Interactions amongst rabies virus nucleoprotein, phosphoprotein and genomic RNA in virus-infected and transfected cells

Previous in vitro studies have indicated that rabies virus (RV) phosphoprotein (P), by interacting with the nucleoprotein (N), confers the specificity of genomic RNA encapsidation by N. In this study, interactions amongst N, P and the genomic RNA in virus-infected as well as in transfected cells were studied. The results showed that when N was expressed alone, it bound non-specific RNA, particularly the N mRNA. When N and P were co-expressed, they formed N-P complexes that did not bind to non-specific RNA. When N and P were co-expressed together with (mini-)genomic RNA, N-P complexes preferentially bound the (mini-)genomic RNA. This demonstrated that RV P, by binding to N, does indeed confer specificity of genomic RNA encapsidation by N in vivo. Furthermore, the role of N phosphorylation in the N, P and RNA interactions was investigated. It was found that only N that bound to RNA was phosphorylated, while N in the N-P complex prior to RNA encapsidation was not, suggesting that RV P, by binding to nascent N, prevents the immediate phosphorylation of de novo-synthesized N. However, mutation at the phosphorylation site of N did not alter the pattern of N-P and N-RNA interactions, indicating that N phosphorylation per se does not play a direct role in N-P interaction and RNA encapsidation.