Journal of General Virology (1996), 77, 1745-1749. Printedin Great Britain SHORT COMMUNICATION Hutational analysis of the influenza virus A/Victoria/3/75 PA protein: studies of interaction with PB1 protein and identification of a dominant negative mutant Thomas Zercher,t Susana de la Luna, Juan J. Sanz-Ezquerro, Amelia Nieto and Juan Ort(n Centro Nacional de Biotecnologia (CSIC), Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain The RNA polymerase activity and PB1 binding of influenza virus PA mutants were studied using an in vivo-reconstituted polymerase assay and a two hybrid system. Deletions covering the whole PA protein abolished polymerase activity, but the deletion of the 154 N-terminal amino acids allowed PB1 binding, indicating that the PA protein N terminus is not absolutely required for this inter- action. Further internal or C-terminal deletions abolished PB1 interaction, suggesting that most of the protein is involved in this association. As a novel finding we showed that a single amino acid insertion mutant, PAI672, was responsible for a temper- ature-sensitive phenotype. Hutant PAS509, which had a serine insertion at position 509, bound to PBI like wild-type PA but did not show any polymerase activity. Over-expression of PAS509 interfered with the polymerase activity of wild-type PA, identifying PAS509 as a dominant negative mutant. The influenza virus RNA polymerase is composed of three polymerase proteins, PB1, PB2 and PA, and catalyses two distinct types of RNA synthesis: (i) synthesis of mRNA (transcription) and (ii) amplification of the vRNA (replication). For mRNA synthesis, 5'-capped, host cell-derived RNA fragments are used as primers and polyadenylation occurs at a signal located 17-22 nucleotides before the 5' end of the template. Replication occurs without primer and the vRNA template is copied to a full-length positive-stranded RNA (cRNA), which serves as template for vRNA synthesis. It has been shown that free nucleoprotein (NP) might be a control element for anti-termination, but little is known about the Authorfor correspondence: Juan Ortfn. Fax +34 1 585 4506. e-mail jortin@samba.cnb.vam.es 1 Present address: National Institute for Medical Research,The Ridgeway, Mill Hill, London NW7 1AA, UK mechanism of the transcription-replication switch and the detailed role of the components of the RNA polymerase (Krug et al., 1989). Comparative sequence analysis and experimental data suggest that PBI is the polymerase itself (Biswas & Nayak, 1994; Poch et al., I990). PB2 binds CAP1 structures and is probably responsible for the binding of the vRNA promoter (Fodor ef al., 1993; Ulmanen et al., 1981) and the endonucleolytic cleavage of the host cell primers (Licheng et aI., I995). The function of PA is unknown. It is essential for the activity of in aiao-reconstituted polymerase (reviewed in Mena et al., 1995) and genetic evidence suggests a role for PA in replication rather than in transcription (reviewed in Mahy, 1983). PA protein induces a general proteolysis of co- expressed proteins (Sanz-Ezquerro et al., 1995), but it is not clear if this property is essential for polymerase activity. The region of PA responsible for the induction of proteolysis maps to the N-terminal third of PA, as determined by mutational analysis (Sanz-Ezquerro et al., 1996). The expression of the P proteins in Xenopus oocytes showed that complex formation occurs in the absence of virus RNA. The co-immunoprecipi- tation of pairs of P proteins indicated that PB2 and PA can interact independently with PB1, yet cannot form a complex directly with each other (Digard ef al., 1989). In this report we describe the use of two in aivo- reconstitution assays to analyse the RNA polymerase activity and the PBI binding of a large set of PA mutants. Polymerase deficient, but interacting mutants have been characterized as dominant negative and therefore identify regions of the molecule relevant for its biochemical activity. Fig. 1 shows the most relevant mutants used in this study. C-temlinal, N- terminal and internal deletions covered the entire PA protein sequence. In addition, three point mutants in the N terminus (positions 151, 154 and 162) and three insertion mutants in the C-terminal third of PA (PAI550, PAI672 and PASS09) were analysed. Mutant PASS09 contained a serine insertion after position 509 just downstream of a conserved sequence with homology to a nucleotide-binding motif (position 502-509). The construction of all mutants has been described previously (Sanz-Ezquerro et aI., 1996). The subcellular localization and expression of the individual mutants used in this study have been shown previously (Sanz-Ezquerro et al., 1996). 74! 0001-3930 © 1996 SGN