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Journal of Virology, February 1999, p. 1186-1194, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Mutational Analysis of Influenza A Virus Nucleoprotein: Identification of Mutations That Affect RNA Replication

Ignacio Mena,1,dagger Enrique Jambrina,1 Carmen Albo,1 Beatriz Perales,2 Juan Ortín,2 Marta Arrese,1 Dolores Vallejo,1 and Agustín Portela1,*

Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, Majadahonda 28220,1 and Centro Nacional de Biotecnología (CSIC), Campus de Cantoblanco 28049,2 Madrid, Spain

Received 3 August 1998/Accepted 4 November 1998

The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected 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 their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis.

* Corresponding author. Mailing address: Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, Majadahonda 28220, Madrid, Spain. Phone: 34-91-5097904. Fax: 34-91-5097918. E-mail: aportela{at}isciii.es.

dagger Current address: Department of Neuropharmacology, CVN-9, The Scripps Research Institute, La Jolla, CA 92037.

Journal of Virology, February 1999, p. 1186-1194, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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