Mutational analysis of the bovine respiratory syncytial virus nucleocapsid protein using a minigenome system: mutations that affect encapsidation, RNA synthesis, and interaction with the phosphoprotein.
The nucleocapsid (N) protein of bovine respiratory syncytial virus (BRSV) is a multifunctional protein that plays a central role in transcription and replication of viral genomic RNA. To investigate the domains and specific residues involved in different N activities, we generated a total of 27 deletion and 12 point mutants of the N protein. These mutants were characterized using an intracellular BRSV-CAT minigenome replication system for the ability to (1) direct minigenome RNA synthesis, (2) direct minigenome encapsidation, and (3) form a complex with the phosphoprotein (P). The mutations tested were defective in synthesis of RNA from the BRSV-CAT minigenome template with the exception of the following: a deletion involving the first N-terminal amino acid and mutations involving conservative substitution at the second amino acid and at certain internal cysteine residues. Micrococcal nuclease enzyme protection assays showed that mutations involving amino acids 1-364 of the 391-amino-acid N protein prevented minigenome encapsidation. Thus the BRSV N protein has a C-terminal, 27-amino-acid tail that is not required for encapsidation. Interestingly, two of the mutations that ablated encapsidation did not greatly affect RNA synthesis; the mutant involving deletion of the N-terminal amino acid and the mutant involving a substitution at position 2. This finding indicates that the formation of a nucleocapsid sufficient to protect the RNA from nuclease is not required for template function. Coimmunoprecipitation of N and P using N- or P-specific antiserum revealed two regions of the N protein that are important for association with the P protein: a central portion of 244-290 amino acids and a C-terminal portion of 338-364 amino acids.
Khattar
S K
SK
Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland 20742, USA.
Yunus
A S
AS
Collins
P L
PL
Samal
S K
SK
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D016679:Genome, Viral; D015707:HN Protein; D006801:Humans; D008836:Micrococcal Nuclease / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D019251:Nucleocapsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D016372:RNA, Antisense / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D018112:Respiratory Syncytial Virus, Bovine / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000378:metabolism; D019065:Virus Assembly / Q000235:genetics*
D015707:HN Protein; D010750:Phosphoproteins; D016372:RNA, Antisense; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C116204:attachment protein G; D008836:Micrococcal Nuclease; D003545:Cysteine