Mutational analysis of influenza A virus nucleoprotein: Identification of mutations that affect RNA replication

The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide w hich plays a pivotal role in virus replication. To get information on the d omains and specific residues involved in the different NP activities, we de scribe here the preparation and characterization of 20 influenza A virus mu tant NPs. The mutations, mostly single-amino-acid substitutions, were intro duced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, aff ected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and thei r functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentati on properties. The results obtained allowed us to identify both a mutant pr otein that accumulated in the cytoplasm and mutations that altered the func tionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyl transferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. interestingly, four mutant NPs, which sh owed a reduced functionality in synthesizing cRNA molecules From a vRNA tem plate, were fully competent to reconstitute complementary ribonucleoprotein s (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecul es. Based on the phenotype of these mutants and on previously published obs ervations, it is proposed that these mutant NPs have a reduced capability t o interact with the polymerase complex and that this NP-polymerase interact ion is responsible for making vRNPs switch from mRNA to cRNA synthesis.