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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 interaction. 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 temperature-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 + 3 4 1 585 4506. e-mail [email protected] 1 Presentaddress:National Institute for Medical Research,The Ridgeway, Mill Hill, London NW7 1AA, UK 0001-3930 © 1996 SGN 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 coexpressed 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-immunoprecipitation 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 aivoreconstitution 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, Nterminal 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). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 03:51:33 74! iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii iiii iii iiii iiiiiiiiiiiiiii 100 200 300 400 500 600 . ___1___, Wt PA and 700 PA mutant Association with PB1 Polymerase activity wt (716) 100 100 C (683) ND ND St (626) ND ND Bg (509) ND ND u (407) ND ND Sc (342) ND ND H (24~3 ND ND S (186) ND ND ii K (al-85) ND ND 66 + 23 ND BS (A509-626) ND ND BA (A407-511) ND ND SB (A341-407) ND ND HS (A246-342) ND ND SH (A186-247) ND ND 1 672 102 _+26 ND/tS 1 550 57 + 15 20 -+0/ts 1 509 123 + 52 ND ND ND D (A1-154) i 154 (E-~G) Internal deletion Point mutation Single amino acid insertion m ~"/']~/vP16 fusion NDNot detected ts Temperature-sensitive ii Fig. I. PA mutants: PBI association and polymerase activities. The last PA-encoded amino acid of each C-terminal deletion mutant is indicated. Black boxes indicate a frameshift and a short stretch of unspecific sequence until the next stop codon is reached. For the N-terminal and internal mutants the size and location of the deletion is indicated. The insertion mutants are named with the inserted amino acid and the position upstream of the insertion. The PBI-PA interaction was measured using the two hybrid system as described in the text, The PBI association of the interacting PA mutants is indicated as the percentage luciferase activity of that achieved by the wt VPI co-PAfusion protein. The interaction assay of the non-interacting mutants was repeated at least twice and the values for the interacting mutants represent the average of six independent experiments. The polymerase activity of the individual PA mutants was determined by reconstitution of the RNA polymerase in vivo as described. The activity of mutant 1550 is indicated as the percentage CAT activity of that achieved by the wt PA protein, The polymerase assay was repeated two to three times with each mutant, Firstly, we tested whether the P A mutants were able to reconstitute in vivo an active polymerase complex. The expression of the three P proteins and NP using the vaccinia virus-T7 R N A polymerase expression system allowed the expression of an influenza virus-like v R N A containing the 74( CAT gene flanked by the non<oding regions of the NS gene (Piccone et aL 1993) as previously described (Mena e/ aI., 1994). In addition to the mutants shown in Fig. 1, three mutants with smaller deletions in the C-terminal half of PA (PAA463-511, PAzX407-51I and PAA407-425) and one Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 03:51:33 (a) expression of wt PA allowed CAT expression (data not shown). We considered it of interest to examine if any of the "~-dAC-CM mutations introduced a temperature-sensitive (ts) phenotype. Thus, the RNA polymerase activity was reconstituted at 3 7 °C or 33 °C as described (Mena eta]., 1994). Mutant PAI54 and -,.~--Ac-CM Q Q I O e all deletion mutants remained inactive at 33 °C and mutants PA151 and PAI62 showed only slightly increased activity at III OII II ell "~-CM 33 °C compared to 37 °C (data not shown). Nevertheless, interesting results were obtained with the insertion mutants (b) wt I550 I672 $509 wt I550 I672 $509 (Fig. 2). Mutant PAI672, completely inactive at 37 °C, reached 100 BG 20 BG BG 1100 BG 53 4.4 BG Exp. 1 3"4% of wt activity at 33 °C, a level significantly higher than 100 BG 20 BG BG ]100 BG 37 2.3 BG Exp.2 background (0"3%). Mutant PAI550 was also about twofold I 100 BG 20+0 BG BG 1100 BG 45+11 3.4+1.5BG Avg more active at the permissive temperature. Similar insertion mutants with a ts phenotype for RNA polymerase activity Fig. 2. CAT activity and its competition by PA mutants. CAT activity of inseRion mutants at permissive and non-permissive temperatures. The have been observed for the PB2 gene (Perales eta]., 1996). influenza virus transcription-replication system was reconstituted as Interestingly, a ts mutant (ts263) of fowl plague virus carries a described (IVlena et at., t994) and duplicates of transfected COS-1 missense mutation (Ala to Val change) at position 67i of PA cultures were incubated at 37 °C (restrictive temperature) or 33 °C (permissive temperature). Total protein extracts were prepared and (Herget & Scholtissek, I993). Insertion mutants, in contrast to assayed for CAT activity. (a) Thin layer chromatography assay illustrating missense mutations that are in general found to be responsible CAT activities (de la Luna et el., 1993). The CAT activities for the wt for ts mutations, could be genetically quite stable since control and mutant 1550 were not in the range of this assay. The positions of chloramphenicol (CiVl), 3-acetyl-chloramphenicol (Ac-ClM) and 1,3reversion would require the deletion of exactly three nucleoacetyl-chloramphenicol (dAc-Cb4) are indicated. (b) Quantitative analysis tides. Nevertheless mutations in PA or other virus genes might of CAT activities usin 9 a previously described phase extraction assay (de suppress the ts phenotype (Mandler et a]., 1991). Influenza la Luna et al., 1993) in two separate experiments, with the average. The background (BG) in this assay was lower than 0.3°2'0 of the wt activity. The viruses with ts insertion mutations could be rescued using activities are shown as a percentage of the activity of vet PA. Transfected described procedures (for review see Garc{a-Sastre & Palest, PA plasmids: wt, pGEM3PAwt;-, pGEN13; 1550, pGEM3PA1550; 1672, 1993) to generate recombinants with reduced pathogenicity pGEM3PAI672; $509, pGEM3PAS509. and enhanced genetic stability. For the mutants that were negative for CAT activity it could not be distinguished if the active site of PA, complex additional deletion mutant (PAASA) in the N-terminal half formation or just the general folding of PA was affected by the (A186-280) of PA were analysed (data not shown). The mutation. Thus, we further studied the PA-PB1 association of polymerase activities of the PA mutants are summarized in Fig. the mutants using a GAL4/VP16-based two hybrid system 1. None of these deletion mutants was active in the polymerase developed for the transfection of animal cells (Sadowski et al., assay. This result is not surprising since these drastic mutations 1988 and references therein). Ptasmids were constructed might not only affect the structure, the intraceIlular localization expressing the GAL4 DNA-binding domain fused to the N or the active site of PA but also complex formation and thus terminus of PB1, and the VP16 activation domain fused to the indirectly the activity of the other polymerase subunits. The N terminus of PA. For the construction of GAL4 fusions the point mutant PA154, the only cytoplasmic mutant beside multiple cloning site (MCS) of pSG424 (Sadowski & Patshne, PAASA and PAASH, was inactive, whereas the nuclear mutants 1989) was replaced by the MCS of pTM1 (Elroy-Stein eta]., (PA151, PAI62) with mutations in the same region (Sanz1989). PCR fragments of PB1 and PA cDNAs of strain Ezquerro et al., 1996) remained active (data not shown). Of the A/Victoria/75 with a SmaI restriction site upstream of the three insertion mutants only PAI550 showed activity, albeit to ATG and a XbaI site downstream of the stop codon were a reduced level (20%). The expression of the individual PA transferred to the modified pSG424 using the introduced Sinai mutants was analysed by Western blot using a part of the same and XbaI sites. The VP16-PAwt, -PAAD and -PAAK fusion cell extract that was analysed for CAT activity (data not shown), confirming the results previously obtained (Sanzplasmids were constructed by transferring the PA genes of pSG424PA, pGEM3PAAD or pGEM3PAAK into the EcoRIEzquerro et al., 1996). All mutant proteins were of the expected XbaI site of pAASVVP16 (Aso et aL, 1992) whose polylinker size and the majority of the mutants was expressed to similar had been previously replaced by the MCS of pGEM3 or higher levels (PAS, PAASA, PAASH, PA154) than the wild(EcoRI-HindIII). All other pVPPA mutant plasmids were type (wt) control (data not shown). Only the mutants PASc constructed by replacing the EcoNI-XbaI fragment of pVPPA and PAH were reproducibly accumulated at levels lower than with the corresponding fragment from the pGEM3PA mutant wt PA and other mutants. However, the low expression levels plasmid. The expression of the VPPA fusion proteins was of these mutants cannot be the reason for the negative result in confirmed by Western blot analysis using GAL4-VP16- and polymerase activity, since much lower, almost undetectable 37 °C 33 °C ! Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 03:51:33 174; 10000. 1000 i ~- 100 o 1C 1 no competitor 100 % 2 wt PA 3 PAS509 4 PAABA 47 % 0.4 % 36 % Fig. 3. Competition of the reconstituted transcription replication system by over-expression of PA mutants. The influenza virus transcription-replication system was reconstituted at 37 °C as described in the text. COS-1 cells infected with T7 RNA polymerase-expressing vaccinia virus were transfected with 5 ng of pGEM3PAwt, 1O0 ng of pGEM3PB1 and pGEM3PB2, 2 pg of pGEM3NP and an additional 1OO ng of different PA protein-expressing plasmids or control plasmids, as indicated: column 1, pGEM3; column 2, pGEM3PAwt; column 3, pGEIv]3PAS509; column 4, pGEfvl3PAABA. CAT activities were measured using a phase extraction assay (de la Luna et el., 1993). PA-specific antisera (data not shown). All VPPA mutant proteins were of the expected size and accumulated to similar levels as wt VPPA, except for the two shortest mutants expressing C-terminal portions of PA (VPPAH, VPPAS), which were five- to 10-fold less abundant than wt VPPA. The reporter plasmid pGL-G5 (constructed and provided by P. Staheli, Department of Virology, University of Freiburg, Germany) contains five GAL4 DNA-binding sites upstream of an EIB promoter and the luciferase gene. The association of PA and PB1 in vivo was measured by transfection of subconfluent monolayers of COS-I cells (p35 dishes) with 1 pg of pGL-GS, 2"5 IJg of pVPPA (or mutant pVPPA) and 250 or 500 ng of pSG424PBI, using cationic liposomes for transfection. Cells were harvested 48 h posttransfection and assayed for luciferase activity (Brasier et al., 1989). The transactivation mediated by this association of VPPA and GAL4-PB1 resulted in a high level of Iuciferase expression, reaching about I0% of that achieved with GAL4-VP16 by transfection of I p,g of pSGVPA490 (Sadowski & Patshne, 1989; data not shown). When either pSG424PB1 or pVPPA were transfected together with plasmids expressing either the unfused VP16 activation domain or the unfused GAL4 binding domain, only basal level of luciferase activity (1-3 % of wt activity) could be detected. Thus, neither VPPA nor GAL4-PB1 had transactivation ability by itself. The interaction of the PA mutants with PB I is shown in Fig. 1. Deletion of the 154 N-terminal amino acids (mutant '4~ VPPAaD) barely affected the association with PB1, indicating that the N terminus is not absolutely required for such interaction. Since none of the 12 C-terminal and internal PA deletion mutants showed binding to PB1 it might be concluded that the entire C-terminal three quarters of PA are involved in this association. PBI may be bound at several points and/or by a highly structure-dependent element formed by discontinuous regions of PA. The structure is indeed important since a deletion of the 154 N-terminal amino acids did not affect binding to PB1, but a deletion of only 85 amino acids (mutant VPPAaK) or the amino acid substitution at position 154 (mutant VPPA154) were inhibitory. Thus, the sequence located between position 85 and 154 does not contribute to interaction but can inhibit the binding mediated by other sequences. The binding capacity of other mutants might be inhibited similarly. The insertion mutants VPPASS09 and VPPAI672 bound to PB1 with similar affinity to VPPAwt, and VPPAI550 showed about 50% of the binding activity of VPPAwt. In the experiments presented so far, we identified three mutants (PAAD, PASS09 and PAI672) which were negative in polymerase activity, but interacted with PB1 to a similar extent as wt PA. We were interested to know whether these mutants could enter the polymerase complex and thus inhibit its activity in a dominant negative manner. Therefore, we performed in vivo competition experiments at 37 °C (Fig. 3). A low amount of wt pGEM3PA (5 ng per dish) was transfected together with i00 ng of pGEMdPB2 and pGEM3PB1 for the reconstitution of the polymerase complex. In spite of this, the system was saturated with PA protein, since transfection of an additional i00 ng of pGEMdPAwt did not increase CAT activity (Fig. 3). The decrease in CAT activity observed after over-expression of wt PA or the non-interacting mutant PAABA (Fig. 3) can be explained by the proteolytic activity induced by PA protein, which reduced the concentration of the co-expressed proteins (PB1, PB2, NP and CAT; Sanz-Ezquerro et al., 1995). The over-expression of PASS09 (Fig. 3) reduced CAT expression more than 100-fold compared to competition with wt PA or PAhBA proteins, indicating that PASS09 competes for complex formation. It has been shown that the proteolytic activities induced by PAS509, wt PA and PAd~BA are identical (Sanz-Ezquerro et al., 1996) and hence cannot be responsible for this result. Thus, mutation $509 did not alter the structure but might have affected an active site of PA. In this mutant a serine is inserted just downstream of a conserved putative nucleotide-binding motif (Gs0~FIIKGs07Rs0sSs09), which closely resembles the classic GXXXXGKT/S nucleotidebinding motif, and suggests that the function of PA requires the binding of nucleotides. Despite their strong interaction with PB1 in the two hybrid system, PAAD and PAI672 could not compete efficiently under these conditions (data not shown), indicating that their binding properties are not complete in terms of complex formation. In conclusion, two in vivo-reconstituted systems were used to screen a series of PA mutants for their polymerase activity Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 03:51:33 and complex formation capacities. The inability to identify a small region of PA able to interact with PB1 suggests that several distant positions of its amino acid sequence might fold together to form conformation-dependent interaction domains. Several mutations in the C-terminal third of the protein affected its polymerase activity without dramatically altering the PBl-binding properties. In particular, the insertion of a serine downstream of a putative nucleotide-binding domain generated a dominant negative mutant, identifying this region as a potential active site of PA. Further experiments will show if this dominant negative mutant is able to interfere with RNA replication in influenza virus-infected cells, We thank B. Moss, P. Palese and P. St/iheli for providing biological materials. The plasmids used for the two-hybrid system were derived from the laboratory of S. M. Weissman. The technical assistance of J. Fern~indezand M. Pastor is gratefully acknowledged. T. Z. was supported by a fellowship of the Ministerio de Education y Cienciaand from a longterm fellowship of the Human Frontier Science Program (HFSP). This work was supported by Comisi6n Interministerial de Ciencia y Tecnologfa (grant BIO92-1044), Comunidad Aut6noma de Madrid (grant A0063), Programa Sectorial de Promocfon General del Conocimiento (grant PB-1542) and EU HCM Program (grant ERBCHRXCT 949453). References Aso, T., Vasavada, H.A., Kawaguchi, T, Germino, F. J., Ganguly , S., Kitajima, S., Weissman, S. H. & Yasukochi, Y. (1992). Characterization of eDNA for the large subunit of the transcription initiation factor TFIIF. Nature 355, 461-464. Biswas, S.K. & Nayak, D.P. (1994). Mutational analysis of the conserved motifs of influenzaA virus polymerase basic protein 1. Journal of Virology 68, I819-1826. Brasier, A. R., "rate, J. E. & Habener, J. F. (1989). Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. Bio Techniques 7, 1116--1122. de la Luna, S., Hart(n, J., Portela, A. & Ort(n, J. (1993). 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(1981). The role of two of the influenza virus core P proteins in recognizing cap 1 structures (m7GpppNm) on RNAs and in initiating viral RNA transcription. Proceedings of the National Academy of Sciences, USA 78, 7355-7359. Received 6 February 1996; Accepted 23 April 1996 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 03:51:33 '4!