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Journal of Virology, November 2005, p. 13285-13297, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13285-13297.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Major Determinant for Membrane Protein Interaction Localizes to the Carboxy-Terminal Domain of the Mouse Coronavirus Nucleocapsid Protein

Kelley R. Hurst, Lili Kuo, Cheri A. Koetzner, Rong Ye, Bilan Hsue,{dagger} and Paul S. Masters*

Wadsworth Center, New York State Department of Health, Albany, New York 12201

Received 6 June 2005/ Accepted 3 August 2005

The two major constituents of coronavirus virions are the membrane (M) and nucleocapsid (N) proteins. The M protein is anchored in the viral envelope by three transmembrane segments flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. The M endodomain interacts with the viral nucleocapsid, which consists of the positive-strand RNA genome helically encapsidated by N protein monomers. In previous work with the coronavirus mouse hepatitis virus (MHV), a highly defective M protein mutant, M{Delta}2, was constructed. This mutant contained a 2-amino-acid carboxy-terminal truncation of the M protein. Analysis of second-site revertants of M{Delta}2 revealed mutations in the carboxy-terminal region of the N protein that compensated for the defect in the M protein. To seek further genetic evidence corroborating this interaction, we generated a comprehensive set of clustered charged-to-alanine mutants in the carboxy-terminal domain 3 of N protein. One of these mutants, CCA4, had a highly defective phenotype similar to that of M{Delta}2. Transfer of the CCA4 mutation into a partially diploid MHV genome showed that CCA4 was a loss-of-function mutation rather than a dominant-negative mutation. Analysis of multiple second-site revertants of CCA4 revealed mutations in both the M protein and the N protein that could compensate for the original lesion in N. These data more precisely define the region of the N protein that interacts with the M protein. Further, we found that fusion of domain 3 of the N protein to the carboxy terminus of a heterologous protein caused it to be incorporated into MHV virions.

* Corresponding author. Mailing address: David Axelrod Institute, Wadsworth Center, NYSDOH, New Scotland Avenue, P.O. Box 22002, Albany, NY 12201-2002. Phone: (518) 474-1283. Fax: (518) 473-1326. E-mail: masters{at}wadsworth.org.

{dagger} Present address: Stratagene, 11011 N. Torrey Pines Rd., La Jolla, CA 92037.

Journal of Virology, November 2005, p. 13285-13297, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13285-13297.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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