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

根除HCV有望减少或预防HCC的发病率。HCV NS5B， RNA依赖性RNA聚合酶（RdRP），是抑制HCV复制的靶点。我们构建了一系列NS5B的聚类和点丙氨酸替代突变，并检测了突变对NS5B功能的影响。结果如下。1)新发现5个氨基酸残基对RdRP活性至关重要（《肝病学》，出版中）。所有这些都在RdRPs和逆转录酶之间保守的基序之外。该结果可为HCV RdRP特异性抑制剂的设计提供参考。2) NS5B的RdRP活性不需要模板结合，而需要模板/引物结合。除了模板/引物结合和RdRP活性均为阴性的Y276A突变体外，所有模板/引物结合阴性的突变体RdRP活性均为阳性。3) RdRP活性催化中心外的两个残基对NS5B的寡聚化很重要，而NS5B对RdRP活性至关重要。4)过表达NS5B激活AP-1和NF-kB对急性炎症反应至关重要，但无法确定一个NS5B序列负责激活，这受NS5B表达水平的影响。NS5B可能调节炎症反应，但我们的观察是否在慢性HCV感染中具有生物学相关性仍有待解决，在慢性HCV感染中，低量的NS5B与其他NS蛋白的协调表达是预期的。

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.