Identification of in vivo-interacting domains of the murine coronavirus nucleocapsid protein.
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.
Hurst
Kelley R
KR
New York State Department of Health, David Axelrod Institute, Wadsworth Center, Albany, New York 12201-2002, USA.
Koetzner
Cheri A
CA
Masters
Paul S
PS
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000821:virology; D018352:Coronavirus Infections / Q000378:metabolism* / Q000662:veterinary* / Q000821:virology; D000086462:Coronavirus Nucleocapsid Proteins; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012376:Rodent Diseases / Q000378:metabolism* / Q000821:virology; D019065:Virus Assembly
D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins