Among the viral factors that regulate the extent of HIV-1 reverse transcription, the NC protein has been described to play a major role by virtue of its chaperoning activity [45–49]. To further understand the observed reduction in the total amount of tRNA3Lys extended primer as well as the decrease release from +3 pausing site by the K65R/M184V RT compared to wild-type RT, we determined the efficiency of the initiation of the (-)ssDNA synthesis in the presence of NC, thus mimicking a more physiological scenario. Our results indicate that K65R/M184V RT pausing products distribution at position +3 and +5 was notably different from that of wild-type RT at early time points (Fig. 3d). At 5 min in this reaction, wild-type RT displayed 13 and 15% of newly synthesized (-)ssDNA at pausing positions +3 and +5, respectively (Fig. 3e). In contrast, K65R/M184V RT showed 43 and 40% of accumulation at the aforementioned pausing sites (Fig. 3f).
Recently, L74V-, M184V- and L74V/M184V-containing enzymes have been shown to display diminished efficiency of initiation of minus and plus-strand DNA synthesis . The latter finding is consistent with the observations in this paper on the reduced efficiency of initiation of (-)ssDNA synthesis of RTs containing K65R/M184V. Moreover, both K65R/L74V- and K65R/M184V-containing RTs have been associated with diminished ability to use natural dNTPs relative to wild-type RT [25,50]. To study possible correlations between the decreased levels of (-)ssDNA synthesis by the doubly mutated K65R/M184V RT and the described diminished replicative capacity associated with K65R/M184V-containing viruses, we evaluated the efficiency of the reverse transcription reaction using real-time PCR in a single round of infection in MT-4 cells. Quantification of the data showed that wild-type viruses and M184V and K65R-containing viruses produced peak levels of (-)ssDNA at 12 h after infection as previously described , although K65R/M184V-containing viruses were severely impaired in this regard (Fig. 4a). Additionally, the level of (-)ssDNA synthesis at 24 h after infection for mutated viruses compared to wild-type virus was 89% for K65R, 88% for M184V and 15% for K65R/M184V (Fig. 4c).
An even more dramatic effect was found in regard to reverse transcribed DNAs synthesized after the first strand transfer, which were measured at 24 h after infection. Levels of synthesis for mutated viruses compared to wild-type virus were 56% for K65R, 35% for M184V and 14% for K65R/M184V (Fig. 4b and c).
We also evaluated viral replication of wild-type and mutated viruses in a single round of infection in CBMCs, which are known to exhibit reduced intracellular dNTPs pools . Maximal differences were found at 48 h after infection, at which time wild-type viruses produced at least two-fold more (-)ssDNA than any of the mutated viruses (Fig. 4d). The level of (-)ssDNA synthesis for mutated viruses in comparison with wild-type viruses was 60% for K65R, 32% for M184V and 17% for K65R/M184V (Fig. 4f). Moreover, these differences were further augmented in comparison with wild-type for reverse transcribed DNA detected after the first strand transfer, namely 50% for K65R, 22% for M184V, and 5.5% for K65R/M184V (Fig. 4e and f).
We were also interested in studying viral growth kinetics of wild-type and mutated viruses to assess possible correlations with results obtained in our single-round infection assays. Thus, we infected the MT-4 cell line as well as CBMCs. Viral growth kinetics were performed in MT-4 cells to assess the contribution of increased dNTP pools. Unsurprisingly, we found faster viral growth kinetics for both wild-type and mutated viruses compared to those seen in infected CBMCs. Furthermore, wild-type viruses and M184V-containing viruses reached maximum RT activity levels at 4 days after infection; in contrast, K65R-containing viruses showed a delay of 6 days after infection but the RT levels were comparable with those of wild-type viruses. As expected, K65R/M184V-containing viruses displayed the highest decrease in RT activity at that time point. (Fig. 4g).
On the other hand, infections performed in CBMCs show that peak levels of RT activity in culture supernatant were attained with wild-type viruses at 7 days after infection (Fig. 4h). In addition, M184V and K65R-containing viruses replicated very similarly to each other but to a lesser extent than wild-type viruses. Interestingly, only after the addition of freshly obtained CBMCs did K65R/M184V-containing viruses display detectable levels of RT activity in the culture supernatant at 12 days after infection.
Although viruses containing both the K65R and M184V mutations are observed rarely among clinical isolates [16,24], this combination of mutations in HIV-1 RT is not well understood with regard to resistance to relevant NRTIs and viral replicative capacity. Recently, it was shown that RTs containing the K65R, M184V and K65R/M184V mutations displayed 8.6-fold, 30-fold and 180-fold resistance to 3TC-TP, respectively, in comparison with wild-type RT and also displayed 4.4-fold, 0.4-fold and 1.7-fold resistance to TFV-DP, respectively, in comparison with wild-type RT . Moreover, pre-steady-state kinetics have shown that discrimination by K65R RT may be due to a decreased catalytic rate of incorporation (kpol) of NRTI-TP in comparison with wild-type RT, with modest effect on binding affinity (Kd). Alternatively, discrimination by M184V RT may be the result of decreased binding affinity of NRTI-TP in comparison with wild-type RT, with only a small effect on the catalytic rate of incorporation. When combined, these mutations within RT exhibit both decreased binding affinity and decreased catalytic rate of incorporation of NRTI-TP in comparison with wild-type RT. Our data and the greater degree of resistance to most NRTIs displayed by K65R/M184V RT reinforce this point .
In addition, HIV-1 viruses harboring the K65R, L74V and M184V mutations have been shown to display hypersusceptibility to ZDV due to an impaired rescue of ZDV-MP chain-terminated primers compared to wild-type RT [31,42,53,54]. Moreover, K65R RT has been shown to decrease the binding or incorporation of ZDV-TP as well as the unblocking of ZDV-MP, even at physiological concentrations of the next inhibitory nucleotide in comparison with wild-type RT . Here, we have shown that the K65R/M184V RT displayed a major reduction in ZDV-MP unblocking, suggesting that, when present together, these mutations may have an additive effect in this regard. Interestingly, both K65R and thymidine analogue mutations (TAMs) have been shown to exhibit bidirectional phenotypic and genotypic antagonism, which may explain the negative association of these mutations in genotype databases and the infrequent emergence of K65R in patients receiving ZDV [55–57].
The synthesis of (-)ssDNA has been described as a rate-limiting step in the reverse transcription reaction. Our data show that K65R- and M184V-containing RTs display slightly decreased efficiency of (-)ssDNA synthesis, as described [35,40]. We have also shown that K65R/M184V RT displays the lowest efficiency of initiation of (-)ssDNA synthesis, as reflected by accumulation of products at position +3 and +5 as well as a decrease in the total amount of tRNA3Lys extended primer even in the presence of NC. Recent data have shown that the use of NC to promote complex formation between tRNA3Lys primer and the RNA template led to qualitative differences between types of primer/template complexes formed in the presence or absence of saturating concentrations of NC .
Furthermore, RTs containing both the K65R and M184V mutations produced more (-)ssDNA when primed with the dPR DNA primer, suggesting that these enzymes have a decreased ability to use RNA primers, as has been described for L74V/M184V-containing RTs . We suggest that these mechanisms are responsible for the diminished viral replicative capacity observed in tissue culture when K65R/M184V-containing viruses are studied. However, we cannot exclude the possibility that other mechanisms, including host factors, may also play important roles in determining the decreased replicative capacity associated with these viruses. It has been recently shown that mutations that reside at the HIV-1 RT polymerase active site, such as K65R, L74V and M184V, display a high degree of negative correlation between template switching frequency and viral titers .
Clinical data support our observations. The Tonus trial demonstrated that 14 of 14 treatment-naive patients who received once-daily TDF, abacavir (ABC) and 3TC harboured the K65R/M184V mutations and had relatively low viral loads over 48 weeks of treatment . In Gilead Study 903, treatment-naive patients who developed K65R while on TDF/3TC/efavirenz (EFV) did not fully rebound to baseline HIV-1 RNA levels. Conceivably, the additional presence of the M184V mutation in some patients may have contributed to impaired viral fitness .
We now wish to study whether other drug-resistance related mutations in association with K65R or M184V will also lead to decreased efficiency of initiation of (-)ssDNA synthesis as well as decreased viral replicative capacity.
We thank Claudio Smolarz for assistance with digital artwork.
This work was performed by F.A.F. in partial fulfillment of the requirements for a PhD degree from the Faculty of Graduate Studies and Research, McGill University, Montreal, Quebec, Canada.
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