It is important to determine the effects of drug resistance mutations on HIV-1 RT function in order to better understand the consequences of selection for drug resistance on HIV-1 replication fitness and pathogenic potential. NNRTIs are now commonly used as part of combination antiretroviral regimens and appear to provide a similar antiviral effect to protease inhibitors with fewer long-term toxicities (reviewed in reference 28
). A major disadvantage of NNRTIs is that the genetic barrier to the development of drug-resistant HIV is relatively low, and only one to two mutations are usually required for high-level drug resistance. In order to design drug regimens with more durable antiviral effects, it will be necessary to understand what factors impact on the emergence of drug-resistant mutants and how resistance mutations affect HIV replication fitness and RT function.
Mutations conferring drug resistance are unlikely to result in severe impairment of HIV-1 replication fitness, since they develop readily in clinical isolates during therapy. One might anticipate that any biochemical abnormalities associated with such mutations would be subtle and difficult to detect in biochemical assays. Remarkably, we found that measurable biochemical abnormalities in NNRTI-resistant mutants of RT are quite common. The most commonly observed effect of NNRTI resistance mutations is a change in the ratio of RNase H to polymerase activities.
Mutants of HIV-1 resistant to NNRTIs all line the NNRTI binding pocket, which is adjacent to the polymerase active site, and are thought to confer resistance through a reduction in drug binding. Despite their proximity to the polymerase active site, the mutants analyzed in this and our previous studies (29
) demonstrated no significant alterations in RNA- or DNA-dependent DNA polymerization. We found no consistent differences in polymerization activities or processivities of these mutants, although the Y181C mutant did demonstrate a modest increase in Km
for dTTP, a finding consistent with previous observations by another group (56
). The fact that these NNRTI-resistant mutants all demonstrated processivities of DNA polymerization equivalent to wild-type RT strongly suggests that the polymerization reaction cycle, which consists of primer binding, nucleotide addition, and dissociation after primer elongation, is not significantly altered. It should be noted that we did not exhaustively assay other activities associated with the polymerase active site, such as fidelity of nucleotide incorporation or binding affinity for template-primer. Thus, we cannot definitively rule out the possibility that these mutants affect aspects of the polymerization cycle not directly assayed in our studies.
We have demonstrated that four of the five NNRTI-resistant mutants tested in this and a previous study (29
) exhibit reproducible alterations in rates of RNase H cleavage. It should be emphasized that the relative amounts of mutant and wild-type enzymes were carefully normalized to each other, so that any differences in specific activities of the different preparations could not account for the results that were observed.
When analyzing overall substrate degradation, three mutant RTs (K103N, V106A, and P236L) demonstrated a reduction in the ratio of RNase H to polymerase activities. Apparently, HIV-1 can tolerate some alteration in the ratio of RNase H to polymerase activities of RT, although our results suggest that the greater reductions in the ratio of RNase H to polymerase activities seen with the V106A and P236L mutants have an adverse effect on HIV-1 replication fitness as measured in cell culture. Reductions in this ratio could adversely impact viral replication fitness by impairing the degradation of the RNA genome and the initiation of plus-strand DNA synthesis. If there is a disadvantage to reducing the ratio of RNase H to polymerase activities of RT, it does not necessarily follow that increasing this ratio would prove to be an advantage to the virus, since wild-type RT is likely to have evolved an optimal ratio of these activities.
Four of the NNRTI resistance mutations also had specific effects on the different modes of RNase H cleavage. The V106A mutant demonstrated slowing in RNase H cleavage when assayed for both DNA 3′-end- and RNA 5′-end-directed modes of cleavage. The biochemical phenotype of the V106A mutant is similar to that of the P236L mutant, which demonstrated a replication defect in cell culture, relative to both wild-type virus and the K103N mutant (29
). The K103N mutant, which has a more subtle replication abnormality in cell culture than P236L, has slowed DNA 3′-end-directed cleavage but normal RNA 5′-end-directed cleavage (29
). We have postulated that the combined abnormalities in DNA 3′-end- and RNA 5′-end-directed cleavages contribute to the decreased replication fitness of the P236L mutant. Our current replication studies support this hypothesis, suggesting that a combined reduction in both DNA 3′-end- and RNA 5′-end-directed modes of RNase H cleavage is associated with a significant reduction in the replication fitness of HIV-1.
One resistance mutation, Y181C, showed an alteration in the relative distribution of primary and secondary RNase H cleavage products. Previous work has shown that the primary and secondary cleavages occur 14 to 20 nt and 5 to 7 nt, respectively, from the end of the strand directing cleavage (18
). Studies of the rates of accumulation of these cleavage products suggest that the primary cleavage occurs much more rapidly than the secondary cleavage, although there are no published data on whether the primary cleavage event is required for the secondary cleavage to occur.
Y181C, which along with K103N is one of the two most common NNRTI-resistant mutants that develop in clinical isolates, showed a selective increase in the accumulation of the secondary RNase H cleavage product during both RNA 5′-end- and DNA 3′-end-directed RNase H cleavage. The interpretation of these findings depends on whether the primary and secondary cleavage events can occur independently of each other. If the secondary cleavage can only occur after the primary cleavage, then the finding of a decrease in primary and an increase in secondary product formation relative to substrate with no change in the rate of substrate degradation is most compatible with a selective acceleration by the Y181C mutant of the secondary cleavage event, with little effect on the rate of primary cleavage. However, if the secondary cleavage event can occur independently of the primary cleavage, then a slowing of the primary cleavage with an acceleration of the secondary cleavage would be the most likely explanation of these findings.
It is uncertain to what extent these alterations in RNase H activity contribute to the modest reduction in replication fitness of Y181C relative to wild-type virus, in part because this mutant also demonstrates a reduction in affinity for nucleotide substrate. This reduction in substrate affinity is likely to be a disadvantage to the virus and could explain the modest reduction in replication kinetics of Y181C relative to wild-type virus that was seen in our study. In addition, we still do not completely understand what parameters of RNase H cleavage best correlate with HIV replication fitness. The P236L and V106A mutants, which show marked reductions in replication fitness relative to wild-type and other NNRTI-resistant viruses, demonstrate both a reduction in the rate of substrate degradation and a reduction in the rate of secondary product formation. Overall, the Y181C mutant showed no alteration in the rate of RNase H degradation of full-length substrate. If this measure of RNase H activity best correlates with replication fitness, then one might expect that the altered RNase H activity of the Y181C mutant would have little or no impact on replication fitness. However, it is also possible that the formation of the secondary RNase H cleavage product could be the parameter that most influences viral replication fitness, since the secondary product is most likely to spontaneously dissociate from the RNA-DNA hybrid. Potential advantages resulting from an accelerated secondary cleavage event could include increased rates of plus-strand DNA initiation and synthesis resulting from increased dissociation of genomic RNA fragments and/or accelerated rates of minus-strong-stop-DNA transfer due to increased cleavage near the 5′ end of genomic RNA.
Our studies have not yet convincingly demonstrated a biochemical abnormality that could account for the slightly reduced replication fitness of the V179D mutant. Although we are still in the process of establishing correlations between HIV-1 replication fitness in cell culture and RT function, our experience with the Y181C and K103N mutants leads us to expect that the V179D mutant should demonstrate an abnormality in RT function. It is possible that this mutant has an abnormality in RNase H cleavage that is too subtle to consistently detect in our assays (an acceleration of cleavage was observed in only one of four experiments). Alternatively, V179D may have an abnormality in RT function that we have not assayed for, such as DNA-dependent polymerization, fidelity of nucleotide incorporation, or strand transfer.
The magnitude of difference in RNase H activities of these NNRTI-resistant mutants relative to wild-type enzyme was relatively modest (ca. fourfold). These differences, however, were consistently observed in replicate experiments. It is not surprising that the drug-resistant mutants examined here would demonstrate modest alterations in biochemical function, since these mutants are selected for in patients during therapy and likely have relatively well-preserved replication fitness. The presence of detectable biochemical abnormalities in four of these five mutants is likely to have some subtle effect on replication fitness and is consistent with the theory that drug-resistant mutants rarely predominate in the absence of drug selection pressure because they have reduced replication fitness relative to wild-type virus (12
The mechanism of how these NNRTI-resistant mutants, which are remote from the RNase H active site, affect RNase H cleavage remains to be elucidated. There is extensive precedent in the literature that residues in the polymerase domain of retroviral RTs can have significant effects on the positioning of the RNase H domain and therefore affect the efficiency of cleavage. Mutants in the fingers, palm, and thumb subdomains of RT can affect the specificity and/or efficiency of RNase H cleavage (7
). The alterations in RNase H cleavage seen with these NNRTI-resistant mutants may adversely impact on the replication fitness of HIV-1 through a number of mechanisms, including reducing rates of genomic RNA degradation, slowing rates of minus-strong-stop-DNA transfer, and reducing rates of PPT formation and removal. Studies to assess whether these mutants affect these different steps of reverse transcription in vivo and in vitro are in progress in our laboratory.