A major determinant for membrane protein interaction localizes to the carboxy-terminal domain of the mouse coronavirus nucleocapsid protein.
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, MDelta2, was constructed. This mutant contained a 2-amino-acid carboxy-terminal truncation of the M protein. Analysis of second-site revertants of MDelta2 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 MDelta2. 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.
Hurst
Kelley R
KR
David Axelrod Institute, Wadsworth Center, NYSDOH, New Scotland Avenue, P.O. Box 22002, Albany, NY 12201-2002, USA.
Kuo
Lili
L
Koetzner
Cheri A
CA
Ye
Rong
R
Hsue
Bilan
B
Masters
Paul S
PS
D000818:Animals; D002460:Cell Line; D000086862:Coronavirus M Proteins; D006517:Murine hepatitis virus / Q000378:metabolism / Q000502:physiology*; D009154:Mutation; D019251:Nucleocapsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019590:Nucleocapsid Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D014763:Viral Matrix Proteins / Q000378:metabolism*; D019065:Virus Assembly
D000086862:Coronavirus M Proteins; D019590:Nucleocapsid Proteins; D014763:Viral Matrix Proteins; C099600:nucleocapsid protein, Hepatitis virus