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Journal of Virology, July 2007, p. 7086-7098, Vol. 81, No. 13
0022-538X/07/$08.00+0     doi:10.1128/JVI.00049-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Analysis of Murine Hepatitis Virus Strain A59 Temperature-Sensitive Mutant TS-LA6 Suggests that nsp10 Plays a Critical Role in Polyprotein Processing{triangledown}

Eric F. Donaldson,1 Rachel L. Graham,4,5 Amy C. Sims,2 Mark R. Denison,3,4,5 and Ralph S. Baric1,2*

Department of Microbiology and Immunology, School of Medicine,1 Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, North Carolina,2 Departments of Pediatrics,3 Microbiology and Immunology,4 The Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee5

Received 8 January 2007/ Accepted 5 April 2007

Coronaviruses are the largest RNA viruses, and their genomes encode replication machinery capable of efficient replication of both positive- and negative-strand viral RNAs as well as enzymes capable of processing large viral polyproteins into putative replication intermediates and mature proteins. A model described recently by Sawicki et al. (S. G. Sawicki, D. L. Sawicki, D. Younker, Y. Meyer, V. Thiel, H. Stokes, and S. G. Siddell, PLoS Pathog. 1:e39, 2005), based upon complementation studies of known temperature-sensitive (TS) mutants of murine hepatitis virus (MHV) strain A59, proposes that an intermediate comprised of nsp4 to nsp10/11 (~150 kDa) is involved in negative-strand synthesis. Furthermore, the mature forms of nsp4 to nsp10 are thought to serve as cofactors with other replicase proteins to assemble a larger replication complex specifically formed to transcribe positive-strand RNAs. In this study, we introduced a single-amino-acid change (nsp10:Q65E) associated with the TS-LA6 phenotype into nsp10 of the infectious clone of MHV. Growth kinetic studies demonstrated that this mutation was sufficient to generate the TS phenotype at permissive and nonpermissive temperatures. Our results demonstrate that the TS mutant variant of nsp10 inhibits the main protease, 3CLpro, blocking its function completely at the nonpermissive temperature. These results implicate nsp10 as being a critical factor in the activation of 3CLpro function. We discuss how these findings challenge the current hypothesis that nsp4 to nsp10/11 functions as a single cistron in negative-strand RNA synthesis and analyze recent complementation data in light of these new findings.

* Corresponding author. Mailing address: Department of Epidemiology, School of Public Health, University of North Carolina, 2107 MacGavran-Greenberg, CB#7435, 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 11 April 2007.

Journal of Virology, July 2007, p. 7086-7098, Vol. 81, No. 13
0022-538X/07/$08.00+0     doi:10.1128/JVI.00049-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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