Abstract
Hepatitis B virus (HBV) reverse transcriptase (RT) is essential for viral replication and is an important drug target. Nonetheless, the notorious insolubility of HBV RT has hindered experimental structural studies and structure-based drug design. Here, we demonstrate that a Q151M substitution alone at the nucleotide-binding site (N-site) of human immunodeficiency virus type-1 (HIV-1) RT renders HIV-1 highly sensitive to entecavir (ETV), a potent nucleoside analogue RT inhibitor (NRTI) against HBV. The results suggest that Met151 forms a transient hydrophobic interaction with the cyclopentyl methylene of ETV, a characteristic hydrophobic moiety of ETV. We thus solved the crystal structures of HIV-1 RTQ151M:DNA complex with bound dGTP or ETV-triphosphate (ETV-TP). The structures revealed that ETV-TP is accommodated at the N-site slightly apart from the ribose ring of the 3′-end nucleotide, compared to the position of bound dGTP and previously reported NRTI/dNTP. In addition, the protruding methylene group of bound ETV-TP directly pushes the side-chain of Met184 backward. Met184 is a key residue that confers ETV resistance upon substitution with smaller Ile/Val. These results provide novel insights into NRTI binding to the N-site and further provide important clues for the development of novel anti-HBV/HIV-1 RT inhibitors to overcome critical drug resistance.
Highlights
All approved chemotherapeutics for anti-HBV treatment comprise nucleoside analogue reverse transcriptase (RT) inhibitors (NRTIs)[8]
Based on the previously reported ternary complex structures of human immunodeficiency virus type-1 (HIV-1) RT, a total of 20 amino acid residues lying within 7 Å of the deoxyribonucleoside moiety of the bound NRTI/ dNTP were extracted (Fig. 1)
We generated 5 HIV-1 RT mutants to mimic the HBV RT N-site, constructing recombinant HIV-1 containing those HIV-1 RT mutants for viral replication and anti-viral assays, and found that the Q151M mutation of HIV-1 RT alone is critical for conferring high ETV sensitivity on HIV-1
Summary
All approved chemotherapeutics for anti-HBV treatment comprise nucleoside analogue reverse transcriptase (RT) inhibitors (NRTIs)[8]. HBV and HIV-1 RTs share common metal-dependent dNTP-binding and catalytic mechanism of nucleotide addition to the 3′-end of the primer DNA, whereas amino acid differences around the N-site probably result in different NRTI sensitivities. The phenomenon of RT inhibition by ETV occurs in both HIV-1 and HBV14,15 and is referred to as “delayed chain termination” which enables evasion of incorporated NRTI phosphorolytic excision, a major NRTI-resistance mechanism of HIV-1 RT16. It is important to elucidate the interatomic interactions between ETV-triphosphate (ETV-TP) and HBV RT by X-ray crystallography to understand the structural mechanism of HBV RT inhibition and the reported ETV resistance in detail, which may be useful for novel HBV RT inhibitor design. Based on the structures of RTQ151M together with the results of the antiviral assay, possible mechanisms of ETV-TP action on HIV-1/HBV RT and of the reported ETV resistance are discussed
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