Kulkarni Anupriya S, Damha Masad J, Schinazi Raymond F, Mo Hongmei, Doehle Brian, Sagan Selena M, Götte Matthias
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
Department of Chemistry, McGill University, Montreal, Quebec, Canada.
Antimicrob Agents Chemother. 2016 Mar 25;60(4):2018-27. doi: 10.1128/AAC.02436-15. Print 2016 Apr.
The hepatitis C virus (HCV) RNA-dependent RNA-polymerase NS5B is essentially required for viral replication and serves as a prominent drug target. Sofosbuvir is a prodrug of a nucleotide analog that interacts selectively with NS5B and has been approved for HCV treatment in combination with ribavirin. Although the emergence of resistance to sofosbuvir is rarely seen in the clinic, the S282T mutation was shown to decrease susceptibility to this drug. S282T was also shown to confer hypersusceptibility to ribavirin, which is of potential clinical benefit. Here we devised a biochemical approach to elucidate the underlying mechanisms. Recent crystallographic data revealed a hydrogen bond between S282 and the 2'-hydroxyl of the bound nucleotide, while the adjacent G283 forms a hydrogen bond with the 2'-hydroxyl of the residue of the template that base pairs with the nucleotide substrate. We show that DNA-like modifications of the template that disrupt hydrogen bonding with G283 cause enzyme pausing with natural nucleotides. However, the specifically introduced DNA residue of the template reestablishes binding and incorporation of sofosbuvir in the context of S282T. Moreover, the DNA-like modifications of the template prevent the incorporation of ribavirin in the context of the wild-type enzyme, whereas the S282T mutant enables the binding and incorporation of ribavirin under the same conditions. Together, these findings provide strong evidence to show that susceptibility to sofosbuvir and ribavirin depends crucially on a network of interdependent hydrogen bonds that involve the adjacent residues S282 and G283 and their interactions with the incoming nucleotide and complementary template residue, respectively.
丙型肝炎病毒（HCV）的RNA依赖性RNA聚合酶NS5B是病毒复制所必需的，也是一个重要的药物靶点。索磷布韦是一种核苷酸类似物的前体药物，它能选择性地与NS5B相互作用，已被批准与利巴韦林联合用于HCV治疗。虽然临床上很少出现对索磷布韦的耐药性，但S282T突变已被证明会降低对该药物的敏感性。S282T突变还被证明会使病毒对利巴韦林超敏感，这具有潜在的临床益处。在此，我们设计了一种生化方法来阐明其潜在机制。最近的晶体学数据显示，S282与结合核苷酸的2'-羟基之间存在氢键，而相邻的G283与与核苷酸底物碱基配对的模板残基的2'-羟基形成氢键。我们发现，模板的DNA样修饰若破坏与G283的氢键，会导致天然核苷酸作用下的酶暂停。然而，模板中特意引入的DNA残基在S282T背景下能重新建立索磷布韦的结合与掺入。此外，模板的DNA样修饰在野生型酶背景下会阻止利巴韦林的掺入，而S282T突变体在相同条件下能使利巴韦林结合并掺入。总之，这些发现提供了有力证据，表明对索磷布韦和利巴韦林的敏感性关键取决于一个相互依赖的氢键网络，该网络分别涉及相邻残基S282和G283及其与进入的核苷酸和互补模板残基的相互作用。
