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.
New York State Department of Health, David Axelrod Institute, Wadsworth Center, Albany, New York 12201-2002, USA.
Kelley R
KR
Hurst
Cheri A
CA
Koetzner
Paul S
PS
Masters
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