1887

Abstract

Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE-derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The replication efficiency of the NS4B chimeric GPE replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. and data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.000200
2015-09-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jgv/96/9/2623.html?itemId=/content/journal/jgv/10.1099/vir.0.000200&mimeType=html&fmt=ahah

References

  1. Behrens S.E., Grassmann C.W., Thiel H.J., Meyers G., Tautz N. 1998; Characterization of an autonomous subgenomic pestivirus RNA replicon. J Virol 72:2364–2372[PubMed]
    [Google Scholar]
  2. Claros M.G., von Heijne G. 1994; TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci 10:685–686[PubMed]
    [Google Scholar]
  3. Cole C., Barber J.D., Barton G.J. 2008; The Jpred 3 secondary structure prediction server. Nucleic Acids Res 36:W197–W201[PubMed] [CrossRef]
    [Google Scholar]
  4. Dunn K.W., Kamocka M.M., McDonald J.H. 2011; A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol 300:C723–C742 [View Article][PubMed]
    [Google Scholar]
  5. Egger D., Wölk B., Gosert R., Bianchi L., Blum H.E., Moradpour D., Bienz K. 2002; Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol 76:5974–5984 [View Article][PubMed]
    [Google Scholar]
  6. Fernandez-Sainz I., Gladue D.P., Holinka L.G., O'Donnell V., Gudmundsdottir I., Prarat M.V., Patch J.R., Golde W.T., Lu Z., other authors. 2010; Mutations in classical swine fever virus NS4B affect virulence in swine. J Virol 84:1536–1549 [View Article][PubMed]
    [Google Scholar]
  7. Gautier R., Douguet D., Antonny B., Drin G. 2008; heliquest: a web server to screen sequences with specific alpha-helical properties. Bioinformatics 24:2101–2102 [View Article][PubMed]
    [Google Scholar]
  8. Gladue D.P., Gavrilov B.K., Holinka L.G., Fernandez-Sainz I.J., Vepkhvadze N.G., Rogers K., O'Donnell V., Risatti G.R., Borca M.V. 2011; Identification of an NTPase motif in classical swine fever virus NS4B protein. Virology 411:41–49 [View Article][PubMed]
    [Google Scholar]
  9. Gouttenoire J., Roingeard P., Penin F., Moradpour D. 2010; Amphipathic alpha-helix AH2 is a major determinant for the oligomerization of hepatitis C virus nonstructural protein 4B. J Virol 84:12529–12537[PubMed] [CrossRef]
    [Google Scholar]
  10. Gouttenoire J., Montserret R., Paul D., Castillo R., Meister S., Bartenschlager R., Penin F., Moradpour D. 2014; Aminoterminal amphipathic α-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and virus production. PLoS Pathog 10:e1004501 [View Article][PubMed]
    [Google Scholar]
  11. Greiser-Wilke I., Moennig V., Coulibaly C.O., Dahle J., Leder L., Liess B. 1990; Identification of conserved epitopes on a hog cholera virus protein. Arch Virol 111:213–225 [View Article][PubMed]
    [Google Scholar]
  12. Guermeur Y. 1997; Combinaison de classifieurs statistiques, application à la prédiction de la structure secondaire des proteins. PhD thesis Université de Paris 6, Paris, France
    [Google Scholar]
  13. Guermeur Y., Geourjon C., Gallinari P., Deléage G. 1999; Improved performance in protein secondary structure prediction by inhomogeneous score combination. Bioinformatics 15:413–421 [View Article][PubMed]
    [Google Scholar]
  14. Hügle T., Fehrmann F., Bieck E., Kohara M., Kräusslich H.G., Rice C.M., Blum H.E., Moradpour D. 2001; The hepatitis C virus nonstructural protein 4B is an integral endoplasmic reticulum membrane protein. Virology 284:70–81 [View Article][PubMed]
    [Google Scholar]
  15. Jones D.T. 1999; Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292:195–202 [View Article][PubMed]
    [Google Scholar]
  16. Kameyama K., Sakoda Y., Tamai K., Igarashi H., Tajima M., Mochizuki T., Namba Y., Kida H. 2006a; Development of an immunochromatographic test kit for rapid detection of bovine viral diarrhea virus antigen. J Virol Methods 138:140–146[PubMed] [CrossRef]
    [Google Scholar]
  17. Kameyama K., Sakoda Y., Tamai K., Nagai M., Akashi H., Kida H. 2006b; Genetic recombination at different points in the Npro-coding region of bovine viral diarrhea viruses and the potentials to change their antigenicities and pathogenicities. Virus Res 116:78–84[PubMed] [CrossRef]
    [Google Scholar]
  18. King R.D., Sternberg M.J. 1996; Identification and application of the concepts important for accurate and reliable protein secondary structure prediction. Protein Sci 5:2298–2310 [View Article][PubMed]
    [Google Scholar]
  19. Knoops K., Kikkert M., Worm S.H., Zevenhoven-Dobbe J.C., van der Meer Y., Koster A.J., Mommaas A.M., Snijder E.J. 2008; SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol 6:e226[PubMed] [CrossRef]
    [Google Scholar]
  20. Kopek B.G., Perkins G., Miller D.J., Ellisman M.H., Ahlquist P. 2007; Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle. PLoS Biol 5:e220 [View Article][PubMed]
    [Google Scholar]
  21. Lamp B., Riedel C., Wentz E., Tortorici M.A., Rümenapf T. 2013; Autocatalytic cleavage within classical swine fever virus NS3 leads to a functional separation of protease and helicase. J Virol 87:11872–11883 [View Article][PubMed]
    [Google Scholar]
  22. Larkin M.A., Blackshields G., Brown N.P., Chenna R., McGettigan P.A., McWilliam H., Valentin F., Wallace I.M., Wilm A., other authors. 2007; clustal w clustal_x version 2.0. Bioinformatics 23:2947–2948 [View Article][PubMed]
    [Google Scholar]
  23. Leifer I., Ruggli N., Blome S. 2013; Approaches to define the viral genetic basis of classical swine fever virus virulence. Virology 438:51–55 [View Article][PubMed]
    [Google Scholar]
  24. Lindenbach B.D., Murray C.L., Thiel H.J., Rice C.M. 2013; Flaviviridae: the viruses and their replication. In Fields Virology Edited by Knipe D. M., Howley P. M. vol. 1 pp. 712–794, 6th edn. Philadelphia, PA: Lippincott, Williams & Wilkins;
    [Google Scholar]
  25. Mayer D., Thayer T.M., Hofmann M.A., Tratschin J.D. 2003; Establishment and characterisation of two cDNA-derived strains of classical swine fever virus, one highly virulent and one avirulent. Virus Res 98:105–116 [View Article][PubMed]
    [Google Scholar]
  26. Miller S., Sparacio S., Bartenschlager R. 2006; Subcellular localization and membrane topology of the Dengue virus type 2 Non-structural protein 4B. J Biol Chem 281:8854–8863 [View Article][PubMed]
    [Google Scholar]
  27. Mittelholzer C., Moser C., Tratschin J.D., Hofmann M.A. 1997; Generation of cytopathogenic subgenomic RNA of classical swine fever virus in persistently infected porcine cell lines. Virus Res 51:125–137 [View Article][PubMed]
    [Google Scholar]
  28. Mittelholzer C. I, Moser C. II, Tratschin J.D., Hofmann M.A. 2000; Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains. Vet Microbiol 74:293–308 [View Article][PubMed]
    [Google Scholar]
  29. Moser C., Stettler P., Tratschin J.D., Hofmann M.A. 1999; Cytopathogenic and noncytopathogenic RNA replicons of classical swine fever virus. J Virol 73:7787–7794[PubMed]
    [Google Scholar]
  30. Pollastri G., McLysaght A. 2005; Porter: a new, accurate server for protein secondary structure prediction. Bioinformatics 21:1719–1720 [View Article][PubMed]
    [Google Scholar]
  31. Qu L., McMullan L.K., Rice C.M. 2001; Isolation and characterization of noncytopathic pestivirus mutants reveals a role for nonstructural protein NS4B in viral cytopathogenicity. J Virol 75:10651–10662 [View Article][PubMed]
    [Google Scholar]
  32. Raghava G.P.S. 2002; APSSP2: a combination method for protein secondary structure prediction based on neural network and example based learning. CASP5 A 132
  33. Reed L., Muench H. 1938; A simple method of estimating fifty per cent endpoints. Am J Hyg 27:493–497
    [Google Scholar]
  34. Risager P.C., Fahnøe U., Gullberg M., Rasmussen T.B., Belsham G.J. 2013; Analysis of classical swine fever virus RNA replication determinants using replicons. J Gen Virol 94:1739–1748 [View Article][PubMed]
    [Google Scholar]
  35. Romero-Brey I., Merz A., Chiramel A., Lee J.Y., Chlanda P., Haselman U., Santarella-Mellwig R., Habermann A., Hoppe S., other authors. 2012; Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS Pathog 8:e1003056 [View Article][PubMed]
    [Google Scholar]
  36. Rost B. 1996; phd: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol 266:525–539[PubMed] [CrossRef]
    [Google Scholar]
  37. Sakoda Y., Hikawa M., Tamura T., Fukusho A. 1998; Establishment of a serum-free culture cell line, CPK-NS, which is useful for assays of classical swine fever virus. J Virol Methods 75:59–68 [View Article][PubMed]
    [Google Scholar]
  38. Schmeiser S., Mast J., Thiel H.J., König M., Sandri-Goldin R.M. 2014; Morphogenesis of pestiviruses: new insights from ultrastructural studies of strain Giraffe-1. J Virol 88:2717–2724 [View Article][PubMed]
    [Google Scholar]
  39. Sharma R., Ghasparian A., Robinson J.A., McCullough K.C. 2012; Synthetic virus-like particles target dendritic cell lipid rafts for rapid endocytosis primarily but not exclusively by macropinocytosis. PLoS One 7:e43248 [View Article][PubMed]
    [Google Scholar]
  40. Spuul P., Salonen A., Merits A., Jokitalo E., Kääriäinen L., Ahola T. 2007; Role of the amphipathic peptide of Semliki forest virus replicase protein nsP1 in membrane association and virus replication. J Virol 81:872–883[PubMed] [CrossRef]
    [Google Scholar]
  41. Tamura T., Sakoda Y., Yoshino F., Nomura T., Yamamoto N., Sato Y., Okamatsu M., Ruggli N., Kida H. 2012; Selection of classical swine fever virus with enhanced pathogenicity reveals synergistic virulence determinants in E2 and NS4B. J Virol 86:8602–8613 [View Article][PubMed]
    [Google Scholar]
  42. Tamura T., Nagashima N., Ruggli N., Summerfield A., Kida H., Sakoda Y. 2014; Npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type I interferon induction at local replication sites. Vet Res 45:47[PubMed] [CrossRef]
    [Google Scholar]
  43. Weiskircher E., Aligo J., Ning G., Konan K.V. 2009; Bovine viral diarrhea virus NS4B protein is an integral membrane protein associated with Golgi markers and rearranged host membranes. Virol J 6:185 [View Article][PubMed]
    [Google Scholar]
  44. Welsch S., Miller S., Romero-Brey I., Merz A., Bleck C.K., Walther P., Fuller S.D., Antony C., Krijnse-Locker J., Bartenschlager R. 2009; Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe 5:365–375 [View Article][PubMed]
    [Google Scholar]
  45. Yamasaki T., Suzuki A., Shimizu T., Watarai M., Hasebe R., Horiuchi M. 2012; Characterization of intracellular localization of PrP(Sc) in prion-infected cells using a mAb that recognizes the region consisting of aa 119-127 of mouse PrP. J Gen Virol 93:668–680 [View Article][PubMed]
    [Google Scholar]
  46. Yu G.Y., Lee K.J., Gao L., Lai M.M. 2006; Palmitoylation and polymerization of hepatitis C virus NS4B protein. J Virol 80:6013–6023 [View Article][PubMed]
    [Google Scholar]
  47. Zhong W., Gutshall L.L., Del Vecchio A.M. 1998; Identification and characterization of an RNA-dependent RNA polymerase activity within the nonstructural protein 5B region of bovine viral diarrhea virus. J Virol 72:9365–9369[PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.000200
Loading
/content/journal/jgv/10.1099/vir.0.000200
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error