Localisation of the RNA-Binding Domain on the Respiratory Syncytial Virus Nucleocapsid Protein

The purpose of the study presented herein was to investigate the RNA-binding properties of the nucleocapsid (N) protein of respiratory syncytial virus (RSV). The position of the RNA binding-site within the protein was investigated. In addition, the ability of N protein to assemble into nucleocapsid-like structures was studied, thus determining whether these facets were distinguishable. The sequence specificity of RNA-binding by the N protein was investigated using several RNA probes, one of which represented the RSV leader sequence. Various in vitro assays, such as the yeast three-hybrid system, gel mobility shift assay and northwestern blots, were employed to this end. In the absence of other viral proteins, N possessed no sequence preference in the manner of its association with RNA. The locale of the RNA-binding site within the N protein was addressed by employing two methodologies. The first approach employed a UV crosslinking assay with subsequent analysis by mass spectrometry. A single N-derived peptide was identified within the carboxy-domain of N (amino acids 352 to 358) as being potentially modified by RNA-binding. Secondly, the use of carboxy-terminal deletion mutants of the N protein demonstrated the presence of an RNA-binding domain within the first 92 amino acids of the N protein. The two individual binding regions identified demonstrate the conformational nature of the RNA-binding domain. However, the role of the carboxy terminus was shown to be minimal, as deletion of this region did not prevent RNA- binding. Concurrent with the binding of RNA is the formation of nucleocapsids, helical structures formed from N proteins encapsulating the viral genomic RNA. The ability of recombinant N to assemble into such structures was investigated by EM. Histidine-tag N demonstrated the capacity to form nucleocapsid-like structures and N:RNA rings, the latter representing a single turn of the nucleocapsid helix. The rings were shown to consist of 10 N protein monomers, a situation different from Sendai virus (13 monomers) and more similar to that of rabies virus (10 monomers). A mutant representing amino acids 1 to 92 of the RSV N protein formed RNase-resistant structures that were nucleocapsid-like in appearance. This suggests that characteristics of RNA-binding and nucleocapsid assembly are situated within the same domain. A potential map of the domain structure of RSV N protein, derived from this work and from studies on-going in the laboratory, is presented