We measured the relative fitness of NNRTI-resistant viruses compared to the wild type using a flow cytometry-based cell culture assay (17
) and then looked for biochemical correlates of reduced fitness for the V106A, G190A, G190S, and P236L mutant RT strains. Previous studies from our research group using a sequenced-based fitness assay are consistent with the fitness hierarchy of the panel of 6 mutants that we tested in this study: WT ≥ Y181C ≥ K103N > G190A > V106A > P236L ≥ G190S (4
Previous biochemical studies of recombinant RTs revealed that RNase H activity correlates with fitness and that tested NNRTI-resistant RTs have normal polymerization activity (4
). However, these studies were performed with recombinant RTs in which the p66 and p51 subunits were both His tagged and mixed after separate column chromatography purification. This method produced less active protein and had a smaller amount of p66/p51 heterodimers than that for the method that we used to purify the RT for the current studies. Here, recombinant heterodimer RT purified by mixing the His-tagged p51 and untagged p66 bacterial lysates before column chromatography resulted in four to eight times more active enzyme at the same protein concentration. Since fractions with equal molar amounts of p66 and p51 were chosen for study, the amount of p66/p51 heterodimer versus p51 homodimer is almost certainly larger as well.
In this study we show that the pre-steady-state and steady-state polymerization activities of recombinant NNRTI-resistant mutant RTs are similar to what we previously published, except that there was no difference in
primer extension. Previous studies showed that G190A and G190S mutants had reduced rates of polymerization from a tRNA primer despite having normal rates of polymerization for a DNA PBS primer (38
). In this study the rate of polymerization from the tRNA primer was similar to that of the wild type for all mutant RTs tested. These discordant results are likely explained by the purification method, since the older preparation had a much lower proportion of active p66/p51 heterodimer. Catalytically inactive RT or alternative RT structural forms may have produced the observed result. Specifically, the packaging and annealing of tRNA into virions involve a complex set of interactions between the tRNA, lysyl-tRNA synthetase, and Gag and Gag/Pol polyproteins (7
). It is known that
can stimulate polymerization by p66 homodimers, induce conformational changes, and increase proteolytic cleavage of the RNase H domain (3
). In addition the initiation from a tRNA primer involves complex enzyme and substrate interactions that change as nucleotides are added (28
). Therefore, heterodimers and homodimers of RT may have different kinetic properties that are mutant specific when a tRNA primer is used. Although previously published methods for purifying RT, which did not control for the formation of p66 and p51 homodimers, may have been adequate for measuring most kinetic parameters, they were apparently not adequate specifically for tRNA initiation.
Consistent with our previous work, the RNase H activities of V106A, G190A, G190S, and P236L recombinant RTs were reduced relative to those of wild-type, K103N, and Y181C RTs. The relative level of RNase H activities of recombinant proteins correlated with fitness of the corresponding virus. The P236L mutant RT was consistently more defective in RNase H than indicated in previous studies as confirmed with independent protein preparations. The effect of the P236L mutation on RNase H activity is consistent with known structural information on RT. P236 is located at the base of the thumb in the p51 subunit, which supports the RNase H domain of p66 and could therefore influence domain activity (26
During reverse transcription the minus-strand strong-stop DNA (−SSDNA) that is synthesized from the tRNA primer strand transfers to the 3′ end of the RNA genome to complete minus-strand synthesis (5
). Studies of tRNA initiation and minus-strand transfer in vitro
have shown that the −SSDNA can fold back to create a self-primed primer/template (P/T) (22
). When the template that we used for the tRNA initiation studies, D199, is copied, it produces −SSDNA, which self-primes. The extension of this self-primed P/T is dependent on the removal of the original RNA template, D199, by RT RNase H activity (Fig. ). Consistent with previous studies, P236L, which has greatly reduced RNase H activity, did not form the extended self-primed product because it cannot degrade the RNA template.
To confirm that defects in RNase H activity of recombinant RTs are responsible for the reduced fitness of NNRTI-resistant mutant viruses, we measured the virion-associated polymerization and RNase H activities. Virion-associated polymerization activity also correlated with fitness, with the exception of the virus with P236L RT. This mutant had polymerization activity that was similar to that of the K103N mutant, a fit mutant, with a polymerization level that was 70% of the wild-type level. Since the polymerization activity of recombinant protein was normal for each mutant, we hypothesized that the absolute amount of RT in the mutant variants must be reduced in order for the virion-associated RT activity to correlate with fitness. When we quantified the RT content in the virions, this was indeed the case. However, the P236L mutant had 30% of the amount of RT in the virion whereas the G190S and V106A mutants had 6% and 11%, respectively. This explains why the polymerization activity of the P236L mutant was higher than expected based on its relative fitness.
In contrast, the virion-associated RNase H activity uniformly correlated with recombinant protein activity and with fitness. K103N and Y181C RT viruses, which replicated at a rate similar to that of the wild type in cell culture, had virion-associated RNase H activity equivalent to that of the wild type. The remaining mutant viruses had reduced RNase H activity, although the RNase H activity of the P236L mutant was slightly higher than that of the G190S mutant. This is most likely explained by the increased amount of RT in P236L viruses. However, the RNase H activities of the unfit NNRTI mutant viruses were ≤50% of that of the wild type, indicating that RNase H is greatly impaired and that this is a likely explanation for reduced fitness.
HIV-1 p66/p51 heterodimer RT formation in virions requires the activity of protease such that mature RT is formed by the proteolytic cleavage of the Gag-Pol polyprotein (Pr160Gag-Pol
) during virion maturation (reviewed in reference 23
). The order and timing of Gag-Pol precursor processing are well established, with cleavage shown to occur in a sequential manner. Studies of the fitness, RT content, and Gag-Pol processing of G190 mutant viruses showed that reduced RT content apparently resulted from reduced Gag and Gag-Pol processing due to reduced Gag-Pol incorporation into virions (25
). Our data clearly show that diminished RT content does not completely correlate with Gag processing of NNRTI-resistant mutants. P236L virus had RT levels similar to those of Y181C and K103N viruses despite having defective Gag processing. In addition the mutants in our study had normal amounts of integrase, indicating normal Gag-Pol incorporation into virions. Studies of NNRTI mutations showed that some mutants such as the K103N, Y181C, and G190A mutants increased heterodimer stability (18
). This suggests that the P236L mutation promotes RT stability, resulting in an overall increase in RT content despite reduced proteolytic cleavage. However, it is not clear whether increased stability of heterodimers results in proteolytic stability of RT, since mutations in the protease cleavage site which prevent cleavage of p66 into p51 result in reduced amounts of RT in the virion (1
). Our results suggest that NNRTI resistance mutants do not affect Gag-Pol incorporation and that all NNRTI resistance mutants affect proteolytic stability to different extents, resulting in different amounts of RT in the virion.
What have we learned here about the mechanisms that reduce fitness of NNRTI-resistant viruses? The effects of the P236L mutation and those of reduced RT content have an obvious commonality. The P236L mutation severely impairs 3′-end-dependent RNase H primary and secondary cleavages. With the P236L mutation in RT, as minus-strand DNA is made, almost none of the template cleavages that that would normally accompany synthesis can actually occur. Since cleavages during synthesis produce the RNA oligomer substrates for later 5′-end-directed cleavage, the defects in polymerization-associated cleavage alone would be sufficient to greatly reduce RNA template degradation. Additionally, although P236L RT exhibits significant 5′-end-directed primary cleavage activity, it is defective for 5′-end secondary cleavage activity. The latter defect would additionally reduce whatever 5′-end cleavage activity can occur at the infrequent cleavage sites generated during synthesis.
The corresponding effect of RT content reduction is that overall RNase H activity is also impaired. Details of the mechanism, however, are different from the effects of mutations that impair RNase H in each RT. There are normally 50 to 70 RT molecules per virion (for a review, see reference 9
). Since only one RT is needed for primer extension, the likely reason for the excess is that the additional RTs are employed to carry out 5′-end-directed RNase H activity. Cuts made during synthesis should produce hundreds of RNA oligomers that likely require many RTs for efficient degradation. Depletion of RT is unlikely to affect polymerization or polymerization-dependent RNase H. However, it should reduce the rate at which the resultant RNA oligomers are degraded. While this is clearly different mechanistically from the effects of the RNase H defect in P236L RT, the impact on reverse transcription should be similar. That is, the long-lived RNA oligomers would hinder strand transfers and second-strand RNA synthesis, slowing viral replication.
The effect of the reduction of DNA polymerase active sites caused by RT depletion is difficult to predict and cannot be approached experimentally by the techniques used here, since polymerization and RNase H are reduced concomitantly. One view is that since only one RT is needed for polymerization, the effects should be minimal. However, reduction of polymerization activity in virions has previously been reported to decrease substantially the rate of viral replication (27
Another possibility is that maintaining the natural activity ratio between the polymerization and RNase H, as occurs when RT content is reduced, is less disruptive of reverse transcription than is the great imbalance caused by the P236L mutation. This again would be hard to verify experimentally.
Finally, the differences in characteristics of the 3′-end-directed RNase H and the 5′-end-directed RNase H activities of the P236L mutant RT are noteworthy. While the positioning of the RT for these two RNase H functions is determined by two different structures, the orientations of the RT with respect to the RNA and DNA strands are much the same (10
). Nevertheless, the 3′-end-directed primary cleavages are strongly suppressed by the mutation while the 5′-end-directed cleavages are not. This suggests that a difference in protein-nucleic acid contacts between one binding mode and the other is capable of activating the RNase H. Understanding the structural basis of this RNase H regulation could have therapeutic potential.
In conclusion, our current results indicate that the reduction of fitness in viruses with NNRTI resistance mutations is based on either of two possible mechanisms. We had previously reported that the mutant RTs shared the property that they are deficient in RNase H activity. We showed here that the P236L HIV-1 has a normal content of RT but that the RT is severely deficient in RNase H function. We propose that the observed fitness defect derives from the inability of the P236L RT to effectively degrade the viral RNA to allow strand transfer and second-strand DNA synthesis. While the defect in cleavage most severely affects polymerization-dependent RNase H activity, this activity creates the substrate for polymerization-independent activity, so that overall RNase H activity is impaired. Viruses with the G190S, G190A, and V106A RTs have substantially reduced RT content. This situation also reduces the amount of RT available for 5′-end-directed RNase H activity and would also impair proper degradation of the viral RNA. The problem is further exacerbated by moderate intrinsic defects in the RNase H of the mutant RTs. While the correlation between poor fitness and RNase H deficiency is unmistakable, the effect of lowered polymerization activity in those mutants with low RT content still needs to be evaluated.