During the induction experiments on HIV-1s harboring I135X, the emergence of E138K, which is usually a rare mutation, was often observed. To analyze the effects of E138K alone and its combination with I135X on NNRTI susceptibility, a panel of recombinant HIV-1 clones was constructed and their IC50 values for EFV, NVP, and ETR were determined. As expected, I135X alone did not confer significant NNRTI resistance (Table 2). The combination of I135T and E138K (I135T/E138K) conferred significant resistance to EFV, NVP, and ETR, though E138K alone did not change NNRTI susceptibility as reported previously (Su et al. 16th International HIV Drug Resistance Workshop, 2007) [22,23]. I135L/E138K and I135R/E138K conferred significant resistance to NVP and ETR, respectively. In summary, E138K conferred significant resistance when combined with some of the I135X mutations, especially I135T, which is the most prevalent in treatment-naive individuals in Japan (Table 1).
The in-vitro drug susceptibility assay described above showed that I135T/E138K conferred the most efficient resistance to EFV and significant resistance to NVP and ETR. To analyze the molecular mechanisms by which E138K combined with I135T alter NNRTI susceptibility, we conducted a structural analysis that included computational methods. A total of 12 structural models of reverse transcriptase-NNRTI complexes were constructed with four reverse transcriptases (wild-type, I135T, E138K, and I135T/E138K) and three NNRTIs (EFV, NVP, and ETR). We first calculated the binding energies between reverse transcriptase and NNRTI. Differences in the binding energies between mutant and wild-type complexes (ΔΔGb) were calculated using the models. The ΔΔGb value correlated positively with the logarithm of fold resistance value obtained by our in-vitro drug susceptibility assay described above: a greater reduction in the binding energy correlated with a greater resistance (r = 0.77, P < 0.02) , suggesting that our modeling appropriately reflects the actual binding mode between the reverse transcriptase molecule and NNRTI. In the 12 models tested, the ΔΔGb value of the I135T/E138K RT-NNRTI complex was persistently larger than wild-type and single mutation reverse transcriptases, indicating that I135T/E138K caused a larger loss of interactions between reverse transcriptase and NNRTI than the single mutations. We then examined the structural changes in the loss of interactions by I135T/E138K. In the wild-type reverse transcriptase, the E138 positioned relatively closely to the EFV, which could contribute to the generation of van der Waals and electrostatic interactions between reverse transcriptase and NNRTI (Fig. 3a). The I135T single substitution caused no significant changes in the steric position of the E138 side chain (Fig. 3b). E138K substitution caused significant changes in the steric position of the E138 side chain (Fig. 3c), whereas the calculated van der Waals energy was similar to that of wild-type reverse transcriptase. I135T/E138K also caused significant changes in the steric position of the K138 side chain, but the orientation of the side chain was different from that of the E138K single mutant reverse transcriptase, possibly due to the interactions between T135 and K138 (Fig. 3d). The K138 conformation in the RTI135T/E138K generated a steric gap between K138 and EFV, and significantly reduced van der Waals energy. In addition, the conformational change necessitated increased electrostatic energy of the reverse transcriptase–EFV complex. These data suggest that an appropriate steric position of the 138th residue is critical for the generation of an optimal EFV binding pocket, and that I135T/E138K, but not the single mutations, effectively break the binding pocket for EFV.
As the HIV-1 pandemic progresses, viral genetic diversity is increasing and becoming geographically heterogeneous [25,26]. We recently indicated that HIV-1 adapts to CTL by acquiring escape mutations in the CTL epitopes, and that such escape variants are increasing in the populations at an alarming high rate of corresponding HLA alleles . When escape mutations occur in drug target proteins, they may alter the mutation patterns of drug resistance even if they do not confer drug resistance themselves. In this study, we focused on I135X in reverse transcriptase, which are escape mutations of HLA-B*51-restricted CTL, because I135X are the prevailing mutations and accumulating in Japan, where the frequency of HLA-B*51 is high (∼20%). Cultural passages of HIV-1 sequences harboring I135X in the presence of increasing concentrations of EFV induced the emergence of E138K, which is not listed as a resistance mutation in the IAS-USA Table. The analysis of recombinant HIV-1 sequences showed that the combination of E138K and some of the I135X, especially I135T, which is most frequent, conferred significant resistance to NNRTI, though solo E138K did not alter drug susceptibility significantly. However, E138K did not always emerge in triplicate experiments of EFV-resistance induction from HIV-1 sequences harboring I135X, whereas the already known NNRTI-resistance mutations emerged. Importantly, variable mutation patterns emerged under the same conditions of resistance induction experiments, indicating that the drug selective pressure is one of the driving forces making the genetic diversity of HIV-1 at population levels as CTL pressure does (HLA-B*51-restricted CTL pressure selects not only I135T but also other I135Xs).
Evidences for the interactions between CTL and drug resistance mutations are accumulating [30–34]. Considering that HIV-1 adapts to particular human HLA alleles and evolves among infected individuals, drug mutation patterns may be affected and altered in currently prevailing viruses. Analysis of drug resistance mutations and development of new antiretroviral agents against laboratory HIV-1 strains derived from isolates obtained decades ago may not always be a suitable strategy. The use of recently obtained clinical isolates may be critical and indispensable in some studies.
This work was supported in part by a Grant-in Aid for AIDS research from the Ministry of Health, Labor, and Welfare (H20-AIDS-002), and the Global Center of Excellence Program (Global Education and Research Center Aiming at the Control of AIDS) from the Ministry of Education, Science, Sports and Culture of Japan.
H.G. designed and executed the study, analyzed the data and wrote the manuscript. H.O. and H.S. performed computational analysis and wrote the manuscript. A.H. and T.H. executed the study and collected data. M.T. provided the hypothesis and participated in discussion and review. S.O. participated in discussion and review and supervised the study.
There are no conflicts of interest.
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