去污剂诱导丙型肝炎病毒 1b 型 RNA 聚合酶的激活。

Detergent-induced activation of the hepatitis C virus genotype 1b RNA polymerase.

机构信息

Unit of Viral Genome Regulation, Institut Pasteur of Shanghai, Key Laboratory of Molecular Virology & Immunology, Chinese Academy of Sciences, Shanghai, PR China.

出版信息

Gene. 2012 Apr 1;496(2):79-87. doi: 10.1016/j.gene.2012.01.044. Epub 2012 Jan 28.

DOI:10.1016/j.gene.2012.01.044

PMID:22306265

Abstract

Recently, we found that sphingomyelin bound and activated hepatitis C virus (HCV) 1b RNA polymerase (RdRp), thereby recruiting the HCV replication complex into lipid raft structures. Detergents are commonly used for resolving lipids and purifying proteins, including HCV RdRp. Here, we tested the effect of detergents on HCV RdRp activity in vitro and found that non-ionic (Triton X-100, NP-40, Tween 20, Tween 80, and Brij 35) and twitterionic (CHAPS) detergents activated HCV 1b RdRps by 8-16.6 folds, but did not affect 1a or 2a RdRps. The maximum effect of these detergents was observed at around their critical micelle concentrations. On the other hand, ionic detergents (SDS and DOC) completely inactivated polymerase activity at 0.01%. In the presence of Triton X-100, HCV 1b RdRp did not form oligomers, but recruited more template RNA and increased the speed of polymerization. Comparison of polymerase and RNA-binding activity between JFH1 RdRp and Triton X-100-activated 1b RdRp indicated that monomer RdRp showed high activity because JFH1 RdRp was a monomer in physiological conditions of transcription. Besides, 502H plays a key role on oligomerization of 1b RdRp, while 2a RdRps which have the amino acid S at position 502 are monomers. This oligomer formed by 502H was disrupted both by high salt and Triton X-100. On the contrary, HCV 1b RdRp completely lost fidelity in the presence of 0.02% Triton X-100, which suggests that caution should be exercised while using Triton X-100 in anti-HCV RdRp drug screening tests.

摘要

最近，我们发现鞘磷脂能结合并激活丙型肝炎病毒（HCV）1b RNA 聚合酶（RdRp），从而将 HCV 复制复合物募集到脂筏结构中。去污剂常用于溶解脂质和纯化蛋白质，包括 HCV RdRp。在这里，我们测试了去污剂对 HCV RdRp 体外活性的影响，发现非离子（Triton X-100、NP-40、Tween 20、Tween 80 和 Brij 35）和两性离子（CHAPS）去污剂能使 HCV 1b RdRp 的活性增强 8-16.6 倍，但不影响 1a 或 2a RdRp。这些去污剂的最大效果出现在其临界胶束浓度附近。另一方面，离子去污剂（SDS 和 DOC）在 0.01%时完全使聚合酶失活。在 Triton X-100 存在的情况下，HCV 1b RdRp 不会形成寡聚体，但会募集更多的模板 RNA 并提高聚合速度。JFH1 RdRp 和 Triton X-100 激活的 1b RdRp 的聚合酶和 RNA 结合活性比较表明，单体 RdRp 具有高活性，因为 JFH1 RdRp 在转录的生理条件下是单体。此外，502H 在 1b RdRp 的寡聚化中起关键作用，而在位置 502 具有氨基酸 S 的 2a RdRps 是单体。这种由 502H 形成的寡聚体既被高盐也被 Triton X-100 破坏。相反，HCV 1b RdRp 在 0.02% Triton X-100 的存在下完全失去保真度，这表明在抗 HCV RdRp 药物筛选试验中使用 Triton X-100 时应谨慎。