Skip to main content
SpringerLink
Log in

The classic swine fever virus (CSFV) core protein can enhance de novo-initiated RNA synthesis by the CSFV polymerase NS5B

  • Published:
Virus Genes Aims and scope Submit manuscript

Abstract

Study of the classical swine fever virus (CSFV) replication is challenging because it is a BSL-3-Ag pathogen that requires specialized facilities. We developed a cell-based assay in human embryonic kidney 293T cells that can quantify the activities of NS5B, the CSFV RNA-dependent RNA polymerase. The 5BR assay uses transiently-expressed CSFV NS5B to produce RNAs that activate the RIG-I-mediated signaling pathway to result in reporter protein production. Upon co-expression of the CSFV core protein, we observed enhancement of the CSFV RdRp activity. The CSFV core and NS5B proteins could co-immunoprecipitate with each other and co-localize in cells, when visualized by confocal microscopy. Analyses of combinations of RdRps and capsid proteins from different viruses demonstrated that the CSFV core could enhance the CSFV NS5B activity in a virus species-specific manner. Studies of truncated versions of CSFV core demonstrated that the first 30 residues of core protein are dispensable for interaction with the CSFV NS5B. Purified core protein could enhance RNA synthesis by the purified NS5B in vitro, with the increase being in the synthesis of the de novo-initiated RNA. These results demonstrate that the CSFV core protein can regulate the mechanism of RNA synthesis by the CSFV RdRp.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. C. Tu, Z. Lu, H. Li, X. Yu, X. Liu, Y. Li, H. Zhang, Z. Yin, Phylogenetic comparison of classical swine fever virus in China. Virus Res. 81, 29–37 (2001)

    Article  CAS  PubMed  Google Scholar 

  2. V. Kaden, U. Ziegler, E. Lange, J. Dedek, Classical swine fever virus: clinical, virological, serological and hematological findings after infection of domestic pigs and wild boars with the field isolate “Spante” originating from wild boar. Berl Munch Tierarztl Wochenschr 113, 412–416 (2000)

    CAS  PubMed  Google Scholar 

  3. D.J. Paton, I. Greiser-Wilke, Classical swine fever—an update. Res. Vet. Sci. 75, 169–178 (2003)

    Article  CAS  PubMed  Google Scholar 

  4. L. He, Y.M. Zhang, Z. Lin, W.W. Li, J. Wang, H.L. Li, Classical swine fever virus NS5A protein localizes to endoplasmic reticulum and induces oxidative stress in vascular endothelial cells. Virus Genes 45, 274–282 (2012)

    Article  CAS  PubMed  Google Scholar 

  5. Q.H. Tang, Y.M. Zhang, Y.Z. Xu, L. He, C. Dai, P. Sun, Up-regulation of integrin beta3 expression in porcine vascular endothelial cells cultured in vitro by classical swine fever virus. Vet. Immunol. Immunopathol. 133, 237–242 (2010)

    Article  CAS  PubMed  Google Scholar 

  6. Q.H. Tang, Y.M. Zhang, L. Fan, G. Tong, L. He, C. Dai, Classic swine fever virus NS2 protein leads to the induction of cell cycle arrest at S-phase and endoplasmic reticulum stress. Virol. J. 7, 4 (2010)

    Article  PubMed Central  PubMed  Google Scholar 

  7. S.M. Kang, J.K. Choi, S.J. Kim, J.H. Kim, D.G. Ahn, J.W. Oh, Regulation of hepatitis C virus replication by the core protein through its interaction with viral RNA polymerase. Biochem. Biophys. Res. Commun. 386, 55–59 (2009)

    Article  CAS  PubMed  Google Scholar 

  8. C.V. Subba-Reddy, I. Goodfellow, C.C. Kao, VPg-primed RNA synthesis of norovirus RNA-dependent RNA polymerases by using a novel cell-based assay. J. Virol. 85, 13027–13037 (2011)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. C.V. Subba-Reddy, M.A. Yunus, I.G. Goodfellow, C.C. Kao, Norovirus RNA synthesis is modulated by an interaction between the viral RNA-dependent RNA polymerase and the major capsid protein, VP1. J. Virol. 86, 10138–10149 (2012)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Appendix D—agriculture pathogen biosafety. pp. 350–351. http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_appendixD.pdf. Accessed 8 May 2014

  11. C.T. Ranjith-Kumar, Y. Wen, N. Baxter, K. Bhardwaj, C. Cheng Kao, A cell-based assay for RNA synthesis by the HCV polymerase reveals new insights on mechanism of polymerase inhibitors and modulation by NS5A. PLoS One 6, e22575 (2011)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. B. Fan, K.Y. Lu, F.X. Sutandy, Y.W. Chen, K. Konan, H. Zhu, C.C. Kao, C.S. Chen, A human proteome microarray identifies that the heterogeneous nuclear ribonucleoprotein K (hnRNP K) recognizes the 5 ‘ terminal sequence of the hepatitis C virus RNA. Mol. Cell Proteomics 13(1), 84–92 (2013)

    Article  PubMed  Google Scholar 

  13. D. Li, H. Dong, S. Li, M. Munir, J. Chen, Y. Luo, Y. Sun, L. Liu, H.J. Qiu, Hemoglobin subunit beta interacts with the capsid protein and antagonizes the growth of classical swine fever virus. J. Virol. 87, 5707–5717 (2013)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. R. Yan, D. Xu, J. Yang, S. Walker, Y. Zhang, A comparative assessment and analysis of 20 representative sequence alignment methods for protein structure prediction. Sci. Rep. 3, 2619 (2013)

    PubMed Central  PubMed  Google Scholar 

  15. D.T. Jones, Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292, 195–202 (1999)

    Article  CAS  PubMed  Google Scholar 

  16. G.H. Yi, C.Y. Zhang, S. Cao, H.X. Wu, Y. Wang, De novo RNA synthesis by a recombinant classical swine fever virus RNA-dependent RNA polymerase. Eur. J. Biochem. 270, 4952–4961 (2003)

    Article  CAS  PubMed  Google Scholar 

  17. S. Chinnaswamy, I. Yarbrough, S. Palaninathan, C.T. Kumar, V. Vijayaraghavan, B. Demeler, S.M. Lemon, J.C. Sacchettini, C.C. Kao, A locking mechanism regulates RNA synthesis and host protein interaction by the hepatitis C virus polymerase. J. Biol. Chem. 283, 20535–20546 (2008)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. S. Chinnaswamy, W.B. Lott, The adenine-rich tract in the 5-end of the hepatitis C virus ORF encodes a peptide regulating the binding of the C protein to RNA. Acta Virol. 56, 217–226 (2012)

    Article  CAS  PubMed  Google Scholar 

  19. S. Chinnaswamy, A. Murali, P. Li, K. Fujisaki, C.C. Kao, Regulation of de novo-initiated RNA synthesis in hepatitis C virus RNA-dependent RNA polymerase by intermolecular interactions. J. Virol. 84, 5923–5935 (2010)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. G. Chinnaswamy, M. Cole, A.V. Boddy, M. Keir, L. Price, A. Parry, M. English, G.J. Veal, Estimation of renal function and its potential impact on carboplatin dosing in children with cancer. Br. J. Cancer 99, 894–899 (2008)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. C.C. Kao, J.H. Sun, Initiation of minus-strand RNA synthesis by the brome mosaicvirus RNA-dependent RNA polymerase: use of oligoribonucleotide primers. J. Virol. 70, 6826–6830 (1996)

    CAS  PubMed Central  PubMed  Google Scholar 

  22. J. Schmidt-Mende, E. Bieck, T. Hugle, F. Penin, C.M. Rice, H.E. Blum, D. Moradpour, Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase. J. Biol. Chem. 276, 44052–44063 (2001)

    Article  CAS  PubMed  Google Scholar 

  23. D. Egger, B. Wolk, R. Gosert, L. Bianchi, H.E. Blum, D. Moradpour, K. Bienz, Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J. Virol. 76, 5974–5984 (2002)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. C.C. Kao, P. Singh, D.J. Ecker, De novo initiation of viral RNA-dependent RNA synthesis. Virology 287, 251–260 (2001)

    Article  CAS  PubMed  Google Scholar 

  25. C.T. Ranjith-Kumar, J.L. Santos, L.L. Gutshall, V.K. Johnston, J. Lin-Goerke, M.J. Kim, D.J. Porter, D. Maley, C. Greenwood, D.L. Earnshaw, A. Baker, B. Gu, C. Silverman, R.T. Sarisky, C. Kao, Enzymatic activities of the GB virus-B RNA-dependent RNA polymerase. Virology 312, 270–280 (2003)

    Article  CAS  PubMed  Google Scholar 

  26. D. Paul, R. Bartenschlager, Architecture and biogenesis of plus-strand RNA virus replication factories. World J. Virol. 2, 32–48 (2013)

    Article  PubMed Central  PubMed  Google Scholar 

  27. J.A. den Boon, P. Ahlquist, Organelle-like membrane compartmentalization of positive-strand RNA virus replication factories. Annu. Rev. Microbiol. 64, 241–256 (2010)

    Article  Google Scholar 

  28. V. Lohmann, A. Roos, F. Korner, J.O. Koch, R. Bartenschlager, Biochemical and structural analysis of the NS5B RNA-dependent RNA polymerase of the hepatitis C virus. J. Viral. Hepat. 7, 167–174 (2000)

    Article  CAS  PubMed  Google Scholar 

  29. V. Lohmann, F. Korner, U. Herian, R. Bartenschlager, Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. J. Virol. 71, 8416–8428 (1997)

    CAS  PubMed Central  PubMed  Google Scholar 

  30. R. Bartenschlager, V. Lohmann, F. Penin, The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nat. Rev. Microbiol. 11, 482–496 (2013)

    Article  CAS  PubMed  Google Scholar 

  31. R. Bartenschlager, Candidate targets for hepatitis C virus-specific antiviral therapy. Intervirology 40, 378–393 (1997)

    Article  CAS  PubMed  Google Scholar 

  32. A. Iwasaki, A virological view of innate immune recognition. Annu. Rev. Microbiol. 66, 177–196 (2012)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. M. Xiao, Y. Bai, H. Xu, X. Geng, J. Chen, Y. Wang, B. Li, Effect of NS3 and NS5B proteins on classical swine fever virus internal ribosome entry site-mediated translation and its host cellular translation. J. Gen. Virol. 89, 994–999 (2008)

    Article  CAS  PubMed  Google Scholar 

  34. Y. Chen, J. Xiao, C. Sheng, J. Wang, L. Jia, Y. Zhi, G. Li, J. Chen, M. Xiao, Classical swine fever virus NS5A regulates viral RNA replication through binding to NS5B and 3′UTR. Virology 432, 376–388 (2012)

    Article  CAS  PubMed  Google Scholar 

  35. P. Ni, C. Cheng, Kao. Non-encapsidation activities of the capsid proteins of positive-strand RNA viruses. Virology 446, 123–132 (2013)

    Article  CAS  PubMed  Google Scholar 

  36. L. Neeleman, R.C. Olsthoorn, H.J. Linthorst, J.F. Bol, Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA. Proc. Natl. Acad. Sci. USA. 98, 14286–14291 (2001)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. M. Wolf, M. Dimitrova, T.F. Baumert, C. Schuster, The major form of hepatitis C virus alternate reading frame protein is suppressed by core protein expression. Nucleic Acids Res. 36, 3054–3064 (2008)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. T. Shimoike, S. Mimori, H. Tani, Y. Matsuura, T. Miyamura, Interaction of hepatitis C virus core protein with viral sense RNA and suppression of its translation. J. Virol. 73, 9718–9725 (1999)

    CAS  PubMed Central  PubMed  Google Scholar 

  39. G. Yi, C. Kao, Brome mosaic virus capsid protein regulates accumulation of viral replication proteins by binding to the replicase assembly RNA element. RNA 15, 615–626 (2009)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. C.S. Ilkow, V. Mancinelli, M.D. Beatch, T.C. Hobman, Rubella virus capsid protein interacts with poly(a)-binding protein and inhibits translation. J. Virol. 82, 4284–4294 (2008)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. M.H. Chen, J.P. Icenogle, Rubella virus capsid protein modulates viral genome replication and virus infectivity. J. Virol. 78, 4314–4322 (2004)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. J.T. Juuti, D.H. Bamford, Protein P7 of phage phi6 RNA polymerase complex, acquiring of RNA packaging activity by in vitro assembly of the purified protein onto deficient particles. J. Mol. Biol. 266, 891–900 (1997)

    Article  CAS  PubMed  Google Scholar 

  43. Q. Ye, R.M. Krug, Y.J. Tao, The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature 444, 1078–1082 (2006)

    Article  CAS  PubMed  Google Scholar 

  44. L.L. Newcomb, R.L. Kuo, Q. Ye, Y. Jiang, Y.J. Tao, R.M. Krug, Interaction of the influenza a virus nucleocapsid protein with the viral RNA polymerase potentiates unprimed viral RNA replication. J. Virol. 83, 29–36 (2009)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. J.K. Marklund, Q. Ye, J. Dong, Y.J. Tao, R.M. Krug, Sequence in the influenza A virus nucleoprotein required for viral polymerase binding and RNA synthesis. J. Virol. 86, 7292–7297 (2012)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. K.M. Guglielmi, S.M. McDonald, J.T. Patton, Mechanism of intraparticle synthesis of the rotavirus double-stranded RNA genome. J. Biol. Chem. 285, 18123–181289 (2010)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  47. J.T. Patton, R. Vasquez-Del Carpio, M.A. Tortorici, Z.F. Taraporewala, Coupling of rotavirus genome replication and capsid assembly. Adv. Virus. Res. 69, 167–201 (2007)

    Article  CAS  PubMed  Google Scholar 

  48. B. Bhattacharya, R.J. Noad, P. Roy, Interaction between Bluetongue virus outer capsid protein VP2 and vimentin is necessary for virus egress. Virol. J. 4, 1–12 (2007)

    Article  Google Scholar 

  49. E. De Clercg, Current race in the development of DAAs (direct-acting antivirals) against HCV. Biochem. Pharmacol. (2014). doi:10.1016/j.bcp.2014.04.005

    Google Scholar 

Download references

Acknowledgments

We thank members of the Kao lab for advice and reagents used during this work, especially Y. Wen, G. Yi and X. Lin and B. Fan. We thank Laura Kao for editing the manuscript. W. Li is supported in part by a fellowship from China Council Scholarship and Northwest A&F University. This work used funds from the Indiana Economic Development Corporation to C. K.

Author information

Authors and Affiliations

  1. College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China

    Weiwei Li & Yanming Zhang

  2. Department of Molecular & Cellular Biochemistry, Indiana University, Bloomington, IN, 47405, USA

    Weiwei Li & C. Cheng Kao

Authors
  1. Weiwei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yanming Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C. Cheng Kao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C. Cheng Kao.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, W., Zhang, Y. & Kao, C.C. The classic swine fever virus (CSFV) core protein can enhance de novo-initiated RNA synthesis by the CSFV polymerase NS5B. Virus Genes 49, 106–115 (2014). https://doi.org/10.1007/s11262-014-1080-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11262-014-1080-x

Keywords

Search

Navigation