1.

Background: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.Methods:Gel-based nucleotide extension assays were used to study the homopolymeric sequence surrounding K65.Results: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.Conclusions: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.

Background: We have shown that the K65R mutation is selected more rapidly in subtype C than in subtype B HIV-1 isolates in both cell culture and clinical studies. Biochemical comparisons between subtype B and C-derived reverse transcriptase (RT) enzymes revealed similar molecular characteristics that do not explain the more rapid selection of K65R with subtype C viruses. This study attempts to establish the mechanistic basis for the difference. Methods: Recombinant subtype C and B HIV-1 RT enzymes were expressed and purified in E. coli. Gel-based nucleotide extension assays were used to study DNA synthesis from various natural and synthetic DNA and RNA templates that spanned regions of the pol gene responsible for the K65R and M184V mutations. Cell based experiments were performed using MT2 cells infected with mutated subtype B HIV-1 pNL4-3 viruses. Results: The propensity for the more rapid selection of K65R with subtype C enzymes is due to the mechanism of DNA synthesis from a subtype C template. The use of templates containing the 64, 65 and 66 codons of the pol gene led to different patterns of DNA synthesis. When subtype C RT was employed to synthesize DNA from subtype C templates, preferential pausing was seen at the nucleotide position responsible for the AAG to AGG mutation on codon 65 which gives rise to K65R. In contrast, the use of subtype B RT together with a subtype B template reveals a different pattern of DNA synthesis. When subtype B RT was employed with a subtype C template, DNA synthesis stopped at the exact nucleotide position responsible for K65R. This phenomenon was not observed when subtype C RT was used with a subtype B template. A similar method was employed to investigate if differences exist in the appearance of M184V between subtypes. The results suggest that M184V is not favoured due to its coding sequence and that the propensity for the development of M184V remains the same in subtype B and C HIV. In cell culture, K65R was detected faster in subtype B that has been mutated to include the 64/65 codons of subtype C, when compared to wild-type subtype B HIV. Conclusions: The more rapid emergence of K65R but not M184V in subtype C RT appears to be based on the pol gene coding sequence. These results urge for the analysis of resistance mechanisms to be studied in all HIV subtypes separately and have clinical relevance in regard to the management of subtype C infections.

Methods We compared mutations of the subtype B NL4-3 (wt) sequence at sites 64, 65, 70, 210, 219 with silent polymorphisms found in the subtype C consensus sequence. Sitedirected mutagenesis of the NL4-3 (wt) plasmid was used to generate all mutant plasmids. Selections in MT-2 cells were performed with each of 3TC, FTC, TDF, ABC, ATC, d4T, and ddI either alone or in combination. Recombinant RT enzymes of both subtype B and C origin were also studied in cell-free reactions.