Functional analysis of Hepatitis C Virus replicase NS5B and a regulatory factor NS5A

丙型肝炎病毒复制酶NS5B和调节因子NS5A的功能分析

• 批准号: 14370054
• 负责人: MURAKAMI Seishi
• 金额: $ 7.62万
• 依托单位: KANAZAWA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370054/
• 关键词: HCV; NS5B; NS5A; RNA replicon; Nucleolin; RNA dependent RNA polymerase (RdRP); library of clustered alanine substitution mutant; HCV NS5B; アラニンスキャニング; 鋳型; プライマー結合; HCV複製; オリゴマー

Eradication of HCV persistent infection may prevent or delay incidence of hepatocellular carcinoma. HCV NS5B is RdRP that is a strong target to eradicate HCV replication. In hope to find novel strategies, we had identified 5 residues of NS5B critical for RdRP activity by introducing clustered alanine substitution (Cm) and point alanine substitution. This grant aims to get further insight of structure and function of RdRP enzyme, to characterize NS5B-interacting partners, NS5A and a host factor nucleolin, and finally to evaluate these interactions and mutations of NS5B on HCV RNA replication using a HCV RNA replicon system. Followings are the results we obtained.1)Oligomer formation of NS5B was observed and two residues were defined by scanning the Cm and Pm libraries to be critical for oligomer formation. Interestingly the two residues are among the residues indispensable for RdRP activity, strongly suggesting that oligomeric interaction might be prerequisite for RdRP activity. 2)Speci … More fic interaction between NS5B and NS5A was found. The critical regions of the two factors for the interaction were mapped, 3)We found NS5B truncated at C-terminal was enriched in nucleoli. Nucleolin was defined to the interacting factor with NS5B. The NS5B-binding fragments did not affected much RdRP activity, although the interaction affected the subcellular localization of NS5B. 4)We evaluated whether the critical 5 residues for RdRP in vitro are indispensable for HCV replication using MlLE HCV RNA replicon. Actually all the residues are absolutely required for HCV replication. 5)Using HCV replicon harboring mutations of NS5A that are defective in NS5B-binding, we evaluated the role of the NS5A-NS5B interaction in HCV replication. Cm mutants and some Pm mutants within the regions necessary for NS5B-binding could not support HCV replication, strongly suggesting the importance of the inter action. 6)We evaluated the interaction of nucleolin and NS5B in the HCV RNA replicon system. Cm mutant of NS5B defective in nucleolin-binding could not support efficient HCV replication. Transient knock-down of nucleolin with siRNA greatly reduced the G418-forming foci, strongly suggesting that the interaction of nucleolin and NS5B is critical for HCV replication. Less

根除HCV持续感染可预防或延缓肝细胞癌的发生。HCV NS 5 B是RdRP，其是根除HCV复制的强靶标。为了寻找新的策略，我们通过引入簇状丙氨酸取代（Cm）和点丙氨酸取代，鉴定了对RdRP活性至关重要的NS 5 B的5个残基。该基金旨在进一步了解RdRP酶的结构和功能，表征NS 5 B相互作用伙伴，NS 5A和宿主因子核仁素，并最终使用HCV RNA复制子系统评估NS 5 B对HCV RNA复制的这些相互作用和突变。主要结果如下：1）通过扫描Cm和Pm文库，发现NS 5 B有两个残基对寡聚体的形成至关重要。有趣的是，这两个残基是RdRP活性不可或缺的残基之一，强烈表明寡聚相互作用可能是RdRP活性的先决条件。2）规格 ...更多信息 发现NS 5 B与NS 5A之间存在着强的相互作用，并对两个因子相互作用的关键区域进行了定位。核仁素被定义为与NS 5 B相互作用的因子。NS 5 B结合片段并没有影响RdRP活性，但相互作用影响了NS 5 B的亚细胞定位。（4）利用MlLE HCV RNA复制子，我们评估了RdRP的关键5个残基是否是HCV体外复制所必需的。事实上，所有的残基都是HCV复制所必需的。5)使用携带NS 5A突变的HCV复制子，这些突变在NS 5 B结合中有缺陷，我们评估了NS 5A-NS 5 B相互作用在HCV复制中的作用。在NS 5 B结合所必需的区域内的Cm突变体和一些Pm突变体不能支持HCV复制，强烈地表明相互作用的重要性。6）研究了核仁素与NS 5 B在HCV RNA复制子系统中的相互作用。Cm突变的NS 5 B在核仁结合缺陷不能支持有效的HCV复制。瞬时敲低核仁素的siRNA大大减少了G418形成的焦点，强烈表明核仁素和NS 5 B的相互作用是HCV复制的关键。少

项目成果

Arai, K., et al.: "Two independent regions of human telomerase reverse transcriptase (hTERT) are important for their oligomerization and telomerase activity"J. Biol. Chem.. 277. 8536-8544 (2002)

Arai, K. 等人：“人端粒酶逆转录酶 (hTERT) 的两个独立区域对于寡聚化和端粒酶活性非常重要”。

Interaction with general transcription factor IIF(TFIIF) is Required for the suppression of activated transcription by RPB5-mediating protein RMP.

与通用转录因子 IIF (TFIIF) 的相互作用是抑制 RPB5 介导蛋白 RMP 激活转录的必要条件。

• 发表时间: 2003
• 期刊: Cell Research 13
• 作者: H.Fujita;C.Nishitani;K.Ogawa;W.Wei et al.
• 通讯作者: W.Wei et al.

Direct interaction between nucleolin and hepatitis C virus NS5B

• DOI: 10.1074/jbc.m207629200
• 发表时间: 2003-02-14
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Hirano, M;Kaneko, S;Murakami, S
• 通讯作者: Murakami, S

Hepatitis C Virus NS5A Bind RNA-Dependent RNA polymerase NS5B and modulates RdRP activity.

丙型肝炎病毒 NS5A 结合 RNA 依赖性 RNA 聚合酶 NS5B 并调节 RdRP 活性。

• 发表时间: 2002
• 期刊: J.Biol.Chem. 277-13
• 作者: N.Hayashi et al.;STMI;K.Arai et al.;K.Masutomi et al.;C.Maida et al.;Y.Shirota et al.
• 通讯作者: Y.Shirota et al.

Wei, W., et al.: "Interaction with general transcription factor IIF(TFIIF) is required for the suppression as activated transcription by RPB5-mediating protein RMP"CellResearch. 13. 111-120 (2003)

Wei, W. 等人：“需要与通用转录因子 IIF (TFIIF) 相互作用才能抑制 RPB5 介导蛋白 RMP 激活的转录”Cell Research。