- Email: [email protected]
Fine mapping of the subunit binding sites of influenza virus RNA polymerase
International Congress Series 1219 (2001) 463 – 469
Fine mapping of the subunit binding sites of influenza virus RNA polymerase Yasushi Ohtsua, Yoshikazu Hondab, Tetsuya Toyodaa,* a
Department of Virology, Kurume University School of Medicine, 67 Asahimachi, Kurume, Fukuoka 830-0011, Japan b International Livestock Research Institute, Nairobi, Kenya
Abstract Background: Influenza virus RNA polymerase consists of three subunits, PB1, PB2 and PA, and catalyzes both transcription and replication of the RNA genome. PB1 is a catalytic subunit of RNA polymerization and a core of subunit assembly. Previously, we mapped the PB1 – PB2 binding sites (1 – 259 of PB2 and 501 – 757 of PB1), and the PB1 – PA binding sites (1 – 140 of PB1 and 201 – 716 of PA). In order to determine the fine map of the subunit binding sites, we continued the same line of experiments. Methods: Serial deletion mutants of each subunit were expressed with N-terminal HAtag, and co-immunoprecipitated with the non-tagged wild-type subunits by anti-HA or subunitspecific antibodies. Results: PB1N599 and PB1C10 were co-immunoprecipitated with PB2wt, but PB1N625 and PB1N700 were weakly co-immunoprecipitated with PB2wt. PB1C25 or PB1C40 were not co-immunoprecipitated with PB2wt. PB2N50 and PB2N75 were co-immunoprecipitated with PB1wt. However, PB2N104 or PB2N150 were not co-immunoprecipitated with PB1wt. PB1C535 and PB1C617 were co-immunoprecipitated with PAwt, but PB1N25 was not. PAN300, PAN400, PAN500 and PA501 – 692 were co-immunoprecipitated with PB1wt, but PAC49 was not. Conclusions: PB1 – PB2 binding sites are mapped in PB1(600 – 747) and PB2(76 – 104), and PB1 – PA binding sites are in PB1(1 – 25) and PA(668 – 692). D 2001 Elsevier Science B.V. All rights reserved. Keywords: Immunoprecipitation; HA-tag; Anti-HA; T7 RNA polymerase
*
Corresponding author. Tel.: +81-942-31-7549; fax: +81-942-32-0903. E-mail address: [email protected] (T. Toyoda).
0531-5131/01/$ – see front matter D 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 3 9 5 - 8
464
Y. Ohtsu et al. / International Congress Series 1219 (2001) 463–469
1. Introduction Influenza virus RNA polymerase catalyzes both transcription and replication of the RNA genome [1– 5]. The viral RNA polymerase also catalyzes polyadenylation at the 3e termini of mRNA [6], and performs template-dependent capped RNA cleavage [7,8] and apparent proofreading of nascent RNA chains [9]. The RNA polymerase purified from influenza virus consists of one part each of the three subunits, PB1, PB2 and PA [10]. In vitro reconstitution studies of enzymatically active RNA polymerase using individual P proteins purified either from baculovirus infected cells [11], Pichia pastoris cells [12] or by SDS-polyacrylamide gel electrophoresis of virions [13] confirmed the subunit structure. Recently, we and other groups mapped the subunit binding sites of the influenza virus RNA polymerase, which demonstrated that PB1 is the core subunit of polymerase assembly [14 –18]. However, the results are somehow contradictory to each other and the subunit binding sites remain still ambiguous. For detailed analysis of the biding sites, we continued the same co-immunoprecipitation experiments of the subunits in various combinations.
2. Materials and methods 2.1. Cell culture RK-13 cells and COS-7 cells were maintained in Dulbecco’s modified minimal essential medium (DMEM; Liftech) containing 5% fetal bovine serum (FBS; JRC Scientific). 2.2. Construction of T7pro/HA-tagged polymerase mutants A nested set of cDNA for PB1, PB2 and PA mutants was constructed by PCR amplification using pAPR206 (EMBL, GenBank, DDBJ Accession no. J02151), pAPR102 (J02152) and pAPR303 (J02153) as templates (Fig. 1) [19]. The resulting Nterminal HA-tagged mutants of each subunit were constructed. They were cloned into BlueScript II KS (+) (Stratagene) or pSP72 (Promega) under the control of T7 promoter. For construction of non-tagged wild-type subunits, the PCR fragments were cloned into BlueScript II SK (+).
Fig. 1. Construction scheme and the binding activity of deletion mutants of the influenza virus RNA polymerase subunits. Maps of the deletion mutants of PB1 and PB2 (A), and PB1 and PA (B), and the results of coimmunoprecipitation experiments are indicated on the right. Numbers on the bars indicate the position of the first and the last amino acid. Only the fragments which were co-immunoprecipitated by both antibodies were indicated as ‘‘ + + ’’, and those were not co-immunoprecipitated by either antibodies were indicated as ‘‘ ’’. ‘‘ + ’’ indicated weak interaction of the subunits.
Y. Ohtsu et al. / International Congress Series 1219 (2001) 463–469
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Y. Ohtsu et al. / International Congress Series 1219 (2001) 463–469
2.3. Recombinant vaccinia virus expressing the bacteriophage T7 RNA polymerase Recombinant vaccinia virus, rVVT7, expressing bacteriophage T7 RNA polymerase was generated using pHA vector, which contained the vaccinia virus HA gene and modified 7.5-kDa promoter [20] and T7 RNA polymerase gene (M38308) from pAR1173 [21]. rVVT7 was obtained by homologous recombination from vaccinia virus, Lister 16m0 strain [22].
Fig. 2. Co-immunoprecipitation of the influenza virus RNA polymerase subunits. Co-immunoprecipitation of PB1 deletion mutants and PAwt (A), PA deletion mutants and PB1wt (B), PB1 deletion mutants and PB2wt (C), and PB2 deletion mutants and PB1wt (D) are shown. Expressed subunits were co-immunoprecipitated with antiHA (odd lanes) and subunit-specific antibodies (even lanes). The positions of the intact (non-tagged) wild-type subunits were indicated on the right and those of deletion mutants were indicated by arrowheads.
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2.4. Co-transfection and co-immunoprecipitation COS-7 cells were first infected with vvT7 (m.o.i = 2) for 1 h. The cells were incubated for 4 h in Opti-MEM I containing 2% Lipofectin (Lifetech Orientals), combinations of the P protein expression plasmids and 40 mg/ml of cytosine-1-b-D(+)-arabinofuranoside (AraC, Wako). After transfection, the cells were incubated overnight in DMEM containing 5% FBS and 40 mg/ml Ara-C. The cells were pulse-labeled with 1 mCi/ml of [35S]methionine and [35S]cystin (Amersham) for 4 h in methionine-free MEM containing 40 mg/ml Ara-C. The pulse-labeled cells were processed for co-immunoprecipitation with anti-HA or antisubunit specific antibodies as previously mentioned [17].
3. Results 3.1. PB1 – PA binding sites PB1C535 and PB1C617 were co-immunoprecipitated with PAwt, but PB1N25 and PAwt were not co-immunoprecipitated (Fig. 2A,B). PAN300, PAN400, PAN500 and PA501-692 were co-immunoprecipitated with PB1wt, but PAC49 was not co-immunoprecipitated with PB1wt. 3.2. PB1 – PB2 binding sites PB1N599 and PB2wt were co-immunoprecipitated (Fig. 2C,D). However, the PB2 bands co-immunoprecipitated with PB1N625 and PB1N700 were weak. The signals of PB1N625 and PB1N700 co-immunoprecipitated with PB2 were also weak. PB1C10 and PB2wt were co-immunoprecipitated, but PB1C25 or PB1C40 were not co-immunoprecipitated with PB2wt. Then, the PB1 binding site on PB2 N-terminal 1 – 259 was determined. PB2N50 and PB2N75 were co-immunoprecipitated with PB1wt, but PB2N104 or PB2N150 were not co-immunoprecipitated with PB1wt.
4. Discussion In the subunit binding sites determined by four groups, only the fact that PB1 binds with PB2 and PA is consistent [14 – 18]. From the co-immunoprecipitation experiment using the N-terminal deletion mutants of PB2 and PB1wt, the binding domain was mapped in 76 – 104 amino acids of PB2 (Fig. 2D). However, internal deletion mutant PB2del76 – 104 bound with PB1 (data not shown), indicating that the N-terminal region of PB2 also contributed to the binding or the conformation of the N-terminal structure of PB2, which affected the structure of the subunit. PB1N599 and PB1C10 bound with PB2, but PB1N625 and PB1N700 weakly bound. PB1C25 did not bind with PB2 (Fig. 2C). These results indicated that critical region of PB2 binding was localized on 600 –747 amino acids of PB1 and 732– 747 of PB1. Amino
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Y. Ohtsu et al. / International Congress Series 1219 (2001) 463–469
Fig. 3. Fine mapping of the subunit binding sites of influenza virus RNA polymerase.
acids 626 – 747 of PB1 could weakly bind with PB2, while amino acids 600 –625 bound strongly. From the co-immunoprecipitation experiment (Fig. 2A), we concluded that the PB1 binding region of PA mapped in the C-terminal 49 amino acids (668 – 692). When the Nterminal 25 amino acids were deleted from PB1, PB1 did not bind with PA (Fig. 2B). However, we failed to identify the responsible amino acid(s) for binding in this region (data not shown). The subunit binding sites of influenza virus RNA polymerase were mapped in Fig. 3. The subunit binding sites were mapped in independent regions of the other functional domains of each subunit. PB1 and PB2 bound using relatively wide region(s) and PB1 and PA bound to each other alone a 25-amino acid length. This may be the reason why the PB1 – PB2 binary complex was tightly formed in a short period (less than 10 m), but the recruitment of PA into the PB1 – PB2 complex took more than 3 h when the three subunits were expressed and pulse-labeled in transfected cells [17].
Acknowledgements The authors are grateful to Dr. H. Shida and Dr. Studier for pHA and pAR1173, respectively. This work was supported partly by a grant-in aid from the Naito Foundation, the Ishibashi Foundation, the Fukuoka Cancer Society and Yakult Honsha Co. Ltd.
References [1] A. Honda, A. Ishihama, The molecular anatomy of influenza virus RNA polymerase, Biol. Chem. 378 (1997) 483 – 488.
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[2] A. Ishihama, A multi-functional enzyme with RNA polymerase and RNase activities: molecular anatomy of influenza virus RNA polymerase, Biochimie 78 (1997) 1097 – 1102. [3] A. Ishihama, K. Nagata, Viral RNA polymerases, CRC Crit. Rev. Biochem. 23 (1988) 27 – 76. [4] R.M. Krug, F.V. Alonso-Caplen, I. Julkunen, M.G. Katze, Expression and replication of the influenza virus genome, in: RM. Krug (Ed.), The Influenza Viruses, Plenum, New York, NY, 1989, pp. 89 – 152. [5] T. Toyoda, Y. Ohtsu, K. Hara, K. Masunaga, N. Hamada, M. Koga, K. Kashiwagi, J. Iwahashi, Molecular dissection of influenza virus RNA polymerase, Rec. Res. Dev. Virol. 1 (1999) 839 – 847. [6] D.C. Pritlove, L.L. Poon, E. Fodor, J. Shares, G.G. Brownlee, Poladenylation of influenza virus m RNA transcribed in vitro from model virion RNA templates: requirement for 50 conserved sequences, J. Virol. 72 (1998) 1280 – 1286. [7] K. Kawakami, K. Mizumoto, A. Ishihama, RNA polymerase of influenza virus, IV. Catalytic properties of the capped RNA endonuclease associated with the RNA polymerase, Nucleic Acids Res. 11 (1983) 3637 – 3649. [8] S.J. Plotch, M. Bouloy, I. Ulmanen, R.M. Krug, Initiation of influenza viral RNA transcription by capped RNA primers: a unique cap (m7GpppXm)-dependent virion endonuclease generates 50 terminal RNA fragment that prime transcription, Cell 23 (1981) 847 – 858. [9] A. Ishihama, K. Mizumoto, K. Kawakami, A. Kato, A. Honda, Proofreading function associated with the RNA-dependent RNA polymerase from influenza virus, J. Biol. Chem. 261 (1986) 10417 – 10421. [10] A. Honda, J. Mukaigawa, A. Yokoiyama, A. Kato, S. Ueda, K. Nagata, M. Krystal, D.P. Nayak, A. Ishihama, Purification and molecular structure of RNA polymerase from influenza virus A/PR8, J. Biochem. 107 (1990) 624 – 628. [11] M. Kobayashi, K. Tuchiya, K. Nagata, A. Ishihama, Reconstitution of influenza virus RNA polymerase from three subunits expressed using recombinant baculovirus system, Virus Res. 22 (1992) 235 – 245. [12] J.-H. Hwang, K. Yamada, A. Honda, K. Nakade, A. Ishihama, Expression of functional influenza virus RNA polymerase in the methylotropic yeast Pichia pastoris, J. Virol. 74 (2000) 4074 – 4084. [13] B. Szewczyk, W.G. Laver, D.F. Summers, Purification, thioredoxin renaturation and reconstituted activity of the three subunits of the influenza A virus RNA polymerase, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 7907 – 7911. [14] S.K. Biswas, D.P. Nayak, Influenza virus polymerase basic protein 1 interacts with influenza virus polymerase basic protein at multiple sites, J. Virol. 70 (1996) 6716 – 6722. [15] S. Gonza´lez, T. Zu¨rcher, J. Ortı´n, Identification of two separate domains in the influenza virus PB1 protein involved in the interaction with the PB2 and PA subunits: a model for the viral RNA polymerase structure, Nucleic Acids Res. 24 (1996) 4456 – 4463. [16] D. Pe´rez, R.O. Donis, A 48-amino-acid region of influenza A virus PB1 protein is sufficient for complex formation with PA, J. Virol. 69 (1995) 6932 – 6939. [17] T. Toyoda, D.M. Adyshev, M. Kobayashi, A. Iwata, A. Ishihama, Molecular assembly of the influenza virus RNA polymerase: determination of the subunit – subunit contact sites, J. Gen. Virol. 77 (1996) 2149 – 2157. [18] T. Zu¨rcher, S. de la Luna, J.J. Sanz-Ezquerro, A. Nieto, J. Ortı´n, Mutational analysis of the influenza virus A/Victoria/3/75 PA protein L studies of interaction with PB1 protein and identification of a dominant negative mutant, J. Gen. Virol. 77 (1996) 1745 – 1749. [19] J.F. Young, U. Desselberger, U. Graves, P. Palese, A. Shatzman, M. Rosenburg, Cloning and expression of influenza virus genes, in: W.G. Laver (Ed.), The Origin of Pandemic Influenza Viruses, Elsevier, New York, NY, 1983, pp. 129 – 138. [20] H. Shida, T. Tochikura, T. Sato, T. Konno, K. Hirayoshi, Y. Ito, M. Hatanaka, Y. Hinuma, M. Sugimoto, F. Takahashi-Nishimaki, T. Maruyama, K. Miki, K. Suzuki, M. Morita, H. Sashiyama, N. Yoshimura, M. Hayami, Effect of the recombinant vaccinia viruses that express HTLV-I envelop gene on HTLV-I infection, EMBO J. 6 (1987) 3379 – 3384. [21] J.J. Dunn, B. Krippl, K.E. Bernstein, H. Westphal, F.W. Studier, Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus, Gene 68 (1988) 259 – 266. [22] H. Shida, Y. Hinuma, M. Hatanaka, M. Morita, M. Kidokoro, K. Suzuki, T. Maruyama, F. Takahashi-Nishimaki, M. Sugimoto, R. Kitamura, Effects and virulences of recombinant vaccinia viruses derived from attenuated strains that express the human T-cell leukemia virus type I envelope gene, J. Virol. 62 (1988) 4474 – 4480.
Fine mapping of the subunit binding sites of influenza virus RNA polymerase Yasushi Ohtsua, Yoshikazu Hondab, Tetsuya Toyodaa,* a
Department of Virology, Kurume University School of Medicine, 67 Asahimachi, Kurume, Fukuoka 830-0011, Japan b International Livestock Research Institute, Nairobi, Kenya
Abstract Background: Influenza virus RNA polymerase consists of three subunits, PB1, PB2 and PA, and catalyzes both transcription and replication of the RNA genome. PB1 is a catalytic subunit of RNA polymerization and a core of subunit assembly. Previously, we mapped the PB1 – PB2 binding sites (1 – 259 of PB2 and 501 – 757 of PB1), and the PB1 – PA binding sites (1 – 140 of PB1 and 201 – 716 of PA). In order to determine the fine map of the subunit binding sites, we continued the same line of experiments. Methods: Serial deletion mutants of each subunit were expressed with N-terminal HAtag, and co-immunoprecipitated with the non-tagged wild-type subunits by anti-HA or subunitspecific antibodies. Results: PB1N599 and PB1C10 were co-immunoprecipitated with PB2wt, but PB1N625 and PB1N700 were weakly co-immunoprecipitated with PB2wt. PB1C25 or PB1C40 were not co-immunoprecipitated with PB2wt. PB2N50 and PB2N75 were co-immunoprecipitated with PB1wt. However, PB2N104 or PB2N150 were not co-immunoprecipitated with PB1wt. PB1C535 and PB1C617 were co-immunoprecipitated with PAwt, but PB1N25 was not. PAN300, PAN400, PAN500 and PA501 – 692 were co-immunoprecipitated with PB1wt, but PAC49 was not. Conclusions: PB1 – PB2 binding sites are mapped in PB1(600 – 747) and PB2(76 – 104), and PB1 – PA binding sites are in PB1(1 – 25) and PA(668 – 692). D 2001 Elsevier Science B.V. All rights reserved. Keywords: Immunoprecipitation; HA-tag; Anti-HA; T7 RNA polymerase
*
Corresponding author. Tel.: +81-942-31-7549; fax: +81-942-32-0903. E-mail address: [email protected] (T. Toyoda).
0531-5131/01/$ – see front matter D 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 3 9 5 - 8
464
Y. Ohtsu et al. / International Congress Series 1219 (2001) 463–469
1. Introduction Influenza virus RNA polymerase catalyzes both transcription and replication of the RNA genome [1– 5]. The viral RNA polymerase also catalyzes polyadenylation at the 3e termini of mRNA [6], and performs template-dependent capped RNA cleavage [7,8] and apparent proofreading of nascent RNA chains [9]. The RNA polymerase purified from influenza virus consists of one part each of the three subunits, PB1, PB2 and PA [10]. In vitro reconstitution studies of enzymatically active RNA polymerase using individual P proteins purified either from baculovirus infected cells [11], Pichia pastoris cells [12] or by SDS-polyacrylamide gel electrophoresis of virions [13] confirmed the subunit structure. Recently, we and other groups mapped the subunit binding sites of the influenza virus RNA polymerase, which demonstrated that PB1 is the core subunit of polymerase assembly [14 –18]. However, the results are somehow contradictory to each other and the subunit binding sites remain still ambiguous. For detailed analysis of the biding sites, we continued the same co-immunoprecipitation experiments of the subunits in various combinations.
2. Materials and methods 2.1. Cell culture RK-13 cells and COS-7 cells were maintained in Dulbecco’s modified minimal essential medium (DMEM; Liftech) containing 5% fetal bovine serum (FBS; JRC Scientific). 2.2. Construction of T7pro/HA-tagged polymerase mutants A nested set of cDNA for PB1, PB2 and PA mutants was constructed by PCR amplification using pAPR206 (EMBL, GenBank, DDBJ Accession no. J02151), pAPR102 (J02152) and pAPR303 (J02153) as templates (Fig. 1) [19]. The resulting Nterminal HA-tagged mutants of each subunit were constructed. They were cloned into BlueScript II KS (+) (Stratagene) or pSP72 (Promega) under the control of T7 promoter. For construction of non-tagged wild-type subunits, the PCR fragments were cloned into BlueScript II SK (+).
Fig. 1. Construction scheme and the binding activity of deletion mutants of the influenza virus RNA polymerase subunits. Maps of the deletion mutants of PB1 and PB2 (A), and PB1 and PA (B), and the results of coimmunoprecipitation experiments are indicated on the right. Numbers on the bars indicate the position of the first and the last amino acid. Only the fragments which were co-immunoprecipitated by both antibodies were indicated as ‘‘ + + ’’, and those were not co-immunoprecipitated by either antibodies were indicated as ‘‘ ’’. ‘‘ + ’’ indicated weak interaction of the subunits.
Y. Ohtsu et al. / International Congress Series 1219 (2001) 463–469
465
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Y. Ohtsu et al. / International Congress Series 1219 (2001) 463–469
2.3. Recombinant vaccinia virus expressing the bacteriophage T7 RNA polymerase Recombinant vaccinia virus, rVVT7, expressing bacteriophage T7 RNA polymerase was generated using pHA vector, which contained the vaccinia virus HA gene and modified 7.5-kDa promoter [20] and T7 RNA polymerase gene (M38308) from pAR1173 [21]. rVVT7 was obtained by homologous recombination from vaccinia virus, Lister 16m0 strain [22].
Fig. 2. Co-immunoprecipitation of the influenza virus RNA polymerase subunits. Co-immunoprecipitation of PB1 deletion mutants and PAwt (A), PA deletion mutants and PB1wt (B), PB1 deletion mutants and PB2wt (C), and PB2 deletion mutants and PB1wt (D) are shown. Expressed subunits were co-immunoprecipitated with antiHA (odd lanes) and subunit-specific antibodies (even lanes). The positions of the intact (non-tagged) wild-type subunits were indicated on the right and those of deletion mutants were indicated by arrowheads.
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2.4. Co-transfection and co-immunoprecipitation COS-7 cells were first infected with vvT7 (m.o.i = 2) for 1 h. The cells were incubated for 4 h in Opti-MEM I containing 2% Lipofectin (Lifetech Orientals), combinations of the P protein expression plasmids and 40 mg/ml of cytosine-1-b-D(+)-arabinofuranoside (AraC, Wako). After transfection, the cells were incubated overnight in DMEM containing 5% FBS and 40 mg/ml Ara-C. The cells were pulse-labeled with 1 mCi/ml of [35S]methionine and [35S]cystin (Amersham) for 4 h in methionine-free MEM containing 40 mg/ml Ara-C. The pulse-labeled cells were processed for co-immunoprecipitation with anti-HA or antisubunit specific antibodies as previously mentioned [17].
3. Results 3.1. PB1 – PA binding sites PB1C535 and PB1C617 were co-immunoprecipitated with PAwt, but PB1N25 and PAwt were not co-immunoprecipitated (Fig. 2A,B). PAN300, PAN400, PAN500 and PA501-692 were co-immunoprecipitated with PB1wt, but PAC49 was not co-immunoprecipitated with PB1wt. 3.2. PB1 – PB2 binding sites PB1N599 and PB2wt were co-immunoprecipitated (Fig. 2C,D). However, the PB2 bands co-immunoprecipitated with PB1N625 and PB1N700 were weak. The signals of PB1N625 and PB1N700 co-immunoprecipitated with PB2 were also weak. PB1C10 and PB2wt were co-immunoprecipitated, but PB1C25 or PB1C40 were not co-immunoprecipitated with PB2wt. Then, the PB1 binding site on PB2 N-terminal 1 – 259 was determined. PB2N50 and PB2N75 were co-immunoprecipitated with PB1wt, but PB2N104 or PB2N150 were not co-immunoprecipitated with PB1wt.
4. Discussion In the subunit binding sites determined by four groups, only the fact that PB1 binds with PB2 and PA is consistent [14 – 18]. From the co-immunoprecipitation experiment using the N-terminal deletion mutants of PB2 and PB1wt, the binding domain was mapped in 76 – 104 amino acids of PB2 (Fig. 2D). However, internal deletion mutant PB2del76 – 104 bound with PB1 (data not shown), indicating that the N-terminal region of PB2 also contributed to the binding or the conformation of the N-terminal structure of PB2, which affected the structure of the subunit. PB1N599 and PB1C10 bound with PB2, but PB1N625 and PB1N700 weakly bound. PB1C25 did not bind with PB2 (Fig. 2C). These results indicated that critical region of PB2 binding was localized on 600 –747 amino acids of PB1 and 732– 747 of PB1. Amino
468
Y. Ohtsu et al. / International Congress Series 1219 (2001) 463–469
Fig. 3. Fine mapping of the subunit binding sites of influenza virus RNA polymerase.
acids 626 – 747 of PB1 could weakly bind with PB2, while amino acids 600 –625 bound strongly. From the co-immunoprecipitation experiment (Fig. 2A), we concluded that the PB1 binding region of PA mapped in the C-terminal 49 amino acids (668 – 692). When the Nterminal 25 amino acids were deleted from PB1, PB1 did not bind with PA (Fig. 2B). However, we failed to identify the responsible amino acid(s) for binding in this region (data not shown). The subunit binding sites of influenza virus RNA polymerase were mapped in Fig. 3. The subunit binding sites were mapped in independent regions of the other functional domains of each subunit. PB1 and PB2 bound using relatively wide region(s) and PB1 and PA bound to each other alone a 25-amino acid length. This may be the reason why the PB1 – PB2 binary complex was tightly formed in a short period (less than 10 m), but the recruitment of PA into the PB1 – PB2 complex took more than 3 h when the three subunits were expressed and pulse-labeled in transfected cells [17].
Acknowledgements The authors are grateful to Dr. H. Shida and Dr. Studier for pHA and pAR1173, respectively. This work was supported partly by a grant-in aid from the Naito Foundation, the Ishibashi Foundation, the Fukuoka Cancer Society and Yakult Honsha Co. Ltd.
References [1] A. Honda, A. Ishihama, The molecular anatomy of influenza virus RNA polymerase, Biol. Chem. 378 (1997) 483 – 488.
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