Department of Chemical Engineering, Stanford University, Palo Alto, California 94305, United States; Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Palo Alto, California 94305, United States.
Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Palo Alto, California 94305, United States; Department of Microbiology and Immunology, Stanford University School of Medicine, Palo Alto, California 94305, United States.
ACS Cent Sci. 2016 Jul 27;2(7):456-66. doi: 10.1021/acscentsci.6b00112. Epub 2016 Jun 13.
Therapeutic targeting of membrane-associated viral proteins is complicated by the challenge of investigating their enzymatic activities in the native membrane-bound state. To permit functional characterization of these proteins, we hypothesized that the supported lipid bilayer (SLB) can support in situ reconstitution of membrane-associated viral protein complexes. As proof-of-principle, we selected the hepatitis C virus (HCV) NS5B polymerase which is essential for HCV genome replication, and determined that the SLB platform enables functional reconstitution of membrane protein activity. Quartz crystal microbalance with dissipation (QCM-D) monitoring enabled label-free detection of full-length NS5B membrane association, its interaction with replicase subunits NS3, NS5A, and template RNA, and most importantly its RNA synthesis activity. This latter activity could be inhibited by the addition of candidate small molecule drugs. Collectively, our results demonstrate that the SLB platform can support functional studies of membrane-associated viral proteins engaged in critical biological activities.
治疗性靶向膜相关病毒蛋白的一个挑战是研究其在天然膜结合状态下的酶活性。为了对这些蛋白进行功能表征，我们假设支持脂质双层（SLB）可以支持膜相关病毒蛋白复合物的原位重建。作为原理验证，我们选择了丙型肝炎病毒（HCV）NS5B 聚合酶，该酶是 HCV 基因组复制所必需的，并且确定了 SLB 平台能够实现膜蛋白活性的功能重建。石英晶体微天平耗散（QCM-D）监测可实现全长 NS5B 膜结合的无标记检测，其与复制酶亚基 NS3、NS5A 和模板 RNA 的相互作用，以及最重要的是其 RNA 合成活性。通过添加候选小分子药物可以抑制这种活性。总的来说，我们的结果表明，SLB 平台可以支持参与关键生物活性的膜相关病毒蛋白的功能研究。
