Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Functional Characterization and Proteomic Analysis of Porcine Deltacoronavirus Accessory Protein NS7

  • Choi, Subin (Animal Virology Laboratory, School of Life Sciences, BK21 PLUS KNU Creative BioResearch Group, Kyungpook National University) ;
  • Lee, Changhee (Animal Virology Laboratory, School of Life Sciences, BK21 PLUS KNU Creative BioResearch Group, Kyungpook National University)
  • Received : 2019.08.07
  • Accepted : 2019.09.10
  • Published : 2019.11.28
Download PDF
⟨ Previous Next ⟩

Abstract

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus that causes diarrhea in neonatal piglets. Like other coronaviruses, PDCoV encodes at least three accessory or species-specific proteins; however, the biological roles of these proteins in PDCoV replication remain undetermined. As a first step toward understanding the biology of the PDCoV accessory proteins, we established a stable porcine cell line constitutively expressing the PDCoV NS7 protein in order to investigate the functional characteristics of NS7 for viral replication. Confocal microscopy and subcellular fractionation revealed that the NS7 protein was extensively distributed in the mitochondria. Proteomic analysis was then conducted to assess the expression dynamics of the host proteins in the PDCoV NS7-expressing cells. High-resolution two-dimensional gel electrophoresis initially identified 48 protein spots which were differentially expressed in the presence of NS7. Seven of these spots, including two up-regulated and five down-regulated protein spots, showed statistically significant alterations, and were selected for subsequent protein identification. The affected cellular proteins identified in this study were classified into functional groups involved in various cellular processes such as cytoskeleton networks and cell communication, metabolism, and protein biosynthesis. A substantial down-regulation of α-actinin-4 was confirmed in NS7-expressing and PDCoV-infected cells. These proteomic data will provide insights into the understanding of specific cellular responses to the accessory protein during PDCoV infection.

Keywords

References

  1. Woo PC, Lau SK, Lam CS, Lau CC, Tsang AK, Lau JH, et al. 2012. Discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J. Virol. 86: 3995-4008. https://doi.org/10.1128/JVI.06540-11
  2. Li G, Chen Q, Harmon KM, Yoon KJ, Schwartz KJ, Hoogland MJ, et al. 2014. Full-length genome sequence of porcine deltacoronavirus strain USA/IA/2014/8734. Genome Announc. 2: e00278-14.
  3. Marthaler D, Jiang Y, Collins J, Rossow K. 2014. Complete genome sequence of strain SDCV/USA/Illinois121/2014, a porcine deltacoronavirus from the United States. Genome Announc. 2: e00218-14.
  4. Marthaler D, Raymond L, Jiang Y, Collins J, Rossow K, Rovira A. 2014. Rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus. Emerg. Infect. Dis. 20: 1347-1350. https://doi.org/10.3201/eid2008.140526
  5. Wang L, Byrum B, Zhang Y. 2014. Detection and genetic characterization of deltacoronavirus in pigs, Ohio, USA, 2014. Emerg. Infect. Dis. 20: 1227-1230. https://doi.org/10.3201/eid2007.140296
  6. Chen F, Zhu Y, Wu M, Ku X, Yao L, He Q. 2015. Full-length genome characterization of Chinese porcine deltacoronavirus strain CH/SXD1/2015. Genome Announc. 3: e01284-15.
  7. Hu H, Jung K, Vlasova AN, Chepngeno J, Lu Z, Wang Q, et al. 2015. Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States. J. Clin. Microbiol. 53: 1537-1548. https://doi.org/10.1128/JCM.00031-15
  8. Ma Y, Zhang Y, Liang X, Lou F, Oglesbee M, Krakowka S, et al. 2015. Origin, evolution, and virulence of porcine deltacoronaviruses in the United States. MBio 6: e00064.
  9. Jung K, Hu H, Eyerly B, Lu Z, Chepngeno J, Saif LJ. 2015. Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs. Emerg. Infect. Dis. 21: 650-654. https://doi.org/10.3201/eid2104.141859
  10. Lee S. Lee C. 2014. Complete genome characterization of Korean porcine deltacoronavirus strain KOR/KNU14-04/ 2014. Genome Announc. 2: e01191-14.
  11. Dong N, Fang L, Zeng S, Sun Q, Chen H, Xiao S. 2015. Porcine deltacoronavirus in mainland China. Emerg. Infect. Dis. 21: 2254-2255. https://doi.org/10.3201/eid2112.150283
  12. Janetanakit T, Lumyai M, Bunpapong N, Boonyapisitsopa S, Chaiyawong S, Nonthabenjawan N, et al. 2016. Porcine deltacoronavirus, Thailand, 2015. Emerg. Infect. Dis. 2: 757-759.
  13. Jang G, Lee KK, Kim SH, Lee C. 2017. Prevalence, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus in South Korea, 2014-2016. Transbound. Emerg. Dis. 64: 1364-1370. https://doi.org/10.1111/tbed.12690
  14. de Groot RJ, Baker SC, Bari R, Enjuanes L, Gorbalenya AE, Holme KV, et al. 2011. Coronaviridae. pp. 806-828. In King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds.), Virus Taxonomy: 9th Report of the International Committee on Taxonomy of Viruses, Elsevier, Oxford.
  15. Lai MC, Perlman S, Anderson LJ, 2007. Coronaviridae. pp. 1305-1336. In Knipe DM, Howley PM, Griffin DE, Martin M A, L amb RA, Roizman B, a nd S traus SE ( eds.), Fields Virology, 5th Ed. Lippincott Williams & Wilkins, Philadelphia, Pennsylvania.
  16. Fang P, Fang L, Liu X, Hong Y, Wang Y, Dong N, et al. 2016. Identification and subcellular localization of porcine deltacoronavirus accessory protein NS6. Virology 499: 170-177. https://doi.org/10.1016/j.virol.2016.09.015
  17. Fang P, Fang L, Hong Y, Liu X, Dong N, Ma P, et al. 2017. Discovery of a novel accessory protein NS7a encoded by porcine deltacoronavirus. J. Gen. Virol. 98: 173-178. https://doi.org/10.1099/jgv.0.000690
  18. McBride R, Fielding BC. 2012. The role of severe acute respiratory syndrome (SARS)-coronavirus accessory proteins in virus pathogenesis. Viruses 4: 2902-2923. https://doi.org/10.3390/v4112902
  19. Liu DX, Fung TS, Chong KK, Shukla A, Hilgenfeld R. 2014. Accessory proteins of SARS-CoV and other coronaviruses. Antiviral Res. 109: 97-109. https://doi.org/10.1016/j.antiviral.2014.06.013
  20. de Haan CA, Masters PS, Shen X, Weiss S, Rottier PJ. 2002. The group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host. Virology 296: 177-189. https://doi.org/10.1006/viro.2002.1412
  21. Yount B, Roberts RS, Sims AC, Deming D, Frieman MB, Sparks J, et al. 2005. Severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice. J. Virol. 79: 14909-14922. https://doi.org/10.1128/JVI.79.23.14909-14922.2005
  22. Zhang R, Wang K, Ping X, Yu W, Qian Z, Xiong S, et al. 2015. The ns12.9 accessory protein of human coronavirus OC43 is a viroporin involved in virion morphogenesis and pathogenesis. J. Virol. 89: 11383-11395. https://doi.org/10.1128/JVI.01986-15
  23. Menachery VD, Mitchell HD, Cockrell AS, Gralinski LE, Yount BL Jr, Graham RL, et al. 2017. MERS-CoV accessory ORFs play key role for infection and pathogenesis. MBio 8: e00665-17.
  24. Niemeyer D, Zillinger T, Muth D, Zielecki F, Horvath G, Suliman T, et al. 2013. Middle East respiratory syndrome coronavirus accessory protein 4a is a type I interferon antagonist. J. Virol. 87: 2489-2495. https://doi.org/10.1128/JVI.02879-12
  25. Siu KL, Yeung ML, Kok KH, Yuen KS, Kew C, Lui PY, et al. 2014. Middle East respiratory syndrome coronavirus 4a protein is a double-stranded RNA-binding protein that suppresses PACT-induced activation of RIG-I and MDA5 in the innate antiviral response. J. Virol. 88: 4866-4876. https://doi.org/10.1128/JVI.03649-13
  26. Zeng LP, Gao YT, Ge XY, Zhang Q, Peng C, Yang XL, et al. 2016. Bat severe acute respiratory syndrome-like coronavirus WIV1 encodes an extra accessory protein, ORFX, involved in modulation of the host immune response. J. Virol. 90: 6573-6582. https://doi.org/10.1128/JVI.03079-15
  27. Lee S, Lee C. 2015. Functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus. Virus. Res. 208: 136-145. https://doi.org/10.1016/j.virusres.2015.06.013
  28. Jang G, Kim SH, Lee YJ, Kim S, Lee DS, Lee KK, et al. 2018. Isolation and characterization of a Korean porcine deltacoronavirus strain KNU16-07. J. Vet. Sci. 19: 586-590. https://doi.org/10.4142/jvs.2018.19.4.586
  29. Sambrook J, Russell DW. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  30. Creixell P, Schoof EM, Tan CS, Linding R. 2012. Mutational properties of amino acid residues: implications for evolvability of phosphorylatable residues. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 367: 2584-2593. https://doi.org/10.1098/rstb.2012.0076
  31. Lee YJ, Park CK, Nam E, Kim SH, Lee OS, Lee D, et al. 2010. Generation of a porcine alveolar macrophage cell line for the growth of porcine reproductive and respiratory syndrome virus. J. Virol. Methods 163: 410-415. https://doi.org/10.1016/j.jviromet.2009.11.003
  32. Nam E, Lee C. 2010. Contribution of the porcine aminopeptidase N (CD13) receptor density to porcine epidemic diarrhea virus infection. Vet. Microbiol. 144: 41-50. https://doi.org/10.1016/j.vetmic.2009.12.024
  33. Oh J, Lee C. 2012. Proteomic characterization of a novel structural protein ORF5a of porcine reproductive and respiratory syndrome virus. Virus Res. 169: 255-263. https://doi.org/10.1016/j.virusres.2012.08.015
  34. Lee YJ, Lee C. 2018. Porcine deltacoronavirus induces caspase-dependent apoptosis through activation of the cytochrome c-mediated intrinsic mitochondrial pathway. Virus Res. 253: 112-123. https://doi.org/10.1016/j.virusres.2018.06.008
  35. Oakley BR, Kirsch DR, Morris NR. 1980. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105: 361-363. https://doi.org/10.1016/0003-2697(80)90470-4
  36. Shevchenko A, Wil M, Vorm O, Mann M. 1996. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68: 850-858. https://doi.org/10.1021/ac950914h
  37. Fernandez J, Gharahdaghi F, Mische SM. 1998. Routine identification of proteins from sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gels or polyvinyl difluoride membranes using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Electrophoresis 19: 1036-1045. https://doi.org/10.1002/elps.1150190619
  38. Sagong M, Lee C. 2011. Porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-${\beta}$ production by inhibiting IRF3 activation in immortalized porcine alveolar macrophages. Arch. Virol. 156: 2187-2195. https://doi.org/10.1007/s00705-011-1116-7
  39. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402-408. https://doi.org/10.1006/meth.2001.1262
  40. Zhang H, Guo X, Ge X, Chen Y, Sun Q, Yang H. 2009. Changes in the cellular proteins of pulmonary alveolar macrophage infected with porcine reproductive and respiratory syndrome virus by proteomics analysis. J. Proteome. Res. 8: 3091-3097. https://doi.org/10.1021/pr900002f
  41. Alfonso P, Rivera J, Hernaez B, Alonso C, Escriban JM. 2004. Identification of cellular proteins modified in response to African swine fever virus infection by proteomics. Proteomics 4: 2037-2046. https://doi.org/10.1002/pmic.200300742
  42. Brasier AR, Spratt H, Wu Z, Boldogh I, Zhang Y, Garofalo RP, et al. 2004. Nuclear heat shock response and novel nuclear domain 10 reorganization in respiratory syncytial virus-infected A549 cells identified by high-resolution twodimensional gel electrophoresis. J. Virol. 78: 11461-11476. https://doi.org/10.1128/JVI.78.21.11461-11476.2004
  43. Neuman BW, Joseph JS, Saikatendu KS, Serrano P, Chatterjee A, Johnson MA, et al. 2008. Proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3. J. Virol. 82: 5279-5294. https://doi.org/10.1128/JVI.02631-07
  44. Ringrose JH, Jeeninga RE, Berkhout B, Speijer D. 2008. Proteomic studies reveal coordinated changes in T-cell expression patterns upon infection with human immunodeficiency virus type 1. J. Virol. 82: 4320-4330. https://doi.org/10.1128/JVI.01819-07
  45. Sagong M, Lee C. 2010. Differential cellular protein expression in continuous porcine alveolar macrophages regulated by the porcine reproductive and respiratory syndrome virus nucleocapsid protein. Virus Res. 151: 88-96. https://doi.org/10.1016/j.virusres.2010.04.003
  46. Zhang J, Li D, Zheng Y, Cui Y, Feng K, Zhou J, et al. 2008. Proteomic profiling of hepatitis B virus-related hepatocellular carcinoma in China: a SELDI-TOF-MS study. Int. J. Clin. Exp. Pathol. 1: 352-361.
  47. McBride R, van Zyl M, Fielding BC. 2014. The coronavirus nucleocapsid is a multifunctional protein. Viruses 6: 2991-3018. https://doi.org/10.3390/v6082991
  48. Martinez AI, Perez-Arellano I, Pekkala S, Barcelona B, Cervera J. 2010. Genetic, structural and biochemical basis of carbamoyl phosphate synthetase 1 deficiency. Mol. Genet. Metab. 101: 311-323. https://doi.org/10.1016/j.ymgme.2010.08.002
  49. Mignotte B, Vayssiere JL. 1998. Mitochondria and apoptosis. Eur. J. Biochem. 252: 1-15. https://doi.org/10.1046/j.1432-1327.1998.2520001.x
  50. Winder SJ, Ayscough KR. 2005. Actin-binding proteins. J. Cell Sci. 118: 651-654. https://doi.org/10.1242/jcs.01670
  51. An HT, Yoo S, Ko J. 2016. ${\alpha}$-actinin-4 induces the epithelialto-mesenchymal transition and tumorigenesis via regulation of snail expression and $\beta$-catenin stabilization in cervical cancer. Oncogene 35: 5893-5904. https://doi.org/10.1038/onc.2016.117
  52. Agarwal N, Adhikari AS, Iyer SV, Hekmatdoost K, Welch DR, Iwakuma T. 2013. MTBP suppresses cell migration and filopodia formation by inhibiting ACTN4. Oncogene 32: 462-470. https://doi.org/10.1038/onc.2012.69
  53. Aksenova V, Turoverova L, Khotin M, Magnusson KE, Tulchinsky E, Melino G, et al. 2013. Actin-binding protein alpha-actinin 4 (ACTN4) is a transcriptional co-activator of RelA/p65 sub-unit of NF-kB. Oncotarget 4: 362-372. https://doi.org/10.18632/oncotarget.901
  54. Honda K, Yamada T, Endo R, Ino Y, Gotoh M, Tsuda H, et al. 1998. Actinin-4, a novel actin-bundling protein associated with cell motility and cancer invasion. J. Cell Biol. 140: 1383-1393. https://doi.org/10.1083/jcb.140.6.1383
  55. Khurana S, Chakraborty S, Cheng X, Su YT, Kao HY. 2011. The actin-binding protein, actinin alpha 4 (ACTN4), is a nuclear receptor coactivator that promotes proliferation of MCF-7 breast cancer cells. J. Biol. Chem. 286: 1850-1859. https://doi.org/10.1074/jbc.M110.162107
  56. Kumeta M, Yoshimura SH, Harata M, Takeyasu K. 2010. Molecular mechanisms underlying nucleocytoplasmic shuttling of actinin-4. J. Cell Sci. 123: 1020-1030. https://doi.org/10.1242/jcs.059568
  57. Sharma S, Mayank AK, Nailwal H, Tripathi S, Patel JR, Bowzard JB, et al. 2014. Influenza A viral nucleoprotein interacts with cytoskeleton scaffolding protein $\alpha$-actinin-4 for viral replication. FEBS J. 281: 2899-2914. https://doi.org/10.1111/febs.12828
  58. Zhao X, Khurana S, Charkraborty S, Tian Y, Sedor JR, Bruggman LA, et al. 2016. ${\alpha}$-actinin 4 (ACTN4) regulates glucocorticoid receptor-mediated transactivation and transrepression in podocytes. J. Biol. Chem. 292: 1637-1647. https://doi.org/10.1074/jbc.M116.755546
  59. Gross SR. 2013. Actin binding proteins: their ups and downs in metastatic life. Cell Adh. Migr. 7: 199-213. https://doi.org/10.4161/cam.23176
  60. Kemp JP Jr, Brieher WM. 2018. The actin filament bundling protein ${\alpha}$-actinin-4 actually suppresses actin stress fibers by permitting actin turnover. J. Biol. Chem. 293: 14520-14533. https://doi.org/10.1074/jbc.RA118.004345
  61. Burridge K, Guilluy C. 2016. Focal adhesions, stress fibers and mechanical tension. Exp. Cell Res. 343: 14-20. https://doi.org/10.1016/j.yexcr.2015.10.029
  62. Pellegrin S, Mellor H. 2007. Actin stress fibres. J. Cell Sci. 120: 3491-3499. https://doi.org/10.1242/jcs.018473
  63. Berkova Z, Crawford SE, Blutt SE, Morris AP, Estes MK. 2007. Expression of rotavirus NSP4 alters the actin network organization through the actin remodeling protein cofilin. J. Virol. 81: 3545-3553. https://doi.org/10.1128/JVI.01080-06
  64. Taylor M P, Koyuncu OO, Enquist LW. 2011. Subversion o f the actin cytoskeleton during viral infection. Nat. Rev. Microbiol. 9: 427-439. https://doi.org/10.1038/nrmicro2574
  65. Lv X, Li Z, Guan J, Hu S, Zhang J, Lan Y, et al. 2019. Porcine hemagglutinating encephalomyelitis virus activation of the integrin $\alpha$5$\beta$1-FAK-cofilin pathway causes cytoskeletal rearrangement to promote its invasion of N2a Cells. J. Virol. 93: e01736-18.
  66. Stradal TEB, Schelhaas M. 2018. Actin dynamics in host-pathogen interaction. FEBS Lett. 592: 3658-3669. https://doi.org/10.1002/1873-3468.13173
  67. Surjit M, Liu B, Jameel S, Chow VT, Lal SK. 2004. The SARS coronavirus nucleocapsid protein induces actin reorganization and apoptosis in COS-1 cells in the absence of growth factors. Biochem. J. 383: 13-18. https://doi.org/10.1042/BJ20040984
  68. Lomert E, Turoverova L, Kriger D, Aksenov ND, Nikotina AD, Petukhov A, et al. 2018. Co-expression of RelA/p65 and ACTN4 induces apoptosis in non-small lung carcinoma cells. Cell Cycle 17: 616-626.
  69. Huang Q, Li X, Huang Z, Yu F, Wang X, Wang S, et al. 2019. ACTN4 promotes the proliferation, migration, metastasis of osteosarcoma and enhances its invasive ability through the NF-${\kappa}$B pathway. Pathol. Oncol. Res. doi: 10.1007/s12253-019-00637.
  70. Zhao X, Hsu KS, Lim JH, Bruggeman LA, Kao HY. 2015. ${\alpha}$-Actinin 4 potentiates nuclear factor ${\kappa}$-light-chain-enhancer of activated B-cell (NF-${\kappa}$B) activity in podocytes independent of its cytoplasmic actin binding function. J. Biol. Chem. 290: 338-349. https://doi.org/10.1074/jbc.M114.597260
  71. Oeckinghaus A, Ghosh S, 2009. The NF-kappaB family of transcription factors and its regulation. Cold Spring Harb. Perspect. Biol. 1: a000034. https://doi.org/10.1101/cshperspect.a000034
  72. Tornatore L, Thotakura AK, Bennett J, Moretti M, Franzoso G. 2012. The nuclear factor kappa B signaling pathway: integrating metabolism with inflammation. Trends Cell Biol. 22: 557-566. https://doi.org/10.1016/j.tcb.2012.08.001