Rabies virus N, P and RNA interactions in vivo and mapping the functional domains of N in N-P and N-N interactions
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Rabies virus (RV) nucleoprotein (N) is a multifunctional protein that plays a central role in viral RNA transcription and replication. Its functions require interactions with itself and with other viral components. In vitro studies indicated that rabies virus phosphoprotein (P), by interacting with the nucleoprotein (N), confers the specificity of genomic RNA encapsidation by N. In this study, the interactions among N, P, and the RNA was examined in virus-infected cells as well as in transfected cells. When N is expressed alone, it binds to non-specific RNA, particularly the N mRNA. When N and P are co-expressed, the N and P form N-P complexes that do not bind to any RNA. When the N and P are co-expressed together with the minigenomic RNA, the N-P complexes preferentially bind to the mini-genomic RNA. This demonstrated that indeed RV P, by binding to N, confers the specificity of genomic RNA encapsidation by N in vivo. Furthermore, we investigated the role of N phosphorylation in the N, P, and RNA interactions. It was found that only the N that bound to RNA was phosphorylated while the 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 the de novo-synthesized N. However, mutation at the phosphorylation site of the N did not alter the pattern of N-P and NRNA interactions, indicating that N phosphorylation per se does not play a direct role in the N-P interaction and RNA encapsidation. Nevertheless, the fact that N is not phosphorylated prior to RNA encapsidation may suggest that RV P, by binding to N, keeps the N in a unique conformation for specific encapsidation of the genomic RNA. In order to understand the detailed N-P and N-N interactions, a series of terminal and internal deletion mutants of the N were constructed and attempts were made to map the domains on N that are involved in these interactions. It was determined that a central region of the N from amino acids 150 to 285 and a C-terminal region of 360-420 are important for the N-P interactions. Deletion of these regions abolished its ability to bind to P. For self aggregation, amino acids 25 to 30 are required because deletion of the first 25 amino acids did not affect the N-N interactions. However, deletion of the first 30 amino acids completely abolished the ability of N to self-aggregate. However, none of these mutants supported the transcription and/or replication of the minigenomic RNA. Together these data suggest that although different domains on the N are involved in N-N and N-P interactions, the full-length N is absolutely required for optimal viral transcription and replication.