An interaction between the nucleocapsid protein and a component of the replicase-transcriptase complex is crucial for the infectivity of coronavirus genomic RNA.
The coronavirus nucleocapsid (N) protein plays an essential role in virion assembly via interactions with the large, positive-strand RNA viral genome and the carboxy-terminal endodomain of the membrane protein (M). To learn about the functions of N protein domains in the coronavirus mouse hepatitis virus (MHV), we replaced the MHV N gene with its counterpart from the closely related bovine coronavirus (BCoV). The resulting viral mutant was severely defective, even though individual domains of the N protein responsible for N-RNA, N-M, or N-N interactions were completely interchangeable between BCoV and MHV. The lesion in the BCoV N substitution mutant could be compensated for by reverting mutations in the central, serine- and arginine-rich (SR) domain of the N protein. Surprisingly, a second class of reverting mutations were mapped to the amino terminus of a replicase subunit, nonstructural protein 3 (nsp3). A similarly defective MHV N mutant bearing an insertion of the SR region from the severe acute respiratory syndrome coronavirus N protein was rescued by the same two classes of reverting mutations. Our genetic results were corroborated by the demonstration that the expressed amino-terminal segment of nsp3 bound selectively to N protein from infected cells, and this interaction was RNA independent. Moreover, we found a direct correlation between the N-nsp3 interaction and the ability of N protein to stimulate the infectivity of transfected MHV genomic RNA (gRNA). Our results suggest a role for this previously unknown N-nsp3 interaction in the localization of genomic RNA to the replicase complex at an early stage of infection.
Hurst
Kelley R
KR
Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.
Ye
Rong
R
Goebel
Scott J
SJ
Jayaraman
Priya
P
Masters
Paul S
PS
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D000086462:Coronavirus Nucleocapsid Proteins; D017938:Coronavirus, Bovine / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000502:physiology*; D016679:Genome, Viral; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D009154:Mutation; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000502:physiology*; D011995:Recombination, Genetic; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014774:Virulence / Q000235:genetics / Q000502:physiology
D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases