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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.

Conferences on Antiviral Research present a wealth of new information on novel drug development and the 21st annual meeting, held in Montreal between 13–17 April 2008, was no exception. The sessions topics included novel targets for HIV therapy, respiratory and emerging viruses, hepatitis viruses, clinical update on antiviral drugs, retroviruses, and herpes- and poxviruses, among others. In addition, there were a number of themed poster sessions dealing with each of the above areas, as well as novel methods for antiviral analysis, topical microbicides, medicinal chemistry, animal models, veterinary viruses and assorted viral agents. Each of these sessions were well organized and provoked thoughtful discussions. The session on novel targets for HIV therapy featured cutting-edge lectures on interactions between viral proteins and cellular factors. This work has the potential to lead to novel drug discovery as does that on peptide-based approaches to prevent replication of influenza viruses and the development of novel compounds that interfere with the neuraminidase enzymes of both influenza and parainfluenza viruses. Other areas of focus included novel approaches aimed at heightening immune effector mechanisms and the development of gene therapy strategies for certain types of viral infection. Of course, the development of compounds directed at numerous hepatitis C targets also featured prominently at the meeting.

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.

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.

Background We investigated the effects of mutations K65R and K65R plus M184V on enzymatic function and mechanisms of drug resistance in subtype C reverse transcriptase (RT). Methods Recombinant subtype C HIV-1 RTs containing K65R or K65R+M184V were purified from Escherichia coli. Enzyme activities and tenofovir (TFV) incorporation efficiency by wild-type (WT) and mutant RTs of both subtypes were determined in cell-free assays. Efficiency of (-) ssDNA synthesis and initiation by subtype C RTs was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3Lys as primer. Single-cycle processivity was assayed under variable dNTP concentrations. Steady-state analysis was performed to measure the relative inhibitory capacity (ki/km) of TFV-disphosphate (TFV-DP). ATP-dependent excision and rescue of TFV-or ZDV-terminated DNA synthesis was monitored in time-course experiments. Results The efficiency of tRNA-primed (-)ssDNA synthesis by subtype C RTs was: WT > K65R > K65R+M184V RT. At low dNTP concentration, K65R RT exhibited lower activity in single-cycle processivity assays while the K65R+M184V mutant showed diminished processivity independent of dNTP concentration. ATP-mediated excision of TFV-or ZDV-terminated primer was decreased for K65R and for K65R+M184V RT compared to WT RT. K65R and K65R+M184V displayed 9.8-and 5-fold increases in IC50 for TFV-DP compared to WT RT. The Ki/Km of TFV was increased by 4.1-and 7.2-fold, respectively, for K65R and K65R+M184V compared to WT RT. Conclusion The diminished initiation efficiency of K65R-containing RTs at low dNTP concentrations have been confirmed for subtype C as well as subtype B. Despite decreased excision, this decreased binding/incorporation results in diminished susceptibility of K65R and K65R+M184 RT to TFV-DP.

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.