The present study was designed to determine the impact of the M230L mutation in the HIV RT on the antiviral activity of the second-generation NNRTIs ETR and DAP, as well as the first-generation NNRTIs EFV and NVP. M230L was first identified by in vitro
passage of HIV in the presence of the NNRTI delavirdine (DLV) (24
). This mutation has also been detected in clinical isolates from patients and has been shown to reduce the levels of susceptibility to each of NVP, DLV, and EFV by 23- to 58-fold (22
M230 is located within the highly conserved primer grip region of RT, and this position is highly conserved among all HIV isolates. Therefore, the effects of the M230L mutation on virus replication and on the underlying properties of RT should not depend on the particular RT or virus strain that is used in the assays. In particular, defects in replication capacity associated with M230L have previously been documented by other groups (23
) and confirmed here. Moreover, a previous paper by our group has revealed the absence of important differences in enzymatic function between the RT enzymes of HIV-1 subtype B and HIV-1 subtype C (47
Recently, the DUET-1 and DUET-2 clinical trials have identified an array of 17 resistance-associated mutations (RAMs) that confer diminished sensitivity to the second-generation NNRTI ETR; these mutations include V90I, A98G, L100I, K101E/H/P, V106I, E138A, V179D/F/T, Y181C/I/V, G190A/S, and M230L (38
). Our data confirm that M230L confers resistance to all currently available NNRTIs in both phenotypic and biochemical assays. In the former, recombinant viruses harboring M230L displayed similarly moderate levels (5- to 6-fold) of resistance to ETR and DAP and higher-level (>10-fold) resistance to EFV and NVP. The data that we obtained with ETR are consistent with those presented in previous reports, which showed that M230L-containing viruses were ≈3-fold more resistant to ETR than WT RT-containing viruses (38
). However, a different study reported that M230L was not associated with resistance to either ETR or EFV (36
). Our RDDP and DDDP enzyme assays further confirm that M230L causes similar ≈10-fold higher levels of resistance to each of the second-generation NNRTIs ETR and DAP and over 16-fold higher levels of resistance to EFV and NVP. Interestingly, a recent study showed that a newer NNRTI termed rilpivirine (formerly TMC278) was not affected by the presence of most single NNRTI RAMs, including those at positions 100, 103, 106, 138, 179, 188, 190, 221, 236, and 230 (5
The prevalence of the M230L mutation in clinical isolates obtained from patients failing NNRTI-based therapy is low (2
). The emergence of resistance mutations can also depend on the effect of such mutations on viral replicative fitness (10
), with mutations that confer high-level resistance with minimal effects on enzyme activity theoretically developing first. The use of a TZM-bl cell infectivity assay has confirmed that M230L-containing virus replicates less efficiently than WT virus, in agreement with the findings presented previously (23
). The impaired replication capacity of M230L-containing virus might explain the rarity of this mutation in clinical isolates. However, we cannot exclude the possibility that hypersusceptibility to nucleoside and/or nucleotide RT inhibitors might result from the presence of the M230L mutation and that resensitization to NNRTIs might also take place in the presence of other NRTI resistance mutations, including the thymidine analogue mutations (TAMs) K65R and M184V. As a consequence, hypersusceptibility/resensitization effects might contribute to the rarity of M230L in clinical samples. We are investigating the interaction of the M230L mutation with other NRTI resistance mutations. It remains to be determined whether the emergence of this mutation may require other compensatory mutations that might enhance NNRTI resistance and restore viral replicative fitness.
Our biochemical characterization has shown that M230L severely impaired each of minus-strand ssDNA synthesis, both DNA- and RNA-dependent polymerase activity, processivity, and RNase H activity. Previous studies by us and others have documented that the efficiency of each of these reactions can be correlated with viral replication efficiency (11
M230 is located at the tip of the β12-β13 hairpin comprising amino acids 227 to 235 (FLWMGYELH), which defines the so-called primer grip, a highly conserved motif of retroviral RT. The primer grip is involved in maintenance of the primer terminus in an orientation appropriate for nucleophilic attack on an incoming dNTP (25
). Mutational analysis of primer grip residues has shown their influence on various RT functions, including dNTP binding (46
), polypurine tract removal (32
), RNase H activity (31
), template-primer utilization (16
), and the fidelity of DNA synthesis (9
). The biochemical data presented here show that the described impairment in enzyme function associated with M230L contributes to reduced viral replication.
Further research is needed to determine how the M230L mutation might affect the binding kinetics of NNRTIs. We are performing a biochemical investigation of the interplay of M230L with other NNRTI and/or NRTI resistance mutations. We are also studying whether other resistance-related mutations that may appear in association with M230L may be able to help restore viral replication capacity while also potentially increasing the overall levels of drug resistance.