We have developed a model system to evaluate the effect of mutation interactions in viruses generated from recombinant molecular clones containing pol gene sequences derived from patient plasma HIV RNA. This approach is novel in several ways: (1) use of patient-derived virus sequences, with examination of interactions of mutations that actually occur in patients, rather than introducing them into molecular clones of laboratory-derived strains, (2) assessment of specific modifications in molecularly cloned strains in the same viral genetic background, (3) a high level of phenotypic test reproducibility, (4) the ability to assess RC together with resistance for each cloned virus, and (5) assessment of the phenotypic effect of interactions between NRTI and NNRTI mutations that have not been well explored previously.
We demonstrated the effects of interactions of specific RTI drug resistance mutations, which can modulate susceptibility to RT inhibitors, particularly for drugs generally not thought to be affected by the primary mutation. Mutations associated with resistance to single drugs are fairly well characterized, but monotherapy is no longer used; the treatment standard is combination therapy with at least three or four antiretroviral drugs. Combination therapy has been shown to select for more complex mutation patterns, which are commonly seen in treatment-experienced patients. Thus, it is important to understand the consequences of these patterns of mutations.
Our study has confirmed several previous observations and presented some new ones. We have confirmed effects of multiple TAMs on decreasing susceptibility to all NRTI, specifically d4T, ZDV, ABC, and TDF.25,37,38
We also showed that the M184V mutation reduces phenotypic susceptibility to ABC, ddI, and 3TC, but can enhance susceptibility to d4T, ZDV, and TDF, and is associated with decreased RC capacity.11,21,32
Even in the presence of M184V and a marked decrease in phenotypic susceptibility to 3TC and FTC, 3TC appears to contribute to suppression of HIV replication in patients.39
An accumulation of TAMs is associated with broad class resistance by accelerating the rate at which terminal monophosphates (MP) are removed through proteolytic cleavage, particularly for nucleoside analogues, allowing DNA polymerization to proceed.40,41
The appearance of the M184V mutation diminishes the ability of reverse transcriptase harboring TAMs to carry out the phosphorolytic excision of incorporated nucleoside analogs, mediated by ATP or PPi.42–44
We have also extended our understanding of effects of L74V, which are similar to but distinct from those of M184V. The mutation was introduced into molecular clones of laboratory strains by site-directed mutagenesis, and the virus with L74V was shown to have a replicative disadvantage. L74V is a resistance mutation for ddI and ABC. We have shown that L74V reduces susceptibility to ddl and ABC, improves susceptibility to TDF and ZDV, but in contrast to M184V has only very modest effects on 3TC susceptibility; in addition, there is usually decreased susceptibility to d4T. In fact, L74V has been selected in patients failing d4T without coadministration of ddI or ABC.45
The L74V resistance mutation was described for ddI prior to its approval and widespread use.46
In a later study, replication kinetics and reverse transcriptase processivity were examined, and reductions in both measures were found comparable to that seen with the introduction of M184V in the same backbone (HIV-HXB2).47,48
The L74V mutation was shown to counteract the enhanced excision of ZDV MP with reverse transcriptase harboring TAMs.43,49
When M184V and L74V were introduced together, there was more impairment in ZDV-MP excision than seen with either mutation alone.7,43
In addition to affecting excision, these mutations have also been shown to affect RT processivity, with decreases in initiation of primer-driven viral DNA synthesis. These studies were limited by the introduction of only a single predicted amino acid resistance mutation, generated in cell line-adapted virus strains, rather than primary virus isolates. In addition, L74V was found to be a contributing factor to an observed discordance between phenotypic and genotypic NRTI resistance assessments for ZDV.29
We also confirmed that the K103N mutation, while conferring resistance to all NNRTIs, has little effect on NRTI susceptibility or RC.50
Another NNRTI mutation pattern (A98S, K101E, and G190S) selected by NVP therapy and seen previously50
can affect NRTI susceptibility. The L74V mutation can slightly diminish RC when seen in isolation or only with NRTI resistance mutations, but as shown previously, we demonstrated improved RC when L74V was introduced in the presence of G190S. G190S, as well as other mutations at codon 190, typically diminishes susceptibility to NVP and EFV, but generally has little effect on DLV susceptibility.33,34
RT codon 190 mutations are associated with a moderate to marked decrease in replication kinetics33
and have also been found to have less polymerase activity with less processivity than RT enzymes that are wild type at that position.33,34
In these biochemical studies, L74V and V75I appeared to be compensatory in that they restored reverse transcriptase activity to levels similar to that seen with wild-type virus. In fact, L74V and V75L/I were selected in vitro
by selective pressure of an NNRTI.51
Although initial studies identified G190E as a primary NVP resistance mutation that exhibited impairment of reverse transcriptase activity and virus replication,52,53
multiple substitutions at RT position 190 have been observed, including G190A, G190S, G190E, G190Q, and G190V.33,34
The G190A RT variant, while decreasing phenotypic susceptibility to NVP and EVF, has little effect on RT processivity or RC, and in some cases can increase polymerase activity. Viruses with 190S, 190T, 190Q, or 190V have markedly impaired RC when introduced into recombinant virus strains, as well as diminution in RT activity.34
The G190S RT variant is intermediate between wild-type or G190A variants and the markedly impaired variants G190Q, G190V, and G190E. Introduction of L74V, which alone had no measurable effect on the replication efficiency of the reference virus or on the 190A virus, increased replication capacity viruses with 190S, or other variants.33,34,51,54
Review of the Monogram Sciences, Inc. database showed that the RC of viruses with 190E, 190Q, and 190V range from 20% to 60%, although viruses made by site-directed mutagenesis exhibited RC <
1%. This strongly suggests that other mutations can compensate in order to improve RT enzyme activity.
Although not observed with the frequency of M184V, interaction of L74V with other RTI resistance mutations has implications in virus susceptibility as well as replication kinetics. This suggests a theoretical advantage in activity to several NRTIs for viruses that maintain this mutation. Just as ZDV was paired with 3TC because of theoretical advantages, ZDV may exhibit enhanced activity when used with drugs, such as ddI, that maintain L74V, while at the same time negatively impacting replication kinetics. Of note, viruses with K65R has been shown to have an even stronger effect on RC and on resensitization to ZDV, as previously described.55
The K65R mutation is thought to affect susceptibility to all NRTI except ZDV through a mechanism of diminished efficiency of initiation of minus double-stranded HIV-DNA synthesis.44
The decreased efficiency of initiation of HIV-DNA synthesis together with diminished RNA template usage when these mutations are seen together exceeds defects seen with either one alone, and may explain the diminished viral fitness observed in tissue culture when K65R/M184V-containing viruses are evaluated. It is noteworthy that clonal expression of K65R with either T215Y or with L75V is rarely observed.56–58
TAMs appeared to antagonize the phenotypic effects of K65R, reducing resistance to tenofovir, whereas K65R results in resistance to all NRTIs except for ZDV. A further mechanistic explanation for the bidirectional antagonism of K65R and TAMs was shown to result from a decrease in NRTI MP excision activity of RT containing TAMs by K65R. Conversely, TAMs antagonized the ability of K65R RT to discriminate against the nucleotide analog.57
This suggests the potential role for therapies that result in selection for both K65R and TAMs, since one or the other drug is likely to retain some activity, at least initially.
Importantly, we showed the effects of NNRTI resistance mutations on NRTI susceptibility. Although the presence of TAMs can markedly improve susceptibility to NNRTI,23–25
we now show that an NNRTI resistance mutation pattern (K101E and G190S) was shown to generally diminish NRTI susceptibility (). However, this observation did not exhibit the same effects as those seen for changes in NNRTI susceptibility in the presence of TAMs only, where hyper-susceptibility is often observed.
Initial combination therapy is increasing based on an NNRTI and two NRTI drugs. Failure with this regimen results most commonly in the emergence of NNRTI resistance mutations, as well as NRTI resistance mutations,15
and is more prevalent than in patients failing ritonavir-boosted protease inhibitor-based regimens.59
Use of genotypic HIV drug resistance testing at the time of treatment failure has been shown to result in improved short-term outcomes from subsequent therapy.60,61
Better delineation of the effect of complex mutation patterns will help to refine algorithms used to interpret genotypic resistance mutations that arise during antiretroviral therapy. Moreover, since combinations of NRTI and NNRTIs will be frequently used early in therapy, strategies of treatment predicted to result in mutation patterns and their interactions may improve resistance test algorithms and provide better guidance for selection of subsequent drug regimens.