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

Abstract

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.

Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein

“…P is thought to be a connector between L and N. The identification of the sites of interactions between P and its partners thus constitutes a major interest not only for an understanding of the mechanisms of viral RNA synthesis but also for the development of antiviral drug design strategies. The mapping of the P binding site on the RSV N protein was previously investigated by several groups using different approaches (13,22,24,31,32). However, contradictory results were obtained from those studies, in which the nature of N (N-RNA or N°) was not defined.…”

“…For example, Krishnamurthy and Samal used a yeast two-hybrid screen coupled to an immunoprecipitation assay with HeLa cells coexpressing the BRSV P and N proteins and found that an N-P interaction was abolished by internal and C-terminal deletions of N (24). Using a similar approach, i.e., immunoprecipitation in HEp-2 cells coexpressing the BRSV P and truncated N proteins, Khattar et al (22) found that P binding required residues 244 to 290 and 338 to 364 but not C-terminal residues 365 to 391. In the same way, Garcia-Barreno et al (13) showed that the deletion of the last 39 amino acids of N still resulted in coimmunoprecipitation with P but inhibited the formation of IBs.…”

Mutations in the Fusion Protein Cleavage Site of Avian Paramyxovirus Serotype 2 Increase Cleavability and Syncytium Formation but Do Not Increase Viral Virulence in Chickens

“…Radioimmunoprecipitations were performed as described previously (16). Briefly, DF1 cells were infected with parental or mutant APMV-2 at an MOI of 10 for 12 h at 37°C, after which time the cells were switched to methionine-and cysteine-free medium (starvation medium).…”

The 24-Angstrom Structure of Respiratory Syncytial Virus Nucleocapsid Protein-RNA Decameric Rings

“…Domains of N responsible for interactions with associated cofactors have been mapped for several members of Mononegavirales, including the rhabdovirus rabies virus (RV) (17), Sendai virus (SeV) (23), measles virus (MeV), (both members of the Paramyxovirinae [3]), and RSV (13,16,22). Although differences exist among the viruses, there is an overall homology in modular components of the N proteins: the amino terminus of N is largely responsible for the assembly of the nucleocapsid, while the carboxyl terminus contains elements that are necessary for the interaction with the polymerase and M protein (22).…”