RNA-dependent RNA polymerase activity and effect on host inflammatory response of Hepatitis C Virus(HCV)NS5B.

根除丙型肝炎病毒有望减少或预防丙型肝炎的发生。丙型肝炎病毒NS5B，即RNA依赖的RNA聚合酶(RdRP)，是抑制丙型肝炎病毒复制的靶点。我们构建了一系列NS5B的聚集性和点丙氨酸替代突变，并检测了这些突变对NS5B功能的影响。结果如下：1)新鉴定出5个氨基酸残基是RdRP活性所必需的。所有这些都在RdRPs和逆转录酶之间保守的基序之外。2)NS5B的RdRP活性不需要模板结合，而需要模板/引子。除Y276A外，所有模板结合阴性的突变体RdRP活性均为阳性，而Y276A的模板/引物结合和RdRP活性均为阴性。3)RdRP活性中心以外的两个残基对于NS5B的齐聚是重要的，我们可以发现NS5B是RdRP活性所必需的。4)NS5B过表达激活AP-1和对急性炎症反应至关重要的核因子-kB，但不能指定NS5B序列负责激活，而NS5B的表达水平影响NS5B的表达水平。NS5B可能调节炎症反应，但我们的观察在慢性丙型肝炎病毒感染中是否具有生物学意义仍有待解决，因为在慢性丙型肝炎病毒感染中，预期有少量NS5B和其他NS蛋白的协调表达。

Eradication of HCV has been expected to reduce or prevent incidence of HCC.HCV NS5B, RNA-dependent RNA polymerase(RdRP), is a target to inhibit HCV replication. We constructed a series of clustered and point alanine substitution mutations of NS5B and examined the effects of the mutations on functions of NS5B.The results are followings. 1)5 amino acid residues were newly identified to be indispensable for RdRP activity(Hepatology, in press). All are outside of the motifs conserved among RdRPs and reverse transcriptases. The result may provide desigins for specific inhibitors for HCV RdRP.2)RdRP activity of NS5B requires not template binding but template/primer. All mutants negative in the template binding are positive in RdRP activity except Y276A which is both negative in template/primer binding and RdRP activity. 3)The two residues apart from the catalytic center of RdRP activity are important for oligomerization of NS5B which we could find to be essential for RdRP activity. 4)Overexpression of NS5B activated AP-1 and NF-kB critical for acute inflammatory response, but a NS5B sequence could not be specified to be responsible for the activation which is influenced by expression level of NS5B.NS5B may modulate inflammatory responses but it remains addressed whether our observation has biological relevance in chronic HCV infection where a low amount of NS5B with coordinated expression of the other NS proteins is expected.