This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Deming, D. J.
Right arrow Articles by Baric, R. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Deming, D. J.
Right arrow Articles by Baric, R. S.

 Previous Article  |  Next Article 

Journal of Virology, October 2007, p. 10280-10291, Vol. 81, No. 19
0022-538X/07/$08.00+0     doi:10.1128/JVI.00017-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Processing of Open Reading Frame 1a Replicase Proteins nsp7 to nsp10 in Murine Hepatitis Virus Strain A59 Replication{triangledown}

Damon J. Deming,1 Rachel L. Graham,2 Mark R. Denison,3 and Ralph S. Baric1,4*

Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina,1 Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee,2 Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee,3 Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, North Carolina4

Received 3 January 2007/ Accepted 9 July 2007

Coronaviruses express open reading frame 1a (ORF1a) and ORF1b polyproteins from which 16 nonstructural proteins (nsp) are derived. The highly conserved region at the carboxy terminus of ORF1a is processed by the nsp5 proteinase (Mpro) into mature products, including nsp7, nsp8, nsp9, and nsp10, proteins with predicted or identified activities involved in RNA synthesis. Although continuous translation and proteolytic processing of ORF1ab by Mpro is required for replication, it is unknown whether specific cleavage events within the polyprotein are dispensable. We determined the requirement for the nsp7 to nsp10 proteins and their processing during murine hepatitis virus (MHV) replication. Through use of an MHV reverse genetics system, in-frame deletions of the coding sequences for nsp7 to nsp10, or ablation of their flanking Mpro cleavage sites, were made and the effects upon replication were determined. Viable viruses were characterized by analysis of Mpro processing, RNA transcription, and growth fitness. Deletion of any of the regions encoding nsp7 to nsp10 was lethal. Disruption of the cleavage sites was lethal with the exception of that of the nsp9-nsp10 site, which resulted in a mutant virus with attenuated replication. Passage of the attenuated nsp9-nsp10 cleavage mutant increased fitness to near-wild-type kinetics without reversion to a virus capable of processing nsp9-nsp10. We also confirmed the presence of a second cleavage site between nsp7 and nsp8. In order to determine whether a distinct function could be attributed to preprocessed forms of the polyprotein, including nsp7 to nsp10, the genes encoding nsp7 and nsp8 were rearranged. The mutant virus was not viable, suggesting that the uncleaved protein may be essential for replication or proteolytic processing.

* Corresponding author. Mailing address: School of Public Health, Department of Epidemiology, Hooker Research Center, CB, University of North Carolina, Chapel Hill, NC 27599. Phone: (919) 966-3895. Fax: (919) 966-0584. E-mail: rbaric{at}email.unc.edu

{triangledown} Published ahead of print on 18 July 2007.

Journal of Virology, October 2007, p. 10280-10291, Vol. 81, No. 19
0022-538X/07/$08.00+0     doi:10.1128/JVI.00017-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»