The influenza A virus RNA polymerase is a heterotrimeric complex responsible for viral genome transcription and replication in the nucleus of infected cells. We recently carried out a proteomic analysis of purified polymerase expressed in human cells and identified a number of polymerase-associated cellular proteins. Here we characterise the role of one such host factors, SFPQ/PSF, during virus infection. Down-regulation of SFPQ/PSF by silencing with two independent siRNAs reduced the virus yield by 2–5 log in low-multiplicity infections, while the replication of unrelated viruses as VSV or Adenovirus was almost unaffected. As the SFPQ/PSF protein is frequently associated to NonO/p54, we tested the potential implication of the latter in influenza virus replication. However, down-regulation of NonO/p54 by silencing with two independent siRNAs did not affect virus yields. Down-regulation of SFPQ/PSF by siRNA silencing led to a reduction and delay of influenza virus gene expression. Immunofluorescence analyses showed a good correlation between SFPQ/PSF and NP levels in infected cells. Analysis of virus RNA accumulation in silenced cells showed that production of mRNA, cRNA and vRNA is reduced by more than 5-fold but splicing is not affected. Likewise, the accumulation of viral mRNA in cicloheximide-treated cells was reduced by 3-fold. In contrast, down-regulation of SFPQ/PSF in a recombinant virus replicon system indicated that, while the accumulation of viral mRNA is reduced by 5-fold, vRNA levels are slightly increased. In vitro transcription of recombinant RNPs generated in SFPQ/PSF-silenced cells indicated a 4–5-fold reduction in polyadenylation but no alteration in cap snatching. These results indicate that SFPQ/PSF is a host factor essential for influenza virus transcription that increases the efficiency of viral mRNA polyadenylation and open the possibility to develop new antivirals targeting the accumulation of primary transcripts, a very early step during infection.
References
[1]
Palese P, Shaw M (2007) Orthomyxoviridae: the viruses and their replication. In: Knipe DM, Howley PM, editors. Fields Virology 5th edition. Philadelphia: Lippincott Williams & Wilkins. pp. 1647–1689.
[2]
Coloma R, Valpuesta JM, Arranz R, Carrascosa JL, Ortin J, et al. (2009) The structure of a biologically active influenza virus ribonucleoprotein complex. PLoS Pathog 5: e1000491.
[3]
Ortega J, Martín-Benito J, Zürcher T, Valpuesta JM, Carrascosa JL, et al. (2000) Ultrastructural and functional analyses of of recombinant influenza virus ribonucleoproteins suggest dimerization of nucleoprotein during virus amplification. J Virol 74: 156–163.
[4]
Elton D, Digard P, Tiley L, Ortín J (2005) Structure and function of the influenza virus RNP. In: Kawaoka Y, editor. Current Topics in Influenza Virology. Norfolk: Horizon Scientific Press. pp. 1–92.
[5]
Neumann G, Brownlee GG, Fodor E, Kawaoka Y (2004) Orthomyxovirus replication, transcription, and polyadenylation. Curr Top Microbiol Immunol 283: 121–143.
[6]
Resa-Infante P, Jorba N, Coloma R, Ortín J (2011) The influenza virus RNA synthesis machine: Advances in its structure and function. RNA Biology 8: 1–9.
[7]
Biswas SK, Nayak DP (1994) Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1. J Virol 68: 1819–1826.
[8]
Poch O, Sauvaget I, Delarue M, Tordo N (1990) Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J 8: 3867–3874.
[9]
Dias A, Bouvier D, Crepin T, McCarthy AA, Hart DJ, et al. (2009) The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458: 914–918.
[10]
Guilligay D, Tarendeau F, Resa-Infante P, Coloma R, Crepin T, et al. (2008) The structural basis for cap binding by influenza virus polymerase subunit PB2. Nat Struct Mol Biol 15: 500–506.
[11]
Ulmanen I, Broni BA, Krug RM (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. Proc Natl Acad Sci U S A 78: 7355–7359.
[12]
Yuan P, Bartlam M, Lou Z, Chen S, Zhou J, et al. (2009) Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site. Nature 458: 909–913.
[13]
Area E, Martín-Benito J, Gastaminza P, Torreira E, Valpuesta JM, et al. (2004) Three-dimensional structure of the influenza virus RNA polymerase: localization of subunit domains. Proc Natl Acad Sci U S A 101: 308–313.
[14]
Resa-Infante P, Recuero-Checa MA, Zamarre?o N, Llorca O, Ortín J (2010) Structural and functional characterisation of an influenza virus RNA polymerase-genomic RNA complex. J Virol 84: 10477–10487.
[15]
Torreira E, Schoehn G, Fernandez Y, Jorba N, Ruigrok RW, et al. (2007) Three-dimensional model for the isolated recombinant influenza virus polymerase heterotrimer. Nucleic Acids Res 35: 3774–3783.
[16]
Fechter P, Mingay L, Sharps J, Chambers A, Fodor E, et al. (2003) Two aromatic residues in the PB2 subunit of influenza A RNA polymerase are crucial for cap binding. J Biol Chem 278: 20381–20388.
[17]
Fodor E, Crow M, Mingay LJ, Deng T, Sharps J, et al. (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.
[18]
Gastaminza P, Perales B, Falcón AM, Ortín J (2003) Influenza virus mutants in the N-terminal region of PB2 protein are affected in virus RNA replication but not transcription. J Virol 76: 5098–5108.
[19]
Perales B, Ortín J (1997) The influenza A virus PB2 polymerase subunit is required for the replication of viral RNA. J Virol 71: 1381–1385.
[20]
Detjen BM, St Angelo C, Katze MG, Krug RM (1987) The three influenza virus polymerase (P) proteins not associated with viral nucleocapsids in the infected cell are in the form of a complex. J Virol 61: 16–22.
[21]
Huet S, Avilov S, Ferbitz L, Daigle N, Cusack S, et al. (2009) Nuclear import and assembly of the influenza A virus RNA polymerase studied in live cells by Fluorescence Cross Correlation Spectroscopy. J Virol 84: 1254–1264.
[22]
Jorba N, Area E, Ortin J (2008) Oligomerization of the influenza virus polymerase complex in vivo. J Gen Virol 89: 520–524.
[23]
Engelhardt OG, Smith M, Fodor E (2005) Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II. J Virol 79: 5812–5818.
[24]
Gabriel G, Herwig A, Klenk HD (2008) Interaction of Polymerase Subunit PB2 and NP with Importin alpha1 Is a Determinant of Host Range of Influenza A Virus. PLoS Pathog 4: e11.
[25]
Honda A (2008) Role of host protein Ebp1 in influenza virus growth: intracellular localization of Ebp1 in virus-infected and uninfected cells. J Biotechnol 133: 208–212.
[26]
Honda A, Okamoto T, Ishihama A (2007) Host factor Ebp1: selective inhibitor of influenza virus transcriptase. Genes Cells 12: 133–142.
[27]
Huarte M, Sanz-Ezquerro JJ, Roncal F, Ortin J, Nieto A (2001) PA subunit from influenza virus polymerase complex interacts with a cellular protein with homology to a family of transcriptional activators. J Virol 75: 8597–8604.
[28]
Mayer D, Molawi K, Martinez-Sobrido L, Ghanem A, Thomas S, et al. (2007) Identification of Cellular Interaction Partners of the Influenza Virus Ribonucleoprotein Complex and Polymerase Complex Using Proteomic-Based Approaches. J Proteome Res 6: 672–682.
[29]
Momose F, Handa H, Nagata K (1996) Identification of host factors that regulate the influenza virus RNA polymerase activity. Biochimie 78: 1103–1108.
[30]
Momose F, Naito T, Yano K, Sugimoto S, Morikawa Y, et al. (2002) Identification of Hsp90 as a stimulatory host factor involved in influenza virus RNA synthesis. J Biol Chem 277: 45306–45314.
[31]
Resa-Infante P, Jorba N, Zamarreno N, Fernandez Y, Juarez S, et al. (2008) The host-dependent interaction of alpha-importins with influenza PB2 polymerase subunit is required for virus RNA replication. PLoS One 3: e3904.
[32]
Shimizu K, Handa H, Nakada S, Nagata K (1994) Regulation of influenza virus RNA polymerase activity by cellular and viral factors. Nucleic Acids Res 22: 5047–5053.
[33]
Kawaguchi A, Nagata K (2007) De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM. Embo J 26: 4566–4575.
[34]
Jorba N, Juarez S, Torreira E, Gastaminza P, Zamarreno N, et al. (2008) Analysis of the interaction of influenza virus polymerase complex with human cell factors. Proteomics 8: 2077–2088.
[35]
Li G, Zhang J, Tong X, Liu W, Ye X (2011) Heat shock protein 70 inhibits the activity of influenza a virus ribonucleoprotein and blocks the replication of virus in vitro and in vivo. PLoS One 6: e16546.
[36]
Kawaguchi A, Momose F, Nagata K (2011) Replication-coupled and host factor-mediated encapsidation of the influenza virus genome by viral nucleoprotein. J Virol 85: 6197–6204.
[37]
Shav-Tal Y, Zipori D (2002) PSF and p54(nrb)/NonO—multi-functional nuclear proteins. FEBS Lett 531: 109–114.
[38]
Patton JG, Porro EB, Galceran J, Tempst P, Nadal-Ginard B (1993) Cloning and characterization of PSF, a novel pre-mRNA splicing factor. Genes Dev 7: 393–406.
[39]
Akhmedov AT, Lopez BS (2000) Human 100-kDa homologous DNA-pairing protein is the splicing factor PSF and promotes DNA strand invasion. Nucleic Acids Res 28: 3022–3030.
[40]
Bladen CL, Udayakumar D, Takeda Y, Dynan WS (2005) Identification of the polypyrimidine tract binding protein-associated splicing factor.p54(nrb) complex as a candidate DNA double-strand break rejoining factor. J Biol Chem 280: 5205–5210.
[41]
Morozumi Y, Takizawa Y, Takaku M, Kurumizaka H (2009) Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities. Nucleic Acids Res 37: 4296–4307.
[42]
Straub T, Grue P, Uhse A, Lisby M, Knudsen BR, et al. (1998) The RNA-splicing factor PSF/p54 controls DNA-topoisomerase I activity by a direct interaction. J Biol Chem 273: 26261–26264.
[43]
Straub T, Knudsen BR, Boege F (2000) PSF/p54(nrb) stimulates “jumping” of DNA topoisomerase I between separate DNA helices. Biochemistry 39: 7552–7558.
[44]
Peng R, Hawkins I, Link AJ, Patton JG (2006) The splicing factor PSF is part of a large complex that assembles in the absence of pre-mRNA and contains all five snRNPs. RNA Biol 3: 69–76.
[45]
Peng R, Dye BT, Perez I, Barnard DC, Thompson AB, et al. (2002) PSF and p54nrb bind a conserved stem in U5 snRNA. Rna 8: 1334–1347.
[46]
Gozani O, Patton JG, Reed R (1994) A novel set of spliceosome-associated proteins and the essential splicing factor PSF bind stably to pre-mRNA prior to catalytic step II of the splicing reaction. Embo J 13: 3356–3367.
[47]
Emili A, Shales M, McCracken S, Xie W, Tucker PW, et al. (2002) Splicing and transcription-associated proteins PSF and p54nrb/nonO bind to the RNA polymerase II CTD. Rna 8: 1102–1111.
[48]
Dye BT, Patton JG (2001) An RNA recognition motif (RRM) is required for the localization of PTB-associated splicing factor (PSF) to subnuclear speckles. Exp Cell Res 263: 131–144.
[49]
Melton AA, Jackson J, Wang J, Lynch KW (2007) Combinatorial control of signal-induced exon repression by hnRNP L and PSF. Mol Cell Biol 27: 6972–6984.
[50]
Rosonina E, Ip JY, Calarco JA, Bakowski MA, Emili A, et al. (2005) Role for PSF in mediating transcriptional activator-dependent stimulation of pre-mRNA processing in vivo. Mol Cell Biol 25: 6734–6746.
[51]
Marko M, Leichter M, Patrinou-Georgoula M, Guialis A (2010) hnRNP M interacts with PSF and p54(nrb) and co-localizes within defined nuclear structures. Exp Cell Res 316: 390–400.
[52]
Zhang Z, Carmichael GG (2001) The fate of dsRNA in the nucleus: a p54(nrb)-containing complex mediates the nuclear retention of promiscuously A-to-I edited RNAs. Cell 106: 465–475.
[53]
Greco-Stewart VS, Thibault CS, Pelchat M (2006) Binding of the polypyrimidine tract-binding protein-associated splicing factor (PSF) to the hepatitis delta virus RNA. Virology 356: 35–44.
[54]
Harris D, Zhang Z, Chaubey B, Pandey VN (2006) Identification of cellular factors associated with the 3′-nontranslated region of the hepatitis C virus genome. Mol Cell Proteomics 5: 1006–1018.
[55]
Zolotukhin AS, Michalowski D, Bear J, Smulevitch SV, Traish AM, et al. (2003) PSF acts through the human immunodeficiency virus type 1 mRNA instability elements to regulate virus expression. Mol Cell Biol 23: 6618–6630.
[56]
Chase G, Deng T, Fodor E, Leung BW, Mayer D, et al. (2008) Hsp90 inhibitors reduce influenza virus replication in cell culture. Virology 377: 431–439.
[57]
Falcón AM, Marión RM, Zürcher T, Gómez P, Portela A, et al. (2004) Defective RNA replication and late gene expression in temperature-sensitive (A/Victoria/3/75) influenza viruses expressing deleted forms of NS1 protein. J Virol 78: 3880–3888.
[58]
Hay AJ, Lomniczi B, Bellamy AR, Skehel JJ (1977) Transcription of the influenza virus genome. Virology 83: 337–355.
[59]
Hooker L, Sully R, Handa B, Ono N, Koyano H, et al. (2003) Quantitative analysis of influenza virus RNP interaction with RNA cap structures and comparison to human cap binding protein eIF4E. Biochemistry 42: 6234–6240.
[60]
Niedzwiecka A, Marcotrigiano J, Stepinski J, Jankowska-Anyszka M, Wyslouch-Cieszynska A, et al. (2002) Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5′ cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins. J Mol Biol 319: 615–635.
[61]
Worch R, Niedzwiecka A, Stepinski J, Mazza C, Jankowska-Anyszka M, et al. (2005) Specificity of recognition of mRNA 5′ cap by human nuclear cap-binding complex. Rna 11: 1355–1363.
[62]
Li X, Palese P (1994) Characterization of the polyadenilation signal of influenza virus RNA. J Virol 68: 1245–1249.
[63]
Luo GX, Luytjes W, Enami M, Palese P (1991) The polyadenylation signal of influenza virus RNA involves a stretch of uridines followed by the RNA duplex of the panhandle structure. J Virol 65: 2861–2867.
[64]
Poon LL, Fodor E, Brownlee GG (2000) Polyuridylated mRNA synthesized by a recombinant influenza virus is defective in nuclear export. J Virol 74: 418–427.
[65]
Robertson JS, Schubert M, Lazzarini RA (1981) Polyadenylation sites for influenza mRNA. J Virol 38: 157–163.
[66]
Rajesh C, Baker DK, Pierce AJ, Pittman DL (2010) The splicing-factor related protein SFPQ/PSF interacts with RAD51D and is necessary for homology-directed repair and sister chromatid cohesion. Nucleic Acids Res 38:
[67]
DuBridge RB, Tang P, Hsia HC, Leong PM, Miller JH, et al. (1987) Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol Cell Biol 7: 379–387.
[68]
Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, et al. (1973) In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst 51: 1417–1423.
[69]
Ortín J, Nájera R, López C, Dávila M, Domingo E (1980) Genetic variability of Hong Kong (H3N2) influenza viruses: spontaneous mutations and their location in the viral genome. Gene 11: 319–331.
[70]
Tobita K, Sugiura A, Enomoto C, Furuyama M (1975) Plaque-assay and primary isolation of influenza A viruses in an established line of canine kidney cells (MDCK) in the presence of trypsin. Med Microbiol Immunol 162: 9–14.
[71]
Aparicio O, Razquin N, Zaratiegui M, Narvaiza I, Fortes P (2006) Adenovirus virus-associated RNA is processed to functional interfering RNAs involved in virus production. J Virol 80: 1376–1384.
[72]
Bárcena J, Ochoa M, de la Luna S, Melero JA, Nieto A, et al. (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.
[73]
Ochoa M, Bárcena J, de la Luna S, Melero JA, Douglas AR, et al. (1995) Epitope mapping of cross-reactive monoclonal antibodies specific for the influenza A virus PA and PB2 polypeptides. Virus Res 37: 305–315.
[74]
Salvatore M, Basler CF, Parisien JP, Horvarth CM, Bourmakina S, et al. (2002) Effects of Influenza A virus NS1 protein on protein expression: the NS1 protein enhances translation and is not required for shutoff of host protein synthesis. J Virol 76: 1206–1212.
[75]
Marión RM, Zürcher T, de la Luna S, Ortín J (1997) Influenza virus NS1 protein interacts with viral transcription-replication complexes in vivo. J Gen Virol 78: 2447–2451.
[76]
Zürcher T, Marión RM, Ortín J (2000) The protein synthesis shut-off induced by influenza virus infection is independent of PKR activity. J Virol 74: 8781–8784.
[77]
Jorba N, Coloma R, Ortin J (2009) Genetic trans-complementation establishes a new model for influenza virus RNA transcription and replication. PLoS Pathog 5: e1000462.
[78]
Wigler M, Pellicer A, Silverstein S, Axel R, Urlaub G, et al. (1979) DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A 76: 1373–1376.
[79]
Valcárcel J, Portela A, Ortín J (1991) Regulated M1 mRNA splicing in influenza virus-infected cells. J Gen Virol 72: 1301–1308.
[80]
Perales B, de la Luna S, Palacios I, Ortín J (1996) Mutational analysis identifies functional domains in the Influenza A PB2 polymerase subunit. J Virol 70: 1678–1686.