Abstract
A ddRT-PCR analysis was performed to detect cellular genes that are differentially expressed after influenza A virus (H1N1) infection of A549 cells. After ddRT-PCR, eight DNA fragments were identified. PRPF8, one of the cellular genes that were upregulated after virus infection, was further analyzed since it has previously been identified as a cellular factor required for influenza virus replication. The upregulation of PRPF8 gene expression after viral infection was confirmed using real-time RT-PCR for mRNA detection and Western blot analysis for protein detection. Influenza A virus also upregulated the PRPF8 promoter in a reporter assay. In addition to H1N1, influenza A virus H3N2 and influenza B virus could also activate PRPF8 expression. Therefore, upregulation of PRPF8 expression might be important for the replication of different influenza viruses. Indeed, overexpression of PRPF8 gene enhanced virus production, while knockdown of expression of this gene reduced viral production significantly. To determine which viral protein could enhance PRPF8 gene expression, individual viral genes were cloned and expressed. Among the different viral proteins, expression of either the viral NS1 or PB1 gene could upregulate the PRPF8 expression. Our results from this study indicate that influenza A virus upregulates cellular PRPF8 gene expression through viral NS1 and PB1 proteins to increase virus production.
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Brass AL, Huang IC, Benita Y, John SP, Krishnan MN, Feeley EM, Ryan BJ, Weyer JL, van der Weyden L, Fikrig E, Adams DJ, Xavier RJ, Farzan M, Elledge SJ (2009) The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 139:1243–1254
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622
Chakrabarti AK, Vipat VC, Mukherjee S, Singh R, Pawar SD, Mishra AC (2010) Host gene expression profiling in influenza A virus-infected lung epithelial (A549) cells: a comparative analysis between highly pathogenic and modified H5N1 viruses. Virol J 7:219
Chan S, Choi EA, Shi Y (2011) Pre-mRNA 3’-end processing complex assembly and function. Wiley Interdiscip Rev RNA 2:321–335
Chan SC, Lo SY, Liou JW, Lin MC, Syu CL, Lai MJ, Chen YC, Li HC (2011) Visualization of the structures of the hepatitis C virus replication complex. Biochem Biophys Res Commun 404:574–578
Chang CW, Li HC, Hsu CF, Chang CY, Lo SY (2009) Increased ATP generation in the host cell is required for efficient vaccinia virus production. J Biomed Sci 16:80
Chen W, Calvo PA, Malide D, Gibbs J, Schubert U, Bacik I, Basta S, O’Neill R, Schickli J, Palese P, Henklein P, Bennink JR, Yewdell JW (2001) A novel influenza A virus mitochondrial protein that induces cell death. Nat Med 7:1306–1312
Chen Z, Li Y, Krug RM (1999) Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3’-end processing machinery. EMBO J 18:2273–2283
Chung KM, Song OK, Jang SK (1997) Hepatitis C virus nonstructural protein 5A contains potential transcriptional activator domains. Mol Cells 7:661–667
de la Luna S, Fortes P, Beloso A, Ortin J (1995) Influenza virus NS1 protein enhances the rate of translation initiation of viral mRNAs. J Virol 69:2427–2433
Fodor E, Devenish L, Engelhardt OG, Palese P, Brownlee GG, Garcia-Sastre A (1999) Rescue of influenza A virus from recombinant DNA. J Virol 73:9679–9682
Fortes P, Beloso A, Ortin J (1994) Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport. EMBO J 13:704–712
Gack MU, Albrecht RA, Urano T, Inn KS, Huang IC, Carnero E, Farzan M, Inoue S, Jung JU, Garcia-Sastre A (2009) Influenza A virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by the host viral RNA sensor RIG-I. Cell Host Microb 5:439–449
Grainger RJ, Beggs JD (2005) Prp8 protein: at the heart of the spliceosome. RNA 11:533–557
Hale BG, Randall RE, Ortin J, Jackson D (2008) The multifunctional NS1 protein of influenza A viruses. J Gen Virol 89:2359–2376
Karlas A, Machuy N, Shin Y, Pleissner KP, Artarini A, Heuer D, Becker D, Khalil H, Ogilvie LA, Hess S, Maurer AP, Muller E, Wolff T, Rudel T, Meyer TF (2010) Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 463:818–822
Kato N, Lan KH, Ono-Nita SK, Shiratori Y, Omata M (1997) Hepatitis C virus nonstructural region 5A protein is a potent transcriptional activator. J Virol 71:8856–8859
Kawada J, Kimura H, Kamachi Y, Nishikawa K, Taniguchi M, Nagaoka K, Kurahashi H, Kojima S, Morishima T (2006) Analysis of gene-expression profiles by oligonucleotide microarray in children with influenza. J Gen Virol 87:1677–1683
Krug RM, Yuan W, Noah DL, Latham AG (2003) Intracellular warfare between human influenza viruses and human cells: the roles of the viral NS1 protein. Virology 309:181–189
Kurtovic-Kozaric A, Przychodzen B, Singh J, Konarska MM, Clemente MJ, Otrock ZK, Nakashima M, Hsi ED, Yoshida K, Shiraishi Y, Chiba K, Tanaka H, Miyano S, Ogawa S, Boultwood J, Makishima H, Maciejewski JP, Padgett RA (2015) PRPF8 defects cause missplicing in myeloid malignancies. Leukemia 29:126–136
Lamb RA, Takeda M (2001) Death by influenza virus protein. Nat Med 7:1286–1288
Li W, Liu Y, Mukhtar MM, Gong R, Pan Y, Rasool ST, Gao Y, Kang L, Hao Q, Peng G, Chen Y, Chen X, Wu J, Zhu Y (2008) Activation of interleukin-32 pro-inflammatory pathway in response to influenza A virus infection. PLoS One 3:e1985
Lu Y, Qian XY, Krug RM (1994) The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing. Genes Dev 8:1817–1828
Ma HC, Ku YY, Hsieh YC, Lo SY (2007) Characterization of the cleavage of signal peptide at the C-terminus of hepatitis C virus core protein by signal peptide peptidase. J Biomed Sci 14:31–41
Ma HC, Fang CP, Hsieh YC, Chen SC, Li HC, Lo SY (2008) Expression and membrane integration of SARS-CoV M protein. J Biomed Sci 15:301–310
Ma HC, Lin TW, Li H, Iguchi-Ariga SM, Ariga H, Chuang YL, Ou JH, Lo SY (2008) Hepatitis C virus ARFP/F protein interacts with cellular MM-1 protein and enhances the gene trans-activation activity of c-Myc. J Biomed Sci 15:417–425
Muramoto Y, Noda T, Kawakami E, Akkina R, Kawaoka Y (2013) Identification of novel influenza A virus proteins translated from PA mRNA. J Virol 87:2455–2462
Noah DL, Twu KY, Krug RM (2003) Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral NS1A protein via its binding to a cellular protein required for the 3’ end processing of cellular pre-mRNAS. Virology 307:386–395
PaMLS P (2007) Orthomyxoviridae: the viruses and their replication. In: Virology F (ed) DMaPMH K. Lippincott Williams & Wilkins, Philadelphia, pp 1647–1690
Qian XY, Alonso-Caplen F, Krug RM (1994) Two functional domains of the influenza virus NS1 protein are required for regulation of nuclear export of mRNA. J Virol 68:2433–2441
Rozee KR, Williams GL, Van Rooyen CE (1958) Detection and titration of Asian influenza A virus by HeLa cell and monkey kidney cell cultures. Science 128:591–592
Salvatore M, Basler CF, Parisien JP, Horvath CM, Bourmakina S, Zheng H, Muster T, Palese P, Garcia-Sastre A (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
Sarmento L, Afonso CL, Estevez C, Wasilenko J, Pantin-Jackwood M (2008) Differential host gene expression in cells infected with highly pathogenic H5N1 avian influenza viruses. Vet Immunol Immunopathol 125:291–302
Satterly N, Tsai PL, van Deursen J, Nussenzveig DR, Wang Y, Faria PA, Levay A, Levy DE, Fontoura BM (2007) Influenza virus targets the mRNA export machinery and the nuclear pore complex. Proc Natl Acad Sci USA 104:1853–1858
Shapira SD, Gat-Viks I, Shum BO, Dricot A, de Grace MM, Wu L, Gupta PB, Hao T, Silver SJ, Root DE, Hill DE, Regev A, Hacohen N (2009) A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection. Cell 139:1255–1267
Shih SR, Tsao KC, Ning HC, Huang YC, Lin TY (1999) Diagnosis of respiratory tract viruses in 24 h by immunofluorescent staining of shell vial cultures containing Madin-Darby Canine Kidney (MDCK) cells. J Virol Methods 81:77–81
Tanimoto A, Ide Y, Arima N, Sasaguri Y, Padmanabhan R (1997) The amino terminal deletion mutants of hepatitis C virus nonstructural protein NS5A function as transcriptional activators in yeast. Biochem Biophys Res Commun 236:360–364
Tong S, Li Y, Rivailler P, Conrardy C, Castillo DA, Chen LM, Recuenco S, Ellison JA, Davis CT, York IA, Turmelle AS, Moran D, Rogers S, Shi M, Tao Y, Weil MR, Tang K, Rowe LA, Sammons S, Xu X, Frace M, Lindblade KA, Cox NJ, Anderson LJ, Rupprecht CE, Donis RO (2012) A distinct lineage of influenza A virus from bats. Proc Natl Acad Sci USA 109:4269–4274
Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, Yang H, Chen X, Recuenco S, Gomez J, Chen LM, Johnson A, Tao Y, Dreyfus C, Yu W, McBride R, Carney PJ, Gilbert AT, Chang J, Guo Z, Davis CT, Paulson JC, Stevens J, Rupprecht CE, Holmes EC, Wilson IA, Donis RO (2013) New world bats harbor diverse influenza A viruses. PLoS Pathog 9:e1003657
Wright PFGN, Kawaoka Y (2007) Orthomyxoviruses. In: Virology F (ed) DMaPMH K. Lippincott Williams & Wilkins, Philadelphia, pp 1692–1740
Yang CH, Li HC, Jiang JG, Hsu CF, Wang YJ, Lai MJ, Juang YL, Lo SY (2010) Enterovirus type 71 2A protease functions as a transcriptional activator in yeast. J Biomed Sci 17:65
Acknowledgements
We thank Dr. George G. Brownlee for providing 12 plasmids to generate influenza A virus WSN33 (H1N1), and Dr. Shin-Ru Shih for providing MDCK cells and clinical isolates of influenza A virus (H3N2) and influenza B virus. RNAi reagents were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica, supported by grants from the NSC National Research Program for Genomic Medicine (NSC 94-3112-B-001-003 and NSC 94-3112-B-001-018-Y). This work was supported by grants from the Ministry of Science and Technology, R.O.C (MOST 104-2320-B-320-007) to Dr. Shih-Yen Lo and from the Tzu Chi University to Dr. Shih-Yen Lo (TCIRP 101005-03 and TCIRP 103002-03) and to Dr. Hui-Chun Li (TCMRC-P-101015 and TCIRP 103002-02).
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C.-H. Yang and H.-C. Li have contributed equally to this work.
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Yang, CH., Li, HC., Shiu, YL. et al. Influenza A virus upregulates PRPF8 gene expression to increase virus production. Arch Virol 162, 1223–1235 (2017). https://doi.org/10.1007/s00705-016-3210-3
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DOI: https://doi.org/10.1007/s00705-016-3210-3