Oligomerization of the influenza virus nucleoprotein: Identification of positive and negative sequence elements

The RNA genome of influenza virus is encapsidated by the virus nucleoprotei n (NP) to form ribonucleoprotein (RNP) structures of defined morphology. Th ese structures result from the ability of NP to oligomerise and to bind sin gle-strand RNA. To characterise NP oligomerization, we developed a binding assay using immobilised NP fusion proteins and in vitro translated NP. This system was used to estimate a dissociation constant for NP-NP contacts of 2 x 10 M-7. Analysis of NP deletion mutants identified three sequence eleme nts important for oligomerization. Two regions corresponding to the middle and C-terminal thirds of the polypeptide were identified as the minimal seq uences capable of promoting NP-NP contacts. However, the C-terminal 23 amin o-acids of NP inhibited oligomerization, as their removal increased self-as sociation 10-fold. Single codon changes identified amino acids important fo r the function of these regions. Alanine substitution of R199 decreased bin ding affinity threefold, whereas alteration of R416 had a more drastic effe ct, reducing binding 10-fold. In contrast, mutation of F479 increased self- association fivefold. Mutations altering NP oligomerization affected the ab ility of the polypeptides to support influenza virus gene expression in an in vivo assay. Decreased oligomerization activity correlated with decreased transcriptional function. However, mutations that increased self-associati on also decreased transcription competence. This indicates that NP contains both positive and negative sequence elements involved in oligomerization a nd is consistent with the importance of NP-NP contacts for the formation of a transcriptionally active RNP. (C) 1999 Academic Press.