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Journal of Virology, June 2007, p. 6356-6368, Vol. 81, No. 12
0022-538X/07/$08.00+0     doi:10.1128/JVI.02805-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Murine Hepatitis Virus Replicase Protein nsp10 Is a Critical Regulator of Viral RNA Synthesis{triangledown}

Eric F. Donaldson,1 Amy C. Sims,2 Rachel L. Graham,4,5 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 19 December 2006/ Accepted 21 March 2007

Coronavirus replication requires proteolytic processing of the large polyprotein encoded by ORF1a/ab into putative functional intermediates and eventually ~15 mature proteins. The C-terminal ORF1a protein nsp10 colocalizes with viral replication complexes, but its role in transcription/replication is not well defined. To investigate the role of nsp10 in coronavirus transcription/replication, alanine replacements were engineered into a murine hepatitis virus (MHV) infectious clone in place of conserved residues in predicted functional domains or charged amino acid pairs/triplets, and rescued viruses were analyzed for mutant phenotypes. Of the 16 engineered clones, 5 viable viruses were rescued, 3 mutant viruses generated no cytopathic effect but were competent to synthesize viral subgenomic RNAs, and 8 were not viable. All viable mutants showed reductions in growth kinetics and overall viral RNA synthesis, implicating nsp10 as being a cofactor in positive- or negative-strand synthesis. Viable mutant nsp10-E2 was compromised in its ability to process the nascent polyprotein, as processing intermediates were detected in cells infected with this virus that were not detectable in wild-type infections. Mapping the mutations onto the crystal structure of severe acute respiratory syndrome virus nsp10 identified a central core resistant to mutation. Mutations targeting residues in or near either zinc-binding finger generated nonviable phenotypes, demonstrating that both domains are essential to nsp10 function and MHV replication. All mutations resulting in viable phenotypes mapped to loops outside the central core and were characterized by a global decrease in RNA synthesis. These results demonstrate that nsp10 is a critical regulator of coronavirus RNA synthesis and may play an important role in polyprotein processing.

* Corresponding author. Mailing address: 2107 MacGavran-Greenberg, CB#7435, University of North Carolina, Chapel Hill, 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 28 March 2007.

Journal of Virology, June 2007, p. 6356-6368, Vol. 81, No. 12
0022-538X/07/$08.00+0     doi:10.1128/JVI.02805-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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