Previous reports showed that the presence of three or more TAMs (particularly, M41L, T215Y and/or L210W) compromised the efficacy of tenofovir [42–44]. These mutations arise after extensive or suboptimal treatment with nucleoside analogues, particularly with AZT or d4T. In agreement with those findings, our cross-sectional study involving previously treated patients reveals that the presence of TAMs and M184V contributes to failure of salvage therapies containing the combination of tenofovir and emtricitabine, while K65R is relatively rare in those patients. These results were also consistent with the low prevalence of K65R in the presence of TAMs, in patients receiving tenofovir-containing combination therapies [43, 45, 46]. K65R exerts an antagonistic effect on TAMs, by decreasing the ATP-mediated phosphorolytic activity that facilitates removal of NRTIs (i.e. thymidine analogues and tenofovir, among others) from blocked DNA primers ; (reviewed in ref. ).
Several NNRTI resistance mutations were found to be more prevalent in the group of patients failing therapy. In some cases, the presence of these mutations can be attributed to selection with efavirenz (e.g. K103N), since this drug had been co-administered with tenofovir/emtricitabine in 20.2% of the non-responding individuals. Otherwise, NNRTI resistance mutations may have arisen from previous treatments with NNRTIs. The preservation of NNRTI mutations in the viral genome of treated patients can be attributed to their small effect on viral fitness, as demonstrated for V108I, Y181C and G190A [48–51]. Our bivariate analysis also showed that accessory mutations at positions 35, 39, 162, 174, 179, 208, 223, 228, 284, 292, 297 and 333 could be related to therapy failure. Among them, T39A, V179I, H208Y, K223E, L228H/R, R284K and V292I have been previously identified as secondary mutations associated with the accumulation of TAMs and with resistance to nucleoside analogues [21, 22, 35, 37, 52, 53]. On the other hand, L74V, V179I, K223E and L228H/R were previously identified as modulators of NNRTI resistance in a large study carried out in central Italy .
Our analysis clearly shows a separate clustering of TAMs in two different groups that coincides with major selection pathways, previously identified in patients treated with thymidine analogues (i.e. TAM1 and TAM2) [16–18]. Thus, TAM1 includes the amino acid substitutions M41L, L210W and T215Y, while TAM2 contains mutations D67N, K70R, K219E/Q and sometimes T215F. In our data set, the TAM1 pathway includes all three mutations that show high phi values in the correlation analysis. V118I, V179I, M184V and R284K appear to be associated with the TAM1 pathway. V118I and M184V are mutations selected under therapy with lamivudine (reviewed in refs. [1, 4]), while V179I has been associated with resistance to NNRTIs .
Interestingly, the prevalence of R284K seems to increase with the exposure to nucleoside RT inhibitors. In a study involving more than 2,000 patients treated in hospitals in the U.K., Cane et al.  showed that the prevalence of R284K increased with the number of TAMs found in the viral genotype. Only one percent of the isolates lacking TAMs were found to contain the mutation. However, the prevalence of R284K increased up to 4.1%, 6.5% and 9.3% in isolates having 2, 3 or 4 TAMs, respectively. Further evidence of this association was reported by a study of the Swiss Cohort that showed that the frequency of R284K increased from 1.1% in naïve individuals to 5.1% in patients treated with thymidine analogues . Interestingly, that analysis also showed that in those individuals exposed to thymidine analogues and didanosine, the frequency of R284K was 6.0%, while the frequencies of M41L, L210W, T215Y and M184V were 48.3%, 36.8%, 57.7% and 1.0%, respectively. These data together with the strong correlation of R284K and T215Y in our database suggest that the emergence of R284K is not related to the selection of the emtricitabine resistance mutation M184V.
Our results show that R284K has no effect on the viral susceptibility to thymidine analogues and tenofovir of isolates containing the TAM1 complex (i.e. M41L/L210W/T215Y). However, it confers a fitness advantage when the virus is grown in PBMCs in the presence of drug. Arg284 is located in the thumb subdomain close to the template strand and could influence ATP-dependent excision reaction in a similar way as that reported for other amino acid substitutions in the HIV-1 RT thumb subdomain (i.e. P272A/R277K/T286A) . When combined, those three polymorphisms had a negative effect on the efficiency of rescue reactions carried out with blocked primers complexed with RNA, and these effects were due to the lower affinity of the mutant RT for RNA/DNA complexes.
Our biochemical studies showed that in the context of M41L/L210W/T215Y, the substitution of Lys for Arg284 has no effect on ATP-dependent excision. However, R284K increases the efficiency of the rescue reaction and facilitates the elongation of the unblocked primers. This effect cannot be attributed to an influence on nucleic acid binding or processivity but to the higher catalytic efficiency of the M41L/L210W/T215Y/R284K RT in comparison with the M41L/L210W/T215Y enzyme. In combination with TAM1 mutations, R284K produced 60-77% increase in the catalytic rate (k
cat) for nucleotide incorporation. This effect was also consistent with the higher efficiencies of primer extension observed with the M41L/L210W/T215Y/R284K RT, using different concentrations of template-primer. Despite differences in the catalytic parameters, nucleotide discrimination seems to play a minor role in the effects of R284K, since AZTTP and tenofovir-DP inhibition constants (K
i) were similar for both enzymes. Taken together, these results could explain why R284K confers a small fitness advantage in the presence of nucleoside analogues, as shown in the viral replication assays.
Interestingly, the differences in rescue efficiencies between M41L/L210W/T215Y/R284K and M41L/L210W/T215Y RTs were found with DNA/DNA template-primers, but not with RNA/DNA complexes. RNase H assays carried out with both mutants and the wild-type RT revealed the higher endonucleolytic activity of M41L/L210W/T215Y/R284K RT in comparison with WT and mutant M41L/L210W/T215Y RTs. Therefore, the minor differences in rescue efficiencies observed with AZTMP-, d4TMP- or tenofovir-terminated primers complexed with RNA could be explained by the poor stability of the template in rescue assays carried out with the M41L/L210W/T215Y/R284K mutant. It is also possible that the emergence of R284K in RTs having TAM1 mutations could have a negative impact on the stability of the viral RNA during reverse transcription, thereby explaining its small but significant reduction in viral fitness in the absence of inhibitors. Nevertheless, other undetermined factors may also contribute to this effect. For example, the potential effects of thumb subdomain mutations on the stability of the p66/p51 heterodimer [55, 56].
The mechanism by which R284K confers a selective advantage in the context of TAM1 mutations is different from others previously described for mutations in the thumb-connection subdomains and in the RNase H domain. Thus, mutations such as N348I, A360V and Q509L increased chain-terminated primer rescue with RNA/DNA complexes, but not with DNA/DNA template-primers [29, 30, 32–34, 57, 58]. In combination with TAMs, N348I and A360V decreased the efficiency of RNase H cleavage and increased excision of AZT in the presence of ATP . Other mutations, such as the complex P272A/R277K/T286A and G333D had a minor effect on RNase H activity, but increased chain-terminated primer rescue with both RNA/DNA and DNA/DNA template-primers [36, 59]. It has been suggested that secondary mutations such as E40F and K43E  or L214F  that associate with amino acid substitutions of the TAM1 resistance pathway could increase viral fitness by influencing the catalytic efficiency of the RT. However, the suggested effects were not supported by biochemical evidence. In our study we provide an example of a mutation that improves viral fitness in the presence of antiretroviral drugs by increasing its RT DNA polymerase activity, while affecting the RNase H cleavage efficiency.