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Journal of Virology, December 2006, p. 11610-11620, Vol. 80, No. 23
0022-538X/06/$08.00+0     doi:10.1128/JVI.01428-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Replication of Murine Hepatitis Virus Is Regulated by Papain-Like Proteinase 1 Processing of Nonstructural Proteins 1, 2, and 3{triangledown}

Rachel L. Graham2,3 and Mark R. Denison1,2,3*

Departments of Pediatrics,1 Microbiology and Immunology,2 The Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee3

Received 6 July 2006/ Accepted 4 September 2006

Coronaviruses are positive-strand RNA viruses that translate their genome RNA into polyproteins that are co- and posttranslationally processed into intermediate and mature replicase nonstructural proteins (nsps). In murine hepatitis virus (MHV), nsps 1, 2, and 3 are processed by two papain-like proteinase activities within nsp3 (PLP1 and PLP2) to yield nsp1, an nsp2-3 intermediate, and mature nsp2 and nsp3. To determine the role in replication of processing between nsp2 and nsp3 at cleavage site 2 (CS2) and PLP1 proteinase activity, mutations were engineered into the MHV genome at CS2, at CS1 and CS2, and at the PLP1 catalytic site, alone and in combination. Mutant viruses with abolished cleavage at CS2 were delayed in growth and RNA synthesis but grew to wild-type titers of >107 PFU/ml. Mutant viruses with deletion of both CS1 and CS2 exhibited both a delay in growth and a decrease in peak viral titer to ~104 PFU/ml. Inactivation of PLP1 catalytic residues resulted in a mutant virus that did not process at either CS1 or CS2 and was severely debilitated in growth, achieving only 102 PFU/ml. However, when both CS1 and CS2 were deleted in the presence of inactivated PLP1, the growth of the resulting mutant virus was partially compensated, comparable to that of the CS1 and CS2 deletion mutant. These results demonstrate that interactions of PLP1 with CS1 and CS2 are critical for protein processing and suggest that the interactions play specific roles in regulation of the functions of nsp1, 2, and 3 in viral RNA synthesis.

* Corresponding author. Mailing address: Department of Pediatrics, Vanderbilt University Medical Center, D6217 MCN, 1161 21st Ave. S., Nashville, TN 37232-2581. Phone: (615) 343-9881. Fax: (615) 343-9723. E-mail: mark.denison{at}vanderbilt.edu.

{triangledown} Published ahead of print on 13 September 2006.

Journal of Virology, December 2006, p. 11610-11620, Vol. 80, No. 23
0022-538X/06/$08.00+0     doi:10.1128/JVI.01428-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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