In order to determine in which step of the virus life cycle stimulation occurred, we used a single-cycle assay. Since we replaced the nef gene with the mouse Thy 1 gene in our single-cycle vector, the Thy 1 protein was expressed early in infection, and because the env gene was also deleted, no progeny viruses were produced. Since we detected stimulation only when drug was added after the virus was adsorbed to the cells, and not when virus stocks were made during the transfection process, stimulation could only occur during the steps of the life cycle from viral capsid uncoating to protein (i.e., Thy1.2) expression. Therefore, these studies provide evidence that stimulation by NNRTIs occurs during early steps of virus replication. This conclusion is derived from the finding that the relative hierarchy of stimulation measured in the multiple-cycle assay matches that of the single-cycle assay, when drug was present during the infection step. In the single-cycle assay, no stimulation of infectivity was observed when drug was added during the virus production step. We also did not observe an increase in functional RT in virions in the presence of drug, and we did not observe an enhancement of recombinant RT heterodimerization in the presence of drug. We also showed using real-time PCR of reverse transcription intermediates that minus-strand strong-stop DNA synthesis, but not minus-strand transfer efficiency, is stimulated by efavirenz (EFV).
We observed that in the absence of drug, ES, VES, D10, and D10MMTT virions all had reduced RT content compared to the wild type. This result is consistent with our previously published results showing that NNRTI mutants have reduced RT content (37
). The addition of the nucleoside resistance mutation L74V to K101E+G190S (VES) increased the virion RT content compared to K101E+G190S alone. However, the presence of M41L and T215Y in D10 did not increase the RT content compared to D10MMTT, where the M41L and T215Y mutations were back-mutated to the wild type. The finding that L74V and M41L+T215Y eliminate stimulation yet have discordant effects on RT content further supports the conclusion that the mechanism of stimulation does not occur through RT content. However, the presence of L74V may improve the virus fitness cost of K101E+G190S in the absence of drug.
We showed that K101E+G190S has reduced Gag-Pol processing and reduced amount of integrase compared to the wild type. We have previously shown that G190S alone also has reduced Gag-Pol processing (37
). Therefore, the reduction effect of K101E+G190S on Gag-Pol processing is probably through the G190S mutation. However, G190S was not observed to have reduced amounts of integrase. The reduction in the amount of integrase in K101E+G190S virions indicates that this double mutation may have a defect in Gag-Pol incorporation. These defects most likely contribute to the reduced fitness of K101E+G190S. However, since EFV did not correct the processing defect or integrase amounts, these steps of the virus life cycle are not involved in the stimulatory effect of NNRTIs on virus replication.
Stimulation of virus replication was not restricted to EFV, since NVP also shows stimulation for the D10MMTT genotype. However, ETR did not demonstrate stimulation. Etravirine (ETR) is unlike EFV and nevirapine (NVP) in that it has a elastic structure that allows more flexibility in binding to the NNRTI binding pocket (17
). We show here that the ETR IC50
of K101E+G190S is low, indicating that the flexibility of ETR allows it to bind to the NNRTI binding pocket despite the presence of the K101E and G190S mutations.
We show that other NNRTI drug-resistant mutations, such as K101E+V106I and K101E+Y188L, also demonstrate stimulation. However, stimulation was not seen for other combinations of K101E with a second NNRTI resistance mutation. K101E+L100I, K101E+K103N, K101E+G190A, and K101E+Y181C were inhibited to different degrees by EFV but were not stimulated. These results support the conclusion that stimulation is mutation dependent.
Drug-dependent replication of HIV-1 mutants has been demonstrated previously for protease inhibitors and the T20 fusion inhibitor (3
). Protease inhibitors (PIs) prevent the enzyme from cleaving the Gag and Gag-Pol polypeptides (reviewed in reference 16
), but the mechanism by which protease inhibitors can enhance replication of PI-resistant variants is unknown. Fusion inhibitors act by preventing the binding of heptad region 1 (HR1) with heptad region 2 (HR2) in the gp41 protein of the env
gene during virus entry (43
). Fusion inhibitors stimulate replication by acting as a “safety pin,” which prevents the premature binding of HR1 to HR2 of fusion inhibitor-resistant genotypes during entry (reviewed in reference 3
). Since the mechanisms by which NNRTIs, PIs, and fusion inhibitors prevent virus replication are different, it is likely that the mechanisms by which NNRTIs, PIs, and fusion inhibitors stimulate replication are also different.
The specific mechanism by which stimulation occurs for NNRTIs is not yet understood. However, to gain some information about the steps of viral replication involved, real-time PCR measurements of reverse transcription intermediates were performed. These studies show that minus-strand strong-stop DNA synthesis is stimulated by EFV. Therefore, tRNA priming or elongation may be stimulated by the drug. Minus-strand transfer efficiency had variable results with no significant stimulation at 4 or 8 h for 800 nM EFV and 50% stimulation at 8 h only for 400 nM EFV. The magnitude of the stimulation of minus-strand transfer at 400 nM EFV was 10 fold less than the stimulation seen for minus-strand strong-stop DNA synthesis. Since it is known that the RNase H and strand transfer activities of RT are important for efficient minus-strand transfer (5
), we conclude that these activities are not stimulated by NNRTIs. However, we cannot rule out the possibility that the effects of the drug on RNase H activity are more complicated and that specific RNase H cleavages, such as cleavage of the polypurine tract and tRNA primers from the ends of the provirus could be stimulated. Stimulation of receptor binding is likely not involved, since drug is not added until the virus has had time to bind to the cell in the single-cycle assay used here. However, even in light of the real-time PCR results, we cannot rule out the possibility that in addition to reverse transcription, stimulation by the drug could occur during uncoating, integration, or protein expression.
We believe it most likely that the effect of the drug is mediated through a direct interaction with RT, rather than through another viral or cellular protein. This concept is supported by the facts that EFV and NVP, but not ETR, stimulate the relevant drug-resistant genotypes and that the addition of nucleoside resistance mutations in RT reduces or eliminates the stimulation. We postulate that a direct interaction could occur through the creation of a new binding site in a different location than the NNRTI binding pocket, functional only in the presence of the K101E+G190S mutations, and therefore, it would not be present in WT RT. Alternatively, the mutations could alter the binding of the drug to the NNRTI binding pocket, creating new hydrogen bonds and other interactions. This alteration could allow the drug to bind in a different conformation than occurs to inhibit the enzyme, instead causing a stimulatory effect. These mechanisms are allowable even though the drug cannot bind to the NNRTI binding pocket in the same manner as the drug would bind WT RT. Although outside the scope of this work, future studies assessing the structural properties of the K101E+G190S RT and where NNRTIs bind will shed light on the mechanism of stimulation.
Since NNRTIs are widely used to treat HIV-1 infection, virologic failure with mutants containing K101E, V106I, Y188L, and G190S, is likely to develop in significant numbers of patients, despite the low frequency of these mutants relative to K103N. Resistance and stimulation may both play an important role in the development of NNRTI-resistant mutants in patients, particularly when K101E is present. Our results suggest that any newly developed NNRTI should be tested to ensure that it does not stimulate known NNRTI-resistant mutants of HIV-1. Understanding the mechanism by which stimulation by NNRTIs occurs will be necessary to develop drugs that do not select for this phenomenon. Therefore, NNRTI stimulation is clinically relevant, impacts future drug development, and clearly warrants further investigation.