Analysis of the Enzymatic Activity of an NS3 Helicase Genotype 3a Variant Sequence Obtained from a Relapse Patient.
Analysis of the Enzymatic Activity of an NS3 Helicase Genotype 3a Variant Sequence Obtained from a Relapse Patient.
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DOI：
10.1371/journal.pone.0144638
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Rahal P
中科院分区：
文献类型：
--
作者：
Provazzi PJ;Mukherjee S;Hanson AM;Nogueira ML;Carneiro BM;Frick DN;Rahal P
丙型肝炎病毒（HCV）是一种不同基因型的病毒，感染全球超过1.7亿人，引起慢性炎症、肝硬化和肝细胞癌。HCV基因型3a在巴西很常见，它与目前直接作用的抗病毒治疗的反应相对较差有关。HCV NS3蛋白切割部分HCV多蛋白和细胞抗病毒蛋白。因此，它是几种HCV药物的靶点。除了蛋白酶活性外，NS3还是一种RNA解旋酶。以前，发现复发患者中存在的HCV携带已知对HCV基因型1b致命的突变。点突变编码解旋酶RNA结合位点中的氨基酸取代W501R。为了检测W501R取代如何影响基因型3a背景中的NS3解旋酶活性，将野生型和W501R基因型3a的NS3等位基因亚克隆，在E. coli中表达，并对重组蛋白进行纯化和鉴定。还分析了W501R等位基因对基因型2a和3a亚基因组复制子的影响。监测解旋酶催化的DNA和RNA解旋的测定显示，野生型基因型3a NS3解旋酶的催化效率比W501R蛋白高600倍以上。其他测定显示，W501 R蛋白与DNA的结合强度比野生型弱不到2倍，并且两种蛋白质以相似的速率水解ATP。在Huh7.5细胞中，携带W501R等位基因的基因型2a和3a亚基因组HCV复制子均表现出严重的复制缺陷。由于W501R等位基因是作为一个微小变异携带的，因此它的复制需要归因于其他野生型准种的反式互补。
The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people worldwide, causing chronic inflammation, cirrhosis and hepatocellular carcinoma. HCV genotype 3a is common in Brazil, and it is associated with a relatively poor response to current direct-acting antiviral therapies. The HCV NS3 protein cleaves part of the HCV polyprotein, and cellular antiviral proteins. It is therefore the target of several HCV drugs. In addition to its protease activity, NS3 is also an RNA helicase. Previously, HCV present in a relapse patient was found to harbor a mutation known to be lethal to HCV genotype 1b. The point mutation encodes the amino acid substitution W501R in the helicase RNA binding site. To examine how the W501R substitution affects NS3 helicase activity in a genotype 3a background, wild type and W501R genotype 3a NS3 alleles were sub-cloned, expressed in E. coli, and the recombinant proteins were purified and characterized. The impact of the W501R allele on genotype 2a and 3a subgenomic replicons was also analyzed. Assays monitoring helicase-catalyzed DNA and RNA unwinding revealed that the catalytic efficiency of wild type genotype 3a NS3 helicase was more than 600 times greater than the W501R protein. Other assays revealed that the W501R protein bound DNA less than 2 times weaker than wild type, and both proteins hydrolyzed ATP at similar rates. In Huh7.5 cells, both genotype 2a and 3a subgenomic HCV replicons harboring the W501R allele showed a severe defect in replication. Since the W501R allele is carried as a minor variant, its replication would therefore need to be attributed to the trans-complementation by other wild type quasispecies.