Mutational analysis of the bovine respiratory syncytial virus nucleocapsidprotein 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) i s a multifunctional protein that plays a central role in transcription and replication of viral genomic RNA. To investigate the domains and specific r esidues involved in different N activities, we generated a total of 27 dele tion and 12 point mutants of the N protein. These mutants were characterize d using an intracellular BRSV-CAT minigenome replication system for the abi lity to (1) direct minigenome RNA synthesis, (2) direct minigenome encapsid ation, and (3) form a complex with the phosphoprotein (P). The mutations te sted were defective in synthesis of RNA from the BRSV-CAT minigenome templa te with the exception of the following: a deletion involving the first N-te rminal amino acid and mutations involving conservative substitution at the second amino acid and at certain internal cysteine residues. Micrococcal nu clease 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 re quired for encapsidation. Interestingly, two of the mutations that ablated encapsidation did not greatly affect RNA synthesis; the mutant involving de letion 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 f unction. Coimmunoprecipitation of N and P using N- or P-specific antiserum revealed two regions of the N protein that are important for association wi th the P protein: a central portion of 244-290 amino acids and a C-terminal portion of 338-364 amino acids. (C) 2000 Academic Press.