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Journal of Virology, February 2000, p. 1307-1312, Vol. 74, No. 3
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The Replication Activity of Influenza Virus Polymerase Is Linked to the Capacity of the PA Subunit To Induce Proteolysis

Beatriz Perales,1 Juan J. Sanz-Ezquerro,1,dagger Pablo Gastaminza,1 Joaquin Ortega,1 Juan Férnandez Santarén,2 Juan Ortín,1 and Amelia Nieto1,*

Centro Nacional de Biotecnología (CSIC)1 and Centro de Biología Molecular (CSIC-UAM),2 Campus de Cantoblanco, 28049 Madrid, Spain

Received 10 September 1999/Accepted 4 November 1999

The PA subunit of the influenza virus polymerase complex is a phosphorylated protein that induces a proteolytic process that decreases its own accumulation levels and those of coexpressed proteins. The amino-terminal third of the protein is responsible for the induction of proteolysis. We mutated five potential casein kinase II phosphorylation sites located in the amino-terminal third of the protein. Mutations affecting position 157 almost completely abrogated proteolysis induction, whereas a mutation at position 162 produced a moderate decrease and mutations at positions 151, 200, and 224 did not affect proteolysis induction. Reconstitution of the influenza virus polymerase in vivo with viral model RNA containing the chloramphenicol acetyltransferase (CAT) gene indicated that the CAT activity obtained correlated with the capacity of each PA mutant to induce proteolysis. RNA protection assays of the products obtained with viral polymerase, reconstituted in vivo with model RNAs, indicated that mutations at position 157 led to a selective loss of the ability to synthesize cRNA from the viral RNA template but not to transcribe viral RNA, while a mutation affecting position 162 showed an intermediate phenotype. Collectively, these data provide a link between PA-mediated induction of proteolysis and the replication activity of the polymerase.

* Corresponding author. Mailing address: Centro Nacional de Biotecnología, Campus de Cantoblanco, 28049 Madrid, Spain. Phone: 34 91 585 4533. Fax: 34 91 585 4506. E-mail: anmartin{at}cnb.uam.es.

dagger Present address: Wellcome Trust Building, University of Dundee, Dundee DD1 5EH, United Kingdom.

Journal of Virology, February 2000, p. 1307-1312, Vol. 74, No. 3
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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