西尼罗病毒Chin-01株5′非编码区(UTR)中复制相关元件的功能研究

Studies on the function of elements involved in viral replication in 5' untranslated region of West Nile virus strain Chin-01

目的研究西尼罗病毒Chin-01株5′非编码区(UTR)中复制相关元件的功能,为探讨该病毒基因组复制的分子机制提供理论依据。方法通过生物信息学分析,初步筛选出Chin-01株5′UTR中可能的复制相关位点,并构建相应的重组亚基因组突变体,然后以上此为模板,经体外RdRp活性分析及凝胶阻滞试验,观察NS5的RdRp活性及与突变体的结合能力,再将突变位点引入该病毒感染性全长cDNA克隆,初步观察突变体恢复病毒的生物学特性。结果5′UTR中第45-60位核苷酸缺失的突变体无法合成子链,与NS5的结合能力也显著降低。第8和9位核苷酸改变的突变体则丧失了合成子代RNA及与NS5的结合能力,第8、9、69和70位核苷酸突变后形成的结构恢复突变体则可以合成子链RNA,并可与NS5特异性结合。同时,第45-60位核苷酸缺失及第8和9位核苷酸突变的恢复病毒的增殖能力也显著降低。结论Chin-01株5′UTR中的第45-60位及第8、9、69和70位核苷酸维持的结构可能是该病毒基因组复制的关键位点。

Object To study the functions of elements involved in viral replication in the 5＇ untranslated region （5＇UTR） of West Nile virus （WNV） strain Chin-01, so as to further clarify the molecular details of viral replication. Methods A bioinformatic analysis of 51 UTR of strain Chin-01 was performed and several nucleotides were selected as mutations introduced into the 5＇UTR of WNV. Then RdRp activity of NS5pol and NS5 using mutant 5＇UTRs as templates and capability of binding NS5 to mutant templates were observed by RdRp assay in vitro and EMSA, respectively. Finally, the mutations in elements involved in viral replication were introduced into the infectious full-len, gth cDNA clone of strain Chin-01. Then the biological characteristics of the mutant recovered virus were observed. Results Deletion of nucleotides 46--60 in 5＇UTR resulted in deficiency of RdRp activity of NS5pol and NS5F, while mutations at nucleotides 8 and 9 resulted in deficiency of RdRp activity and capability of binding to templates of two proteins using mutants containing 5＇UTR as templates. However, only the mutant with mutations at nucleotides 8, 9, 69 and 70 simultaneously was active for RNA synthesis and showed high affinity for NS5pol and NS5F. Furthermore, the recovered virus from the mutant cDNA clones with mutations at nucleotides 8 and 9 and the deletion of nucleotides 45--60 were almost completely defective replication. Conclusions The nucleotides 8, 9, 69 and 70, and nucleotides 45--60 in 5＇UTR of strain Chin-01 might play a key role in viral RNA replication.