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Construction and genetic analysis of murine hepatitis virus strain A59 Nsp16 temperature sensitive mutant and the revertant virus
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...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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SP Wuhan Institute of Virology, CAS
2011
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7091325/ https://www.ncbi.nlm.nih.gov/pubmed/21331887 http://dx.doi.org/10.1007/s12250-011-3145-x |
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author | Chang, Guo-hui Luo, Bao-jun Lu, Pin Lin, Lei Wu, Xiao-yan Li, Jing Hu, Yi Zhu, Qing-yu |
author_facet | Chang, Guo-hui Luo, Bao-jun Lu, Pin Lin, Lei Wu, Xiao-yan Li, Jing Hu, Yi Zhu, Qing-yu |
author_sort | Chang, Guo-hui |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7091325 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | SP Wuhan Institute of Virology, CAS |
record_format | MEDLINE/PubMed |
spelling | pubmed-70913252020-03-24 Construction and genetic analysis of murine hepatitis virus strain A59 Nsp16 temperature sensitive mutant and the revertant virus Chang, Guo-hui Luo, Bao-jun Lu, Pin Lin, Lei Wu, Xiao-yan Li, Jing Hu, Yi Zhu, Qing-yu Virol Sin Article 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. SP Wuhan Institute of Virology, CAS 2011-02-18 /pmc/articles/PMC7091325/ /pubmed/21331887 http://dx.doi.org/10.1007/s12250-011-3145-x Text en © Wuhan Institute of Virology, CAS and Springer-Verlag Berlin Heidelberg 2011 |
spellingShingle | Article Chang, Guo-hui Luo, Bao-jun Lu, Pin Lin, Lei Wu, Xiao-yan Li, Jing Hu, Yi Zhu, Qing-yu Construction and genetic analysis of murine hepatitis virus strain A59 Nsp16 temperature sensitive mutant and the revertant virus |
title | Construction and genetic analysis of murine hepatitis virus strain A59 Nsp16 temperature sensitive mutant and the revertant virus |
title_full | Construction and genetic analysis of murine hepatitis virus strain A59 Nsp16 temperature sensitive mutant and the revertant virus |
title_fullStr | Construction and genetic analysis of murine hepatitis virus strain A59 Nsp16 temperature sensitive mutant and the revertant virus |
title_full_unstemmed | Construction and genetic analysis of murine hepatitis virus strain A59 Nsp16 temperature sensitive mutant and the revertant virus |
title_short | Construction and genetic analysis of murine hepatitis virus strain A59 Nsp16 temperature sensitive mutant and the revertant virus |
title_sort | construction and genetic analysis of murine hepatitis virus strain a59 nsp16 temperature sensitive mutant and the revertant virus |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7091325/ https://www.ncbi.nlm.nih.gov/pubmed/21331887 http://dx.doi.org/10.1007/s12250-011-3145-x |
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