Carboxy-terminal five amino acids of the nucleocapsid protein of vesicular stomatitis virus are required for encapsidation and replication of genome RNA.
Carboxy-terminal five amino acids of the nucleocapsid protein of vesicular stomatitis virus are required for encapsidation and replication of genome RNA.
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DOI：
10.1006/viro.1999.9768
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发表时间：
1999-06
期刊：
影响因子：
3.7
通讯作者：
T. Das;Bimal K. Chakrabarti;Dhrubajyoti Chattopadhyay;Amiya K. Banerjee
中科院分区：
文献类型：
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作者：
T. Das;Bimal K. Chakrabarti;Dhrubajyoti Chattopadhyay;Amiya K. Banerjee
研究了水泡性口炎病毒（VSV）基因组RNA的包裹作用，这是核衣壳蛋白（N）复制过程的先决条件，通过其在体外以RNA酶抗性形式包装VSV前导RNA的能力来研究。VSV前导RNA来源于SP 6转录载体，而N蛋白在兔网织红细胞裂解物中制备。在32 P标记的前合成的前导RNA的存在下，通过翻译N mRNA进行体外转录。当C-末端5个氨基酸（VEFDK-COOH）被删除时，N蛋白的RNA水解性质被完全废除。对C端5个氨基酸残基的系统突变分析表明，除K --> A和K --> R外，所有突变体均丧失了RNA水解活性，表明C端5个氨基酸残基，特别是赖氨酸残基在基因组RNA水解中起着关键作用。为了将这些突变N蛋白与基因组RNA复制的体外结合能力相关联，我们使用VSV基因组RNA的全长cDNA克隆来从表达L、P和wt或突变N蛋白的细胞中拯救感染性病毒体，并测量空斑形成单位的恢复。结果表明，在前导RNA的体外合成中有缺陷的N突变体不支持复制，建立了在病毒复制中N蛋白的C-末端5个氨基酸的需求。
The encapsidation of vesicular stomatitis virus (VSV) genome RNA, a prerequisite step to the replication process by the nucleocapsid protein (N) was studied by its ability to package VSV leader RNA in vitro in a RNase-resistant form. The VSV leader RNA was derived from the SP6 transcription vector while the N protein was made in rabbit reticulocyte lysate. The in vitro encapsidation was carried out by translating N mRNA in the presence of 32P-labeled presynthesized leader RNA. The RNA encapsidation property of the N protein was completely abrogated when the C-terminal five amino acids (VEFDK-COOH) were deleted. Systematic mutational analyses within the C-terminal five amino acid regions reveal that the RNA encapsidation activity was lost in all mutants except K --> A and K --> R, indicating that C-terminal five amino acids, in particular the lysine residue play critical role in genome RNA encapsidation. To correlate the in vitro encapsidation abilities of these mutant N proteins with genome RNA replication, we have used a full-length cDNA clone of VSV genome RNA to rescue infectious virions from cells expressing L, P, and wt or mutant N proteins and measured the recovery of plaque forming units. The results indicate that the N mutants that are defective in in vitro encapsidation of leader RNA do not support replication, establishing the requirement of C-terminal five amino acids of the N protein in viral replication.