title: Characterization of a critical interaction between the coronavirus nucleocapsid protein and nonstructural protein 3 of the viral replicase-transcriptase complex.
abstract: The coronavirus nucleocapsid protein (N) plays an essential structural role in virions through a network of interactions with positive-strand viral genomic RNA, the envelope membrane protein (M), and other N molecules. Additionally, N protein participates in at least one stage of the complex mechanism of coronavirus RNA synthesis. We previously uncovered an unanticipated interaction between N and the largest subunit of the viral replicase-transcriptase complex, nonstructural protein 3 (nsp3). This was found through analysis of revertants of a severely defective mutant of murine hepatitis virus (MHV) in which the N gene was replaced with that of its close relative, bovine coronavirus (BCoV). In the work reported here, we constructed BCoV chimeras and other mutants of MHV nsp3 and obtained complementary genetic evidence for its association with N protein. We found that the N-nsp3 interaction maps to the amino-terminal ubiquitin-like domain of nsp3, which is essential for the virus. The interaction does not require the adjacent acidic domain of nsp3, which is dispensable. In addition, we demonstrated a complete correspondence between N-nsp3 genetic interactions and the ability of N protein to enhance the infectivity of transfected coronavirus genomic RNA. The latter function of N was shown to depend on both of the RNA-binding domains of N, as well as on the serine- and arginine-rich central region of N, which binds nsp3. Our results support a model in which the N-nsp3 interaction serves to tether the genome to the newly translated replicase-transcriptase complex at a very early stage of infection.
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA.
authors.forename: Kelley R
authors.initials: KR
authors.lastname: Hurst
authors.forename: Cheri A
authors.initials: CA
authors.lastname: Koetzner
authors.forename: Paul S
authors.initials: PS
authors.lastname: Masters
mesh_terms: D000818:Animals; D002460:Cell Line; D017938:Coronavirus, Bovine / Q000235:genetics / Q000502:physiology*; D051379:Mice; D006517:Murine hepatitis virus / Q000235:genetics / Q000502:physiology*; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping; D011995:Recombination, Genetic; D019076:Transgenes; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D019590:Nucleocapsid Proteins; D017361:Viral Nonstructural Proteins; C089383:nsp3 protein, alphavirus