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Journal of Virology, November 2005, p. 13399-13411, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13399-13411.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The nsp2 Replicase Proteins of Murine Hepatitis Virus and Severe Acute Respiratory Syndrome Coronavirus Are Dispensable for Viral Replication

Rachel L. Graham,2,3 Amy C. Sims,4 Sarah M. Brockway,2,3 Ralph S. Baric,4 and Mark R. Denison1,2,3*

Departments of Pediatrics,1 Microbiology and Immunology,2 Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee,3 Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, North Carolina4

Received 21 June 2005/ Accepted 9 August 2005

The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHV{Delta}nsp2 and SARS{Delta}nsp2, respectively). Infectious MHV{Delta}nsp2 and SARS{Delta}nsp2 viruses recovered from electroporated cells had 0.5 to 1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The {Delta}nsp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHV{Delta}nsp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHV{Delta}nsp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function.

* Corresponding author. Mailing address: Department of Pediatrics, Vanderbilt University Medical Center, D6217 MCN, Nashville, TN 37232-2581. Phone: (615) 343-9881. Fax: (615) 343-9723. E-mail: mark.denison{at}vanderbilt.edu.

Journal of Virology, November 2005, p. 13399-13411, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13399-13411.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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