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The interaction of cellular protein ANP32A with influenza A virus polymerase component PB2 promotes vRNA synthesis

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Abstract

The subunits PA, PB1, and PB2 of influenza A virus RNA polymerase are essential for efficient viral RNA synthesis and virus replication because of their role in recruiting multiple nuclear proteins. ANP32A is an acidic leucine-rich nuclear phosphoprotein 32 (ANP32) family member and a crucial cellular protein that determines the species specificity of the influenza virus RNA polymerase activity. However, how ANP32A modulates polymerase activity remains largely unknown. In this study, we showed that viral RNA synthesis was increased in A549 cells overexpressing ANP32A and decreased after treatment with ANP32A RNAi. This decrease in RNA synthesis was reversed by rescued ANP32A expression. The results of docking modeling, co-immunoprecipitation, and yeast two-hybrid assays showed that PB2 was the only subunit of the three that interacted with ANP32A. The C-terminal portion of ANP32A and the middle domains (residues 307-534) of PB2 were required for PB2-ANP32A interaction. Glu189 and Glu196 in ANP32A and Gly450 and Gln447 in PB2 were essential for interaction between ANP32A and PB2. These residues were located in conserved regions of the ANP32A or PB2 protein sequences. These data suggest that ANP32A is recruited to the polymerase through direct interaction with PB2 via critical amino acid residue interactions and promotes viral RNA synthesis. Our findings might provide new insights into the molecular mechanisms underlying influenza virus RNA synthesis and replication in infected human cells.

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References

  1. Te Velthuis AJ, Fodor E (2016) Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis. Nat Rev Microbiol 14:479–493

    Article  CAS  Google Scholar 

  2. Fodor E (2013) The RNA polymerase of influenza a virus: mechanisms of viral transcription and replication. Acta Virol 57:113–122

    Article  CAS  PubMed  Google Scholar 

  3. Bortz E, Westera L, Maamary J, Steel J, Albrecht RA, Manicassamy B, Chase G, Martinez-Sobrido L, Schwemmle M, Garcia-Sastre A (2011) Host- and strain-specific regulation of influenza virus polymerase activity by interacting cellular proteins. MBio 2:e00151-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zhao M, Wang L, Li S (2017) Influenza A virus-host protein interactions control viral pathogenesis. Int J Mol Sci 18:1673

    Article  CAS  PubMed Central  Google Scholar 

  5. Nagata K, Kawaguchi A, Naito T (2008) Host factors for replication and transcription of the influenza virus genome. Rev Med Virol 18:247–260

    Article  CAS  PubMed  Google Scholar 

  6. Momose F, Naito T, Yano K, Sugimoto S, Morikawa Y, Nagata K (2002) Identification of Hsp90 as a stimulatory host factor involved in influenza virus RNA synthesis. J Biol Chem 277:45306–45314

    Article  CAS  PubMed  Google Scholar 

  7. Momose F, Basler CF, O’Neill RE, Iwamatsu A, Palese P, Nagata K (2001) Cellular splicing factor RAF-2p48/NPI-5/BAT1/UAP56 interacts with the influenza virus nucleoprotein and enhances viral RNA synthesis. J Virol 75:1899–1908

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wang Q, Li Q, Liu R, Zheng M, Wen J Zhao G (2016) Host cell interactome of PA protein of H5N1 influenza A virus in chicken cells. J Proteom 136:48–54

    Article  CAS  Google Scholar 

  9. Naito T, Kiyasu Y, Sugiyama K, Kimura A, Nakano R, Matsukage A, Nagata K (2007) An influenza virus replicon system in yeast identified Tat-SF1 as a stimulatory host factor for viral RNA synthesis. Proc Natl Acad Sci USA 104:18235–18240

    Article  PubMed  Google Scholar 

  10. Cao M, Wei C, Zhao L, Wang J, Jia Q, Wang X, Jin Q, Deng T (2014) DnaJA1/Hsp40 is co-opted by influenza A virus to enhance its viral RNA polymerase activity. J Virol 88:14078–14089

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zheng W, Tao YJ (2013) Structure and assembly of the influenza A virus ribonucleoprotein complex. FEBS Lett 587:1206–1214

    Article  CAS  PubMed  Google Scholar 

  12. Dawson WK, Lazniewski M, Plewczynski D (2017) RNA structure interactions and ribonucleoprotein processes of the influenza A virus. Brief Funct Genom 17:402–414

    Google Scholar 

  13. Compans RW, Content J, Duesberg PH (1972) Structure of the ribonucleoprotein of influenza virus. J Virol 10:795–800

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Coloma R, Valpuesta JM, Arranz R, Carrascosa JL, Ortin J, Martin-Benito J (2009) The structure of a biologically active influenza virus ribonucleoprotein complex. PLoS Pathog 5:e1000491

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Pflug A, Lukarska M, Resa-Infante P, Reich S, Cusack S (2017) Structural insights into RNA synthesis by the influenza virus transcription-replication machine. Virus Res 234:103–117

    Article  CAS  PubMed  Google Scholar 

  16. Eisfeld AJ, Neumann G, Kawaoka Y (2015) At the centre: influenza A virus ribonucleoproteins. Nat Rev Microbiol 13:28–41

    Article  CAS  PubMed  Google Scholar 

  17. Engelhardt OG, Fodor E (2006) Functional association between viral and cellular transcription during influenza virus infection. Rev Med Virol 16:329–345

    Article  CAS  PubMed  Google Scholar 

  18. Guilligay D, Tarendeau F, Resa-Infante P, Coloma R, Crepin T, Sehr P, Lewis J, Ruigrok RW, Ortin J, Hart DJ, Cusack S (2008) The structural basis for cap binding by influenza virus polymerase subunit PB2. Nat Struct Mol Biol 15:500–506

    Article  CAS  PubMed  Google Scholar 

  19. Dias A, Bouvier D, Crepin T, McCarthy AA, Hart DJ, Baudin F, Cusack S, Ruigrok RW (2009) The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458:914–918

    Article  CAS  PubMed  Google Scholar 

  20. Crepin T, Dias A, Palencia A, Swale C, Cusack S, Ruigrok RW (2010) Mutational and metal binding analysis of the endonuclease domain of the influenza virus polymerase PA subunit. J Virol 84:9096–9104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Reilly PT, Yu Y, Hamiche A, Wang L (2014) Cracking the ANP32 whips: important functions, unequal requirement, and hints at disease implications. BioEssays 36:1062–1071

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Tsujio I, Zaidi T, Xu J, Kotula L, Grundke-Iqbal I, Iqbal K (2005) Inhibitors of protein phosphatase-2A from human brain structures, immunocytological localization and activities towards dephosphorylation of the Alzheimer type hyperphosphorylated tau. FEBS Lett 579:363–372

    Article  CAS  PubMed  Google Scholar 

  23. Zamora-Caballero S, Siauciunaite-Gaubard L, Bravo J (2015) High-resolution crystal structure of the leucine-rich repeat domain of the human tumour suppressor PP32A (ANP32A). Acta Crystallogr Sect F Struct Biol Commun 71:684–687

    Article  CAS  Google Scholar 

  24. Kadota S, Nagata K (2011) pp32, an INHAT component, is a transcription machinery recruiter for maximal induction of IFN-stimulated genes. J Cell Sci 124:892–899

    Article  CAS  PubMed  Google Scholar 

  25. Sugiyama K, Kawaguchi A, Okuwaki M, Nagata K (2015) pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA. eLife 4:e08939

    Article  PubMed  PubMed Central  Google Scholar 

  26. Long JS, Giotis ES, Moncorge O, Frise R, Mistry B, James J, Morisson M, Iqbal M, Vignal A, Skinner MA, Barclay WS (2016) Species difference in ANP32A underlies influenza A virus polymerase host restriction. Nature 529:101–104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Mehle A (2016) The Avian influenza virus polymerase brings ANP32A home to roost. Cell Host Microbe 19:137–138

    Article  CAS  PubMed  Google Scholar 

  28. Domingues P, Hale BG (2017) Functional insights into ANP32A-dependent influenza a virus polymerase host restriction. Cell Rep 20:2538–2546

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Zhou Z, Cao M, Guo Y, Zhao L, Wang J, Jia X, Li J, Wang C, Gabriel G, Xue Q, Yi Y, Cui S, Jin Q, Wang J, Deng T (2014) Fragile X mental retardation protein stimulates ribonucleoprotein assembly of influenza A virus. Nat Commun 5:3259

    Article  CAS  PubMed  Google Scholar 

  30. Yang J, Zhang Y (2015) I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res 43:W174–181

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Huyton T, Wolberger C (2007) The crystal structure of the tumor suppressor protein pp32 (Anp32a): structural insights into Anp32 family of proteins. Protein Sci 16:1308–1315

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Tuncbag N, Gursoy A, Nussinov R, Keskin O (2011) Predicting protein-protein interactions on a proteome scale by matching evolutionary and structural similarities at interfaces using PRISM. Nat Protoc 6:1341–1354

    Article  CAS  PubMed  Google Scholar 

  33. Hatada E, Hasegawa M, Mukaigawa J, Shimizu K, Fukuda R (1989) Control of influenza virus gene expression: quantitative analysis of each viral RNA species in infected cells. J Biochem 105:537–546

    Article  CAS  PubMed  Google Scholar 

  34. Meyerson NR, Zhou L, Guo YR, Zhao C, Tao YJ, Krug RM, Sawyer SL (2017) Nuclear TRIM25 specifically targets influenza virus ribonucleoproteins to block the onset of RNA chain elongation. Cell Host Microbe 22(627–638):e627

    Article  CAS  Google Scholar 

  35. Zheng X, Wang X, Tu F, Wang Q, Fan Z, Gao G (2017) trim25 is required for the antiviral activity of zinc finger antiviral protein. J Virol 91:e00088-17

    Article  PubMed  PubMed Central  Google Scholar 

  36. Zhu Y, Chen G, Lv F, Wang X, Ji X, Xu Y, Sun J, Wu L, Zheng YT, Gao G (2011) Zinc-finger antiviral protein inhibits HIV-1 infection by selectively targeting multiply spliced viral mRNAs for degradation. Proc Natl Acad Sci USA 108:15834–15839

    Article  PubMed  Google Scholar 

  37. Guo X, Carroll JW, Macdonald MR, Goff SP, Gao G (2004) The zinc finger antiviral protein directly binds to specific viral mRNAs through the CCCH zinc finger motifs. J Virol 78:12781–12787

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Guo X, Ma J, Sun J, Gao G (2007) The zinc-finger antiviral protein recruits the RNA processing exosome to degrade the target mRNA. Proc Natl Acad Sci USA 104:151–156

    Article  CAS  PubMed  Google Scholar 

  39. Zhu Y, Wang X, Goff SP, Gao G (2012) Translational repression precedes and is required for ZAP-mediated mRNA decay. EMBO J 31:4236–4246

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Thierry E, Guilligay D, Kosinski J, Bock T, Gaudon S, Round A, Pflug A, Hengrung N, El Omari K, Baudin F, Hart DJ, Beck M, Cusack S (2016) Influenza polymerase can adopt an alternative configuration involving a radical repacking of PB2 domains. Mol Cell 61:125–137

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Long JX, Wang QZ, Lu JH, Liu YL, Liu XF (2005) [Cloning of full-length genes of H5N1 subtype Avian influenza virus strain A/duck/Shandong/093/2004 and analysis of the sequences]. Wei sheng wu xue bao = Acta microbiologica Sinica 45:690–696

  42. Zhao B, Zhang X, Zhu W, Teng Z, Yu X, Gao Y, Wu D, Pei E, Yuan Z, Yang L, Wang D, Shu Y, Wu F (2014) Novel avian influenza A(H7N9) virus in tree sparrow, Shanghai, China, 2013. Emerg Infect Dis 20:850–853

    Article  PubMed  PubMed Central  Google Scholar 

  43. El Houadfi M, Fellahi S, Nassik S, Guerin JL, Ducatez MF (2016) First outbreaks and phylogenetic analyses of avian influenza H9N2 viruses isolated from poultry flocks in Morocco. Virol J 13:140

    Article  CAS  PubMed  Google Scholar 

  44. Beerens N, Koch G, Heutink R, Harders F, Vries DPE, Ho C, Bossers A, Elbers A (2018) Novel highly pathogenic avian influenza A(H5N6) virus in the Netherlands, December 2017. Emerg Infect Dis 24:770–773

    Article  CAS  PubMed Central  Google Scholar 

  45. Mi Z, Liu W, Fan H, An X, Pei G, Wang W, Xu X, Ma M, Zhang Z, Cao W, Tong Y (2013) Complete genome sequence of avian influenza virus A/chicken/Jiangsu/1001/2013(H5N2), demonstrating continuous reassortance of H5N2 in China. Genome Announc 1:e00469-13

    Article  PubMed  PubMed Central  Google Scholar 

  46. Gao Z, Hu J, Liang Y, Yang Q, Yan K, Liu D, Wang X, Gu M, Liu X, Hu S, Hu Z, Liu H, Liu W, Chen S, Peng D, Jiao XA, Liu X (2017) Generation and comprehensive analysis of host cell interactome of the PA protein of the highly pathogenic H5N1 avian influenza virus in mammalian cells. Front Microbiol 8:739

    Article  PubMed  PubMed Central  Google Scholar 

  47. Baker SF, Ledwith MP, Mehle A (2018) Differential splicing of ANP32A in birds alters its ability to stimulate RNA synthesis by restricted influenza polymerase. Cell Rep 24(2581–2588):e2584

    Google Scholar 

  48. Reich S, Guilligay D, Pflug A, Malet H, Berger I, Crepin T, Hart D, Lunardi T, Nanao M, Ruigrok RW, Cusack S (2014) Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Nature 516:361–366

    Article  CAS  PubMed  Google Scholar 

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Funding

This study was funded by the Faculty Development Grants of Hubei University of Medicine (No. 2016QDJZR03), the Natural Science Foundation of Hubei Province (No. 2018CFB185), the Scientific and Technological Project of Shiyan City (No. 17Y32), and Project of Hubei Provincial Department of Education.

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Author notes

  1. Xiuli Wei and Zhixin Liu contributed equally to this work.

Authors and Affiliations

  1. School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, Hubei, 442000, China

    Xiuli Wei, Zhixin Liu, Jingjie Wang, Jing Yang, Yang Guo & Long Liu

  2. Department of Infectious Diseases, Renmin Hospital, Hubei University of Medicine, Shiyan, 442000, Hubei, China

    Xiuli Wei, Huabing Tan & Long Liu

  3. Biomedical Research Institute, Hubei University of Medicine, Shiyan, 442000, Hubei, China

    Ruiping Yang

  4. College of Life Sciences, Northwest A&F University, Yangling, 712100, Shaanxi, China

    Hongying Chen

  5. The First College of Clinical Medical Science, China Three Gorges University/Yichang Central People’s Hospital, Yichang, 443000, China

    Qiang Liu

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  1. Xiuli Wei
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Wei, X., Liu, Z., Wang, J. et al. The interaction of cellular protein ANP32A with influenza A virus polymerase component PB2 promotes vRNA synthesis. Arch Virol 164, 787–798 (2019). https://doi.org/10.1007/s00705-018-04139-z

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