期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：30
DOI：10.1073/pnas.2203660119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
The enzyme reverse transcriptase (RT) is a key antiviral target, and nonnucleoside RT inhibitors (NNRTIs) are among the frequently used components of antiretroviral therapy for treating HIV-1 infection. The emergence of drug-resistant mutations continues to pose a challenge in HIV treatment. The RT mutations M184I and E138K emerge in patients receiving rilpivirine. We obtained the structural snapshots of rilpivirine, doravirine, and nevirapine inhibited wild-type and M184I/E138K RT/DNA polymerase complexes by cryo-electron microscopy. Key structural changes observed in the rilpivirine- and doravirine-bound structures have implications for understanding NNRTI drug resistance. Additionally, the cryo-EM structure determination strategy outlined in this study can be adapted to aid drug design targeting smaller and flexible proteins.
Structures trapping a variety of functional and conformational states of HIV-1 reverse transcriptase (RT) have been determined by X-ray crystallography. These structures have played important roles in explaining the mechanisms of catalysis, inhibition, and drug resistance and in driving drug design. However, structures of several desired complexes of RT could not be obtained even after many crystallization or crystal soaking experiments. The ternary complexes of doravirine and rilpivirine with RT/DNA are such examples. Structural study of HIV-1 RT by single-particle cryo-electron microscopy (cryo-EM) has been challenging due to the enzyme’s relatively smaller size and higher flexibility. We optimized a protocol for rapid structure determination of RT complexes by cryo-EM and determined six structures of wild-type and E138K/M184I mutant RT/DNA in complexes with the nonnucleoside inhibitors rilpivirine, doravirine, and nevirapine. RT/DNA/rilpivirine and RT/DNA/doravirine complexes have structural differences between them and differ from the typical conformation of nonnucleoside RT inhibitor (NNRTI)–bound RT/double-stranded DNA (dsDNA), RT/RNA–DNA, and RT/dsRNA complexes; the primer grip in RT/DNA/doravirine and the YMDD motif in RT/DNA/rilpivirine have large shifts. The DNA primer 3′-end in the doravirine-bound structure is positioned at the active site, but the complex is in a nonproductive state. In the mutant RT/DNA/rilpivirine structure, I184 is stacked with the DNA such that their relative positioning can influence rilpivirine in the pocket. Simultaneously, E138K mutation opens the NNRTI-binding pocket entrance, potentially contributing to a faster rate of rilpivirine dissociation by E138K/M184I mutant RT, as reported by an earlier kinetic study. These structural differences have implications for understanding molecular mechanisms of drug resistance and for drug design.
