Veterinary Research Forum
Vet Res Forum

Expression and immunogenicity of non-structural protein 8 of porcine epidemic diarrhea virus

Document Type : Original Article

Authors

Hong Chen 1, 2
Jiawu Wan 1, 2
Meihua Wei 1, 2
Ping Liu 1, 2
Lingbao Kong 1, 2
Xiu Xin 1, 2

1 Institute of Pathogenic Microbiology, College of Biological Science and Engineering, Jiangxi Agricultural University, Nanchang, China

2 Nanchang Key Laboratory of Animal Virus and Genetic Engineering, College of Biological Science and Engineering, Jiangxi Agricultural University, Nanchang, China

https://doi.org/10.30466/vrf.2023.2009322.3977
Abstract
The non-structural protein (nsp) 8 of the porcine epidemic diarrhea virus (PEDV) is highly stable across different PEDV strains and plays an important role in PEDV virulence. In current study, nsp8 prokaryotic expression vectors were constructed based on parental vectors pMAL-c2x-maltose binding protein (MBP) and pET-28a (+). Subsequently, the optimization of expression conditions in Escherichia coli, including induced temperature, time and isopropyl β-D-thiogalactopyranoside concentration were performed to obtain a stable expression of MBP-nsp8 and nsp8. The nsp8 fused with MBP increased the water solubility of the expressed products. Target proteins were further purified from E. coli culture and their immunogenicities were evaluated in vivo by mice. The antibody titers of serum from nsp8 immunized mice were up to 1:7,750,000 when measured by indirect enzyme-linked immunosorbent assay; meanwhile, the mice immunized with MBP-nsp8 gave an antibody titer reaching 1:1,000,000. In all, the expression and purification system of PEDV nsp8 and MBP-nsp8 were successfully established in this work and a strong immune response was elicited in mice by both purified nsp8 and MBP-nsp8, providing a basis for the study of the structure and function of PEDV nsp8.

Keywords

Escherichia coli
Immunogenicity
Non-structural protein 8
Porcine epidemic diarrhea virus
Subjects
  1. Pensaert MB, Martelli P. Porcine epidemic diarrhea: a retrospect from Europe and matters of debate. Virus Res 2016; 226: 1-6.
  2. Sun RQ, Cai RJ, Chen YQ, et al. Outbreak of porcine epidemic diarrhea in suckling piglets, China. Emerg Infect Dis 2012; 18(1): 161-163.
  3. Gao X, Zhang L, Jiang X, et al. Porcine epidemic diarrhea: an emerging disease in Tibetan pigs in Tibet, China. Trop Anim Health Prod 2019; 51(2): 491-494.
  4. Lin C-M, Saif LJ, Marthaler D, et al. Evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains. Virus Res 2016; 226: 20-39.
  5. Sun D, Wang X, Wei S, et al. Epidemiology and vaccine of porcine epidemic diarrhea virus in China: a mini-review. J Vet Med Sci 2016; 78(3): 355-363.
  6. Wang E, Guo D, Li C, et al. Molecular characterization of the ORF3 and S1 genes of porcine epidemic diarrhea virus non S-INDEL strains in seven regions of China, 2015. PLoS One 2016; 11(8): e0160561. doi: 10.1371/ journal.pone.0160561.
  7. King AMQ, Lefkowitz EJ, Mushegian AR, et al. Changes to taxonomy and the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2018). Arch Virol 2018; 163(9): 2601-2631.
  8. Kocherhans R, Bridgen A, Ackermann M, et al. Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence. Virus Genes 2001; 23(2): 137-144.
  9. Song D, Park B. Porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. Virus Genes 2012; 44(2): 167-175.
  10. Subissi L, Imbert I, Ferron F, et al. SARS-CoV ORF1b-encoded nonstructural proteins 12-16: replicative enzymes as antiviral targets. Antiviral Res 2014; 101: 122-130.
  11. Luo Y, Zhang J, Deng X, et al. Complete genome sequence of a highly prevalent isolate of porcine epidemic diarrhea virus in South China. J Virol 2012; 86(17): 9551. doi: 10.1128/JVI.01455-12.
  12. Pan Y, Tian X, Li W, et al. Isolation and characterization of a variant porcine epidemic diarrhea virus in China. Virol J 2012; 9: 195. doi: 10.1186/1743-422X-9-195.
  13. Tian Y, Yu Z, Cheng K, et al. Molecular characterization and phylogenetic analysis of new variants of the porcine epidemic diarrhea virus in Gansu, China in 2012. Viruses 2013; 5(8):1991-2004.
  14. Jung K, Saif LJ, Wang Q. Porcine epidemic diarrhea virus (PEDV): An update on etiology, transmission, pathogenesis, and prevention and control. Virus Res 2020; 286: 198045. doi: 10.1016/j.virusres. 2020.198045.
  15. Wathelet MG, Orr M, Frieman MB, et al. Severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain. J Virol 2007; 81(21): 11620-11633.
  16. Züst R, Cervantes-Barragán L, Kuri T, et al. Coronavirus non-structural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines. PLoS Pathog 2007; 3(8): e109. doi: 10.1371/journal.ppat.0030109.
  17. Graham RL, Sims AC, Brockway SM, et al. The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication. J Virol 2005; 79(21): 13399-13411.
  18. Mielech AM, Deng X, Chen Y, et al. Murine coronavirus ubiquitin-like domain is important for papain-like protease stability and viral pathogenesis. J Virol 2015; 89(9): 4907-4917.
  19. Fehr AR, Athmer J, Channappanavar R, et al. The nsp3 macrodomain promotes virulence in mice with coronavirus-induced encephalitis. J Virol 2015; 89(3): 1523-1536.
  20. Deng X, StJohn SE, Osswald HL, et al. Coronaviruses resistant to a 3C-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance. J Virol 2014; 88(20): 11886-11898.
  21. Fehr AR, Channappanavar R, Jankevicius G, et al. The Conserved coronavirus macrodomain promotes virulence and suppresses the innate immune response during severe acute respiratory syndrome coronavirus infection. mBio 2016; 7(6): e01721-16. doi: 10.1128/mbio.01721-16.
  22. Jimenez-Guardeño JM, Nieto-Torres JL, DeDiego ML, et al. The PDZ-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis. PLoS Pathog 2014; 10(8): e1004320. doi: 10.1371/journal.ppat.1004320.
  23. Clementz MA, Kanjanahaluethai A, O'Brien TE, et al. Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles. Virology 2008; 375(1):118-129.
  24. Subissi L, Posthuma CC, Collet A, et al. One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities. Proc Natl Acad Sci U S A 2014; 111(37): E3900-E3909.
  25. Kirchdoerfer RN, Ward AB. Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors. Nat Commun 2019; 10: 2342. doi: 10.1038/s41467-019-10280-3.
  26. te Velthuis AJW, van den Worm SHE, Snijder EJ. The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension. Nucleic Acids Res 2012; 40(4): 1737-1747.
  27. Gorkhali R, Koirala P, Rijal S, et al. Structure and function of major SARS-CoV-2 and SARS-CoV proteins. Bioinform Biol Insights 2021; 15: 11779322 211025876. doi: 10.1177/11779322211025876.
  28. Zhang Q, Ke H, Blikslager A, et al. Type III interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp1 in IRF1 signaling. J Virol 2018; 92(4): e01677-17. doi: 10.1128/JVI.01677-17.
  29. Zhang Q, Ma J, Yoo D. Inhibition of NF-κB activity by the porcine epidemic diarrhea virus nonstructural protein 1 for innate immune evasion. Virology 2017; 510:111-126.
  30. Xing Y, Chen J, Tu J, et al. The papain-like protease of porcine epidemic diarrhea virus negatively regulates type I interferon pathway by acting as a viral deubiquitinase. J Gen Virol 2013; 94(Pt 7): 1554-1567.
  31. Wang D, Fang L, Shi Y, et al. Porcine epidemic diarrhea virus 3C-like protease regulates its interferon antagonism by cleaving NEMO. J Virol 2015; 90(4): 2090-2101.
  32. Zhang Q, Shi K, Yoo D. Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1. Virology 2016; 489: 252-268.
  33. Li Z, Ma Z, Li Y, et al. Porcine epidemic diarrhea virus: molecular mechanisms of attenuation and vaccines. Microb Pathog 2020; 149: 104553. doi: 10.1016/ j.micpath.2020.104553.
  34. Wu Y, Zhang H, Shi Z, et al. Porcine epidemic diarrhea virus nsp15 antagonizes interferon signaling by RNA degradation of TBK1 and IRF3. Viruses 2020; 12(6): 599. doi: 10.3390/v12060599.
  35. Shi P, Su Y, Li R, et al. PEDV nsp16 negatively regulates innate immunity to promote viral proliferation. Virus Res 2019; 265: 57-66.
  36. Hillen HS, Kokic G, Farnung L, et al. Structure of replicating SARS-CoV-2 polymerase. Nature 2020; 584(7819): 154-156.
  37. Wang Q, Wu J, Wang H, et al. Structural basis for RNA replication by the SARS-CoV-2 polymerase. Cell 2020; 182(2): 417-428.
  38. Bouvet M, Imbert I, Subissi L, et al. RNA 3'-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex. Proc Natl Acad Sci U S A 2012; 109(24): 9372-9377.
  39. Krichel B, Falke S, Hilgenfeld R, et al. Processing of the SARS-CoV pp1a/ab nsp7-10 region. Biochem J 2020; 477(5): 1009-1019.
  40. Martin R, Li J, Parvangada A, et al. Genetic conservation of SARS-CoV-2 RNA replication complex in globally circulating isolates and recently emerged variants from humans and minks suggests minimal pre-existing resistance to remdesivir. Antiviral Res 2021; 188: 105033. doi: 10.1016/j.antiviral.2021.105033.
  41. Jung LS, Gund TM, Narayan M. Comparison of binding site of remdesivir and its metabolites with NSP12-NSP7-NSP8, and NSP3 of SARS CoV-2 virus and alternative potential drugs for COVID-19 treatment. Protein J 2020; 39(6): 619-630.
  42. Imbert I, Guillemot J-C, Bourhis J-M, et al. A second, non-canonical RNA-dependent RNA polymerase in SARS coronavirus. EMBO J 2006; 25(20): 4933-4942.
  43. von Brunn A, Teepe C, Simpson JC, et al. Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome. PLoS One 2007; 2(5): e459. doi: 10.1371/journal.pone.0000459.
  44. Jiang Y, Tong K, Yao R, et al. Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV-2 replication. Cell Biosci 2021; 11(1): 140. doi: 10.1186/s13578-021-00644-y.
  45. Niu X, Kong F, Hou YJ, et al. Crucial mutation in the exoribonuclease domain of nsp14 of PEDV leads to high genetic instability during viral replication. Cell Biosci 2021; 11(1): 106. doi: 10.1186/s13578-021-00598-1
  46. Deng X, van Geelen A, Buckley AC, et al. Coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type I and type III interferon responses. J Virol 2019; 93(8): e02000-e2018.
Veterinary Research Forum
Volume 15, Issue 2
February 2024
Pages 65-73

  • Receive Date 16 August 2023
  • Revise Date 19 October 2023
  • Accept Date 05 November 2023

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