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Volume 26 Issue 1
February 2011

    Guo-hui Chang, Bao-jun Luo, Pin Lu, Lei Lin, Xiao-yan Wu, Jing Li, Yi Hu and Qing-yu Zhu. Construction and Genetic Analysis of Murine Hepatitis Virus Strain A59 Nsp16 Temperature Sensitive Mutant and the Revertant Virus[J]. Virologica Sinica, 2011, 26(1): 19-29. doi: 10.1007/s12250-011-3145-x
    Citation: Guo-hui Chang, Bao-jun Luo, Pin Lu, Lei Lin, Xiao-yan Wu, Jing Li, Yi Hu, Qing-yu Zhu. Construction and Genetic Analysis of Murine Hepatitis Virus Strain A59 Nsp16 Temperature Sensitive Mutant and the Revertant Virus .VIROLOGICA SINICA, 2011, 26(1) : 19-29.  http://dx.doi.org/10.1007/s12250-011-3145-x
    shu

    鼠肝炎病毒株MHV-A59温度敏感突变体及其回复突变体的构建及遗传分析

    • 1.

      军事医学科学院微生物流行病研究所, 病原微生物生物安全国家重点实验室, 北京市丰台区东大街20号, 北京100071;2长春长生生物科技有限公司，长春市跃达路1615号, 吉林长春 130103

    • 冠状病毒是一种重要的病原体，主要引起人和畜类感染。SARS冠状病毒的出现再次引起了人们对冠状病毒复制及致病机理研究的关注。鼠肝炎病毒株MHV-A59是一个用来研究冠状病毒结构蛋白功能及病毒复制的良好模型系统。本研究利用以痘苗病毒为载体的反向遗传学系统，通过定点突变和多轮重组筛选，构建了野生型MHV-A59的温度敏感突变株Wu”-ts18(cd)，该突变株对MHV-A59非结构蛋白16（Nsp16）的第12位氨基酸的三个碱基发生了突变，导致该位点氨基酸发生与报道的Wu”-ts18温度敏感突变株相同的氨基酸突变，但降低了在该位点产生回复突变的可能性。在不同培养温度的病毒蚀斑和复制分析结果表明，所构建的温度敏感突变体Wu”-ts18(cd)与报道的Wu”-ts18具有相同的复制特性和温度敏感表型。通过在非容许温度培养Wu”-ts18(cd)，迫使其产生在其它位点发生突变的回复突变体。对多株Wu”-ts18(cd)的回复突变体序列分析结果表明，多数回复突变体在Nsp16的43位氨基酸发生了单个氨基酸位点的突变, 在Nsp16的76位和130位的单个氨基酸突变也可使温度敏感突变株Wu”-ts18(cd)回复为野生型。回复突变体R8和R9在非容许温度39.5°C可形成大小不同的蚀斑，全序列测定结果表明Nsp13的115位氨基酸差异影响了蚀斑的大小。本研究建立了利用冠状病毒温度敏感突变体及其回复突变体研究非结构蛋白功能方法，并确定了影响Nsp16蛋白功能的几个重要氨基酸位点,有利于进一步了解冠状病毒Nsp16蛋白在病毒复制过程中的功能。

    Construction and Genetic Analysis of Murine Hepatitis Virus Strain A59 Nsp16 Temperature Sensitive Mutant and the Revertant Virus

    • 1.

      State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China

    • 2.

      Changchun Changsheng Life Sciences Limited, Changchun 130103, China

    • Corresponding author: Guo-hui Chang, changguohui999@yahoo.com.cn
    • Received Date: 23 April 2010
      Accepted Date: 19 November 2010

      Fund Project: Research Grants from State Key Laboratory of Pathogen and Biosecurity SKLPBS0918

    • Coronaviruses (CoVs) are generally associated with respiratory and enteric infections and have long been recognized as important pathogens of livestock and companion animals. Mouse hepatitis virus (MHV) is a widely studied model system for Coronavirus replication and pathogenesis. In this study, we created a MHV-A59 temperature sensitive (ts) mutant Wu”-ts18(cd) using the recombinant vaccinia reverse genetics system. Virus replication assay in 17C1-1 cells showed the plaque phenotype and replication characterization of constructed Wu”-ts18(cd) were indistinguishable from the reported ts mutant Wu”-ts18. Then we cultured the ts mutant Wu”-ts18(cd) at non-permissive temperature 39.5°C, which “forced” the ts recombinant virus to use second-site mutation to revert from a ts to a non-ts phenotype. Sequence analysis showed most of the revertants had the same single amino acid mutation at Nsp16 position 43. The single amino acid mutation at Nsp16 position 76 or position 130 could also revert the ts mutant Wu”-ts18 (cd) to non-ts phenotype, an additional independent mutation in Nsp13 position 115 played an important role on plaque size. The results provided us with genetic information on the functional determinants of Nsp16. This allowed us to build up a more reasonable model of CoVs replication-transcription complex.
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      Construction and Genetic Analysis of Murine Hepatitis Virus Strain A59 Nsp16 Temperature Sensitive Mutant and the Revertant Virus

        Corresponding author: Guo-hui Chang, changguohui999@yahoo.com.cn
      • 1. State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China
      • 2. Changchun Changsheng Life Sciences Limited, Changchun 130103, China
      • Received Date: 23 April 2010
      • Accepted Date: 19 November 2010
      Fund Project:  Research Grants from State Key Laboratory of Pathogen and Biosecurity SKLPBS0918

      Abstract: Coronaviruses (CoVs) are generally associated with respiratory and enteric infections and have long been recognized as important pathogens of livestock and companion animals. Mouse hepatitis virus (MHV) is a widely studied model system for Coronavirus replication and pathogenesis. In this study, we created a MHV-A59 temperature sensitive (ts) mutant Wu”-ts18(cd) using the recombinant vaccinia reverse genetics system. Virus replication assay in 17C1-1 cells showed the plaque phenotype and replication characterization of constructed Wu”-ts18(cd) were indistinguishable from the reported ts mutant Wu”-ts18. Then we cultured the ts mutant Wu”-ts18(cd) at non-permissive temperature 39.5°C, which “forced” the ts recombinant virus to use second-site mutation to revert from a ts to a non-ts phenotype. Sequence analysis showed most of the revertants had the same single amino acid mutation at Nsp16 position 43. The single amino acid mutation at Nsp16 position 76 or position 130 could also revert the ts mutant Wu”-ts18 (cd) to non-ts phenotype, an additional independent mutation in Nsp13 position 115 played an important role on plaque size. The results provided us with genetic information on the functional determinants of Nsp16. This allowed us to build up a more reasonable model of CoVs replication-transcription complex.

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