The PA Subunit Is Required for Efficient Nuclear Accumulation of the PB1 Subunit of the Influenza A Virus RNA Polymerase Complex (original) (raw)
Abstract
The RNA genome of influenza virus is transcribed and replicated by the viral RNA polymerase complex in the cell nucleus. We have generated green fluorescent protein (GFP)-tagged polymerase subunits to study the assembly of the polymerase complex. Our results show that individually expressed polymerase basic protein 1 (PB1) and polymerase acidic protein (PA) subunits were distributed in both the cytoplasm and the nucleus, while the polymerase basic protein 2 (PB2) subunit accumulated in the nucleus. Although it has been reported that PB1 alone accumulates in the nucleus, we demonstrate that PB1 requires the coexpression of PA for efficient nuclear accumulation. Our results support a model which proposes that PB1 and PA are transported into the nucleus as a complex.
Loading Preview
Sorry, preview is currently unavailable. You can download the paper by clicking the button above.
References (44)
- Akkina, R. K., T. M. Chambers, D. R. Londo, and D. P. Nayak. 1987. Intracellular localization of the viral polymerase proteins in cells infected with influenza virus and cells expressing PB1 protein from cloned cDNA. J. Virol. 61:2217-2224.
- Area, E., J. Martín-Benito, P. Gastaminza, E. Torreira, J. M. Valpuesta, J. L. Carrascosa, and J. Ortín. 2004. 3D structure of the influenza virus polymer- ase complex: localization of subunit domains. Proc. Natl. Acad. Sci. USA 101:308-313.
- Argos, P. 1988. A sequence motif in many polymerases. Nucleic Acids Res. 16:9909-9916.
- Ba ´rcena, J., M. Ochoa, S. de la Luna, J. A. Melero, A. Nieto, J. Ortín, and A. Portela. 1994. Monoclonal antibodies against influenza virus PB2 and NP polypeptides interfere with the initiation step of viral mRNA synthesis in vitro. J. Virol. 68:6900-6909.
- Biswas, S. K., P. L. Boutz, and D. P. Nayak. 1998. Influenza virus nucleo- protein interacts with influenza virus polymerase proteins. J. Virol. 72:5493- 5501.
- Biswas, S. K., and D. P. Nayak. 1994. Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1. J. Virol. 68:1819- 1826.
- Brownlee, G. G., and J. L. Sharps. 2002. The RNA polymerase of influenza A virus is stabilized by interaction with its viral RNA promoter. J. Virol. 76:7103-7113.
- Cormack, B. P., R. H. Valdivia, and S. Falkow. 1996. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173:33-38.
- Digard, P., V. C. Blok, and S. C. Inglis. 1989. Complex formation between influenza virus polymerase proteins expressed in Xenopus oocytes. Virology 171:162-169.
- Fechter, P., L. Mingay, J. Sharps, A. Chambers, E. Fodor, and G. G. Brown- lee. 2003. Two aromatic residues in the PB2 subunit of influenza A RNA polymerase are crucial for cap binding. J. Biol. Chem. 278:20381-20388.
- Fodor, E., and G. G. Brownlee. 2002. Influenza virus replication, p. 1-29. In C. W. Potter (ed.), Influenza. Elsevier, Amsterdam, The Netherlands.
- Fodor, E., M. Crow, L. J. Mingay, T. Deng, J. Sharps, P. Fechter, and G. G. Brownlee. 2002. A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs. J. Virol. 76:8989-9001.
- Fodor, E., L. J. Mingay, M. Crow, T. Deng, and G. G. Brownlee. 2003. A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase promotes the generation of defective interfering RNAs. J. Virol. 77:5017-5020.
- Gonza ´lez, S., T. Zu ¨rcher, and J. Ortín. 1996. Identification of two separate domains in the influenza virus PB1 protein invloved in the interaction with the PB2 and PA subunits: a model for the viral RNA polymerase structure. Nucleic Acids Res. 24:4456-4463.
- Herz, C., E. Stavnezer, R. M. Krug, and T. Gurney. 1981. Influenza virus, an RNA virus, synthesizes its messenger RNA in the nucleus of infected cells. Cell 26:391-400.
- Honda, A., K. Mizumoto, and A. Ishihama. 2002. Minimum molecular ar- chitectures for transcription and replication of the influenza virus. Proc. Natl. Acad. Sci. USA 99:13166-13171.
- FIG. 6. Models for the transport and assembly of the influenza virus RNA polymerase. (A) Subunits PB1, PB2, and PA are transported into the nucleus individually and form a complex in the nucleus. (B) PB1 and PA interact in the cytoplasm and are transported into the nucleus as a dimer. PB2 enters the nucleus as a monomer and binds to PB1-PA in the nucleus. (C) PB1, PB2, and PA interact in the cytoplasm and are transported into the nucleus as a trimeric complex. 9152 FODOR AND SMITH J. VIROL.
- Jackson, D. A., A. J. Caton, S. J. McCready, and P. R. Cook. 1982. Influenza virus RNA is synthesized at a fixed site in the nucleus. Nature 296:366-368.
- Jones, I. M., P. A. Reay, and K. L. Philpott. 1986. Nuclear location of all three influenza polymerase proteins and a nuclear signal in polymerase PB2. EMBO J. 5:2371-2376.
- Lamb, R. A., and R. M. Krug. 2001. Orthomyxoviridae: the viruses and their replication, p. 1487-1530. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Straus (ed.), Fields virology, 4th ed. Lippincott Williams & Wilkins, Philadelphia, Pa.
- Lee, M. T. M., K. Bishop, L. Medcalf, D. Elton, P. Digard, and L. Tiley. 2002. Definition of the minimal components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase. Nucleic Acids Res. 30:429-438.
- Li, M. L., P. Rao, and R. M. Krug. 2001. The active sites of the influenza cap-dependent endonuclease are on different polymerase subunits. EMBO J. 20:2078-2086.
- Medcalf, L., E. Poole, D. Elton, and P. Digard. 1999. Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disrupt RNA binding by the nucleoprotein. J. Virol. 73:7349-7356.
- Mena, I., E. Jambrina, C. Albo, B. Perales, J. Ortín, M. Arrese, D. Vallejo, and A. Portela. 1999. Mutational analysis of influenza A virus nucleoprotein: identification of mutations that affect RNA replication. J. Virol. 73:1186- 1194.
- Mukaigawa, J., and D. P. Nayak. 1991. Two signals mediate nuclear local- ization of influenza virus (A/WSN/33) polymerase basic protein 2. J. Virol. 65:245-253.
- Naffakh, N., P. Massin, and S. van der Werf. 2001. The transcription/repli- cation activity of the polymerase of influenza A viruses is not correlated with the level of proteolysis induced by the PA subunit. Virology 285:244-252.
- Nakagawa, Y., K. Oda, and S. Nakada. 1996. The PB1 subunit alone can catalyze cRNA synthesis, and the PA subunit in addition to the PB1 subunit is required for viral RNA synthesis in replication of the influenza virus genome. J. Virol. 70:6390-6394.
- Nath, S. T., and D. P. Nayak. 1990. Function of two discrete regions is required for nuclear localization of polymerase basic protein 1 of A/WSN/33 influenza virus (H1N1). Mol. Cell. Biol. 10:4139-4145.
- Neumann, G., G. G. Brownlee, E. Fodor, and Y. Kawaoka. 2004. Orthomyxo- virus replication, transcription, and polyadenylation. Curr. Top. Microbiol. Immunol. 283:121-143.
- Nieto, A., S. de la Luna, J. Ba ´rcena, A. Portela, and J. Ortín. 1994. Complex structure of the nuclear translocation signal of influenza virus polymerase PA subunit. J. Gen. Virol. 75:29-36.
- Nieto, A., S. de la Luna, J. Ba ´rcena, A. Portela, J. Valca ´rcel, J. A. Melero, and J. Ortín. 1992. Nuclear transport of influenza virus polymerase PA protein. Virus Res. 24:65-75.
- Ohtsu, Y., Y. Honda, Y. Sakata, H. Kato, and T. Toyoda. 2002. Fine mapping of the subunit binding sites of influenza virus RNA polymerase. Microbiol. Immunol. 46:167-175.
- Perales, B., and J. Ortín. 1997. The influenza A virus PB2 polymerase subunit is required for the replication of viral RNA. J. Virol. 71:1381-1385.
- Perales, B., J. J. Sanz-Ezquerro, P. Gastaminza, J. Ortega, J. F. Santare ´n, J. Ortín, and A. Nieto. 2000. The replication activity of influenza virus polymerase is linked to the capacity of the PA subunit to induce proteolysis. J. Virol. 74:1307-1312.
- Perez, D. R., and R. O. Donis. 2001. Functional analysis of PA binding by influenza A virus PB1: effects on polymerase activity and viral infectivity. J. Virol. 75:8127-8136.
- Pleschka, S., S. R. Jaskunas, O. G. Engelhardt, T. Zu ¨rcher, P. Palese, and A. García-Sastre. 1996. A plasmid-based reverse genetics system for influenza A virus. J. Virol. 70:4188-4192.
- Pombo, A., P. Cuello, W. Schul, J.-B. Yoon, R. G. Roeder, P. R. Cook, and S. Murphy. 1998. Regional and temporal specialization in the nucleus: a tran- scriptionally-active nuclear domain rich in PTF, Oct1 and PIKA antigens associates with specific chromosomes early in the cell cycle. EMBO J. 17: 1768-1778.
- Puig, O., F. Caspary, G. Rigaut, B. Rutz, E. Bouveret, E. Bragado-Nilsson, M. Wilm, and B. Se ´raphin. 2001. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24: 218-229.
- Sanz-Ezquerro, J. J., S. de la Luna, J. Ortín, and A. Nieto. 1995. Individual expression of influenza virus PA protein induces degradation of coexpressed proteins. J. Virol. 69:2420-2426.
- Smith, G. L., J. Z. Levin, P. Palese, and B. Moss. 1987. Synthesis and cellular location of the ten influenza polypeptides individually expressed by recom- binant vaccinia viruses. Virology 160:336-345.
- Subbarao, K., H. Chen, D. Swayne, L. Mingay, E. Fodor, G. G. Brownlee, X. Xu, X. Lu, J. Katz, N. Cox, and Y. Matsuoka. 2002. Evaluation of a genet- ically modified reassortant H5N1 influenza A virus vaccine candidate gen- erated by plasmid-based reverse genetics. Virology 305:192-200.
- Toyoda, T., D. M. Adyshev, M. Kobayashi, A. Iwata, and A. Ishihama. 1996. Molecular assembly of the influenza virus RNA polymerase: determination of the subunit-subunit contact sites. J. Gen. Virol. 77:2149-2157.
- Tsien, R. Y. 1998. The green fluorescent protein. Annu. Rev. Biochem. 67:509-544.
- Zu ¨rcher, T., S. de la Luna, J. J. Sanz-Ezquerro, A. Nieto, and J. Ortín. 1996. Mutational analysis of the influenza virus A/Victoria/3/75 PA protein: studies of interaction with PB1 protein and identification of a dominant negative mutant. J. Gen. Virol. 77:1745-1749.