Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity
Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity
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DOI：
10.1128/jvi.71.11.8416-8428.1997
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发表时间：
1997-11-01
影响因子：
5.4
通讯作者：
Bartenschlager, R
中科院分区：
文献类型：
--
作者：
Lohmann, V;Korner, F;Bartenschlager, R
丙型肝炎病毒（HCV）的NS 5 B蛋白是RNA依赖性RNA聚合酶（RdRp）（S. E.贝伦斯湖Tomei和R. De Francesco，EMBO J. 15：12-22，1996），该酶被认为是病毒基因组复制所需的。为了进一步研究该酶的生物化学和结构特性，用重组杆状病毒在昆虫细胞中表达NS 5 B-六组氨酸融合蛋白，并纯化至接近同质。发现该酶具有引物依赖性RdRp活性，其能够在不存在另外的病毒或细胞因子的情况下复制完整的体外转录的HCV基因组。滤器结合测定和竞争实验表明，纯化的酶结合RNA，对HCV 3 '端序列没有明显的偏好。还检查了与均聚RNA的结合。观察到以下特异性顺序：聚（U）>聚（G）>聚（A）>聚（C）。发现RdRp活性的相反顺序，其使用聚（C）最有效地作为模板，但对聚（U）和聚（G）无活性，这表明聚合酶和模板之间的高结合亲和力干扰了持续合成能力。通过使用突变分析，鉴定了对RdRp活性至关重要的四个氨基酸序列基序，虽然这些基序中保守残基的大多数取代严重降低了酶活性，发现基序D中的单个取代使RdRp活性增强约50%。缺失研究表明，最末端的氨基酸残基，最后，我们发现了与纯化的酶相关的末端转移酶活性。然而，用具有无活性RdRp的NS 5 B蛋白、用以相同方式纯化的NS 4 B蛋白和用野生型杆状病毒也检测到了这种活性，这表明它不是NS 5 B的固有活性。
The NS5B protein of the hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) (S.-E. Behrens, L. Tomei, and R. De Francesco, EMBO J. 15:12-22, 1996) that is assumed to be required for replication of the viral genome, To further study the biochemical and structural properties of this enzyme, an NS5B-hexahistidine fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity, The enzyme was found to have a primer-dependent RdRp activity that was able to copy a complete in vitro-transcribed HCV genome in the absence of additional viral or cellular factors, Filter binding assays and competition experiments showed that the purified enzyme binds RNA with no clear preference for HCV 3'-end sequences, Binding to homopolymeric RNAs was also examined, and the following order of specificity was observed: poly(U) > poly(G) > poly(A) > poly(C). An inverse order was found for the RdRp activity, which used poly(C) most efficiently as a template but was inactive on poly(U) and poly(G), suggesting that a high binding affinity between polymerase and template interferes with processivity, By using a mutational analysis, four amino acid sequence motifs crucial for RdRp activity were identified, While most substitutions of conserved residues within these motifs severely reduced the enzymatic activities, a single substitution in motif D which enhanced the RdRp activity by about 50% was found. Deletion studies indicate that amino acid residues at the very termini, in particular the amino terminus, are important for RdRp activity but not for RNA binding, Finally, we found a terminal transferase activity associated with the purified enzyme, However, this activity was also detected with NS5B proteins with an inactive RdRp, with an NS4B protein purified in the same way, and with wild-type baculovirus, suggesting that it is not an inherent activity of NS5B.