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Journal of Virology, July 2009, p. 7221-7234, Vol. 83, No. 14
0022-538X/09/$08.00+0     doi:10.1128/JVI.00440-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of In Vivo-Interacting Domains of the Murine Coronavirus Nucleocapsid Protein {triangledown}

Kelley R. Hurst, Cheri A. Koetzner, and Paul S. Masters*

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

Received 2 March 2009/ Accepted 27 April 2009

The coronavirus nucleocapsid protein (N), together with the large, positive-strand RNA viral genome, forms a helically symmetric nucleocapsid. This ribonucleoprotein structure becomes packaged into virions through association with the carboxy-terminal endodomain of the membrane protein (M), which is the principal constituent of the virion envelope. Previous work with the prototype coronavirus mouse hepatitis virus (MHV) has shown that a major determinant of the N-M interaction maps to the carboxy-terminal domain 3 of the N protein. To explore other domain interactions of the MHV N protein, we expressed a series of segments of the MHV N protein as fusions with green fluorescent protein (GFP) during the course of viral infection. We found that two of these GFP-N-domain fusion proteins were selectively packaged into virions as the result of tight binding to the N protein in the viral nucleocapsid, in a manner that did not involve association with either M protein or RNA. The nature of each type of binding was further explored through genetic analysis. Our results defined two strongly interacting regions of the N protein. One is the same domain 3 that is critical for M protein recognition during assembly. The other is domain N1b, which corresponds to the N-terminal domain that has been structurally characterized in detail for two other coronaviruses, infectious bronchitis virus and the severe acute respiratory syndrome coronavirus.

* 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

{triangledown} Published ahead of print on 6 May 2009.

Journal of Virology, July 2009, p. 7221-7234, Vol. 83, No. 14
0022-538X/09/$08.00+0     doi:10.1128/JVI.00440-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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