Analysis of classical swine fever virus RNA replication determinants using replicons Free

Abstract

Self-replicating RNAs (replicons), with or without reporter gene sequences, derived from the genome of the Paderborn strain of classical swine fever virus (CSFV) have been produced. The full-length viral cDNA, propagated within a bacterial artificial chromosome, was modified by targeted recombination within . RNA transcripts were produced and introduced into cells by electroporation. The translation and replication of the replicon RNAs could be followed by the accumulation of luciferase (from or ) protein expression (where appropriate), as well as by detection of CSFV NS3 protein production within the cells. Inclusion of the viral E2 coding region within the replicon was advantageous for replication efficiency. Production of chimeric RNAs, substituting the NS2 and NS3 coding regions (as a unit) from the Paderborn strain with the equivalent sequences from the highly virulent Koslov strain or the vaccine strain Riems, blocked replication. However, replacing the Paderborn NS5B coding sequence with the RNA polymerase coding sequence from the Koslov strain greatly enhanced expression of the reporter protein from the replicon. In contrast, replacement with the Riems NS5B sequence significantly impaired replication efficiency. Thus, these replicons provide a system for determining specific regions of the CSFV genome required for genome replication without the constraints of maintaining infectivity.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.052688-0
2013-08-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/94/8/1739.html?itemId=/content/journal/jgv/10.1099/vir.0.052688-0&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. Belsham G. J., Nielsen I., Normann P., Royall E., Roberts L. O. 2008; Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5′ untranslated regions are efficiently translated in cells by a cap-dependent mechanism. RNA 14:1671–1680 [View Article][PubMed]
    [Google Scholar]
  3. Binder M., Quinkert D., Bochkarova O., Klein R., Kezmic N., Bartenschlager R., Lohmann V. 2007; Identification of determinants involved in initiation of hepatitis C virus RNA synthesis by using intergenotypic replicase chimeras. J Virol 81:5270–5283 [View Article][PubMed]
    [Google Scholar]
  4. Deng R., Brock K. V. 1993; 5′ and 3′ untranslated regions of pestivirus genome: primary and secondary structure analyses. Nucleic Acids Res 21:1949–1957 [View Article][PubMed]
    [Google Scholar]
  5. Donnelly M. L., Hughes L. E., Luke G., Mendoza H., ten Dam E., Gani D., Ryan M. D. 2001; The ‘cleavage’ activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring ‘2A-like’ sequences. J Gen Virol 82:1027–1041[PubMed]
    [Google Scholar]
  6. Fletcher S. P., Jackson R. J. 2002; Pestivirus internal ribosome entry site (IRES) structure and function: elements in the 5′ untranslated region important for IRES function. J Virol 76:5024–5033 [View Article][PubMed]
    [Google Scholar]
  7. Frey C. F., Bauhofer O., Ruggli N., Summerfield A., Hofmann M. A., Tratschin J. D. 2006; Classical swine fever virus replicon particles lacking the Erns gene: a potential marker vaccine for intradermal application. Vet Res 37:655–670 [View Article][PubMed]
    [Google Scholar]
  8. Friis M. B., Rasmussen T. B., Belsham G. J. 2012; Modulation of translation initiation efficiency in classical swine fever virus. J Virol 86:8681–8692 [View Article][PubMed]
    [Google Scholar]
  9. Frolov I., McBride M. S., Rice C. M. 1998; cis-acting RNA elements required for replication of bovine viral diarrhea virus-hepatitis C virus 5′ nontranslated region chimeras. RNA 4:1418–1435 [View Article][PubMed]
    [Google Scholar]
  10. Fujimoto R., Osakabe T., Saito M., Kurosawa N., Isobe M. 2009; Minimum length of homology arms required for effective Red/ET recombination. Biosci Biotechnol Biochem 73:2783–2786 [View Article][PubMed]
    [Google Scholar]
  11. Gallei A., Rümenapf T., Thiel H. J., Becher P. 2005; Characterization of helper virus-independent cytopathogenic classical swine fever virus generated by an in vivo RNA recombination system. J Virol 79:2440–2448 [View Article][PubMed]
    [Google Scholar]
  12. Greer L. F. III, Szalay A. A. 2002; Imaging of light emission from the expression of luciferases in living cells and organisms: a review. Luminescence 17:43–74 [View Article][PubMed]
    [Google Scholar]
  13. Jones C. T., Murray C. L., Eastman D. K., Tassello J., Rice C. M. 2007; Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J Virol 81:8374–8383 [View Article][PubMed]
    [Google Scholar]
  14. Leifer I., Hoffmann B., Höper D., Bruun Rasmussen T., Blome S., Strebelow G., Höreth-Böntgen D., Staubach C., Beer M. 2010; Molecular epidemiology of current classical swine fever virus isolates of wild boar in Germany. J Gen Virol 91:2687–2697 [View Article][PubMed]
    [Google Scholar]
  15. Lindenbach B. D., Thiel H., Rice C. M.editors 2007 Fields Virology. Flaviviridae: The Viruses and Their Replication, 5th edn. Philadelphia: Lippincott-Raven;
    [Google Scholar]
  16. Meyers G., Thiel H. J. 1996; Molecular characterization of pestiviruses. Adv Virus Res 47:53–118 [View Article][PubMed]
    [Google Scholar]
  17. 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]
  18. 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]
  19. Muyrers J. P., Zhang Y., Testa G., Stewart A. F. 1999; Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res 27:1555–1557 [View Article][PubMed]
    [Google Scholar]
  20. Oleksiewicz M. B., Rasmussen T. B., Normann P., Uttenthal A. 2003; Determination of the sequence of the complete open reading frame and the 5′NTR of the Paderborn isolate of classical swine fever virus. Vet Microbiol 92:311–325 [View Article][PubMed]
    [Google Scholar]
  21. Pankraz A., Thiel H. J., Becher P. 2005; Essential and nonessential elements in the 3′ nontranslated region of bovine viral diarrhea virus. J Virol 79:9119–9127 [View Article][PubMed]
    [Google Scholar]
  22. Rasmussen T. B., Reimann I., Hoffmann B., Depner K., Uttenthal A., Beer M. 2008; Direct recovery of infectious pestivirus from a full-length RT-PCR amplicon. J Virol Methods 149:330–333 [View Article][PubMed]
    [Google Scholar]
  23. Rasmussen T. B., Reimann I., Uttenthal A., Leifer I., Depner K., Schirrmeier H., Beer M. 2010; Generation of recombinant pestiviruses using a full-genome amplification strategy. Vet Microbiol 142:13–17 [View Article][PubMed]
    [Google Scholar]
  24. Reimann I., Meyers G., Beer M. 2003; Trans-complementation of autonomously replicating Bovine viral diarrhea virus replicons with deletions in the E2 coding region. Virology 307:213–227 [View Article][PubMed]
    [Google Scholar]
  25. Rümenapf T., Unger G., Strauss J. H., Thiel H. J. 1993; Processing of the envelope glycoproteins of pestiviruses. J Virol 67:3288–3294[PubMed]
    [Google Scholar]
  26. Sheng C., Xiao M., Geng X., Liu J., Wang Y., Gu F. 2007; Characterization of interaction of classical swine fever virus NS3 helicase with 3′ untranslated region. Virus Res 129:43–53 [View Article][PubMed]
    [Google Scholar]
  27. Stech J., Stech O., Herwig A., Altmeppen H., Hundt J., Gohrbandt S., Kreibich A., Weber S., Klenk H. D., Mettenleiter T. C. 2008; Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification. Nucleic Acids Res 36:e139 [View Article][PubMed]
    [Google Scholar]
  28. Suter R., Summerfield A., Thomann-Harwood L. J., McCullough K. C., Tratschin J. D., Ruggli N. 2011; Immunogenic and replicative properties of classical swine fever virus replicon particles modified to induce IFN-α/β and carry foreign genes. Vaccine 29:1491–1503 [View Article][PubMed]
    [Google Scholar]
  29. 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]
  30. Tannous B. A., Kim D. E., Fernandez J. L., Weissleder R., Breakefield X. O. 2005; Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo . Mol Ther 11:435–443 [View Article][PubMed]
    [Google Scholar]
  31. Tautz N., Elbers K., Stoll D., Meyers G., Thiel H. J. 1997; Serine protease of pestiviruses: determination of cleavage sites. J Virol 71:5415–5422[PubMed]
    [Google Scholar]
  32. Tautz N., Harada T., Kaiser A., Rinck G., Behrens S., Thiel H. J. 1999; Establishment and characterization of cytopathogenic and noncytopathogenic pestivirus replicons. J Virol 73:9422–9432[PubMed]
    [Google Scholar]
  33. Tautz N., Kaiser A., Thiel H. J. 2000; NS3 serine protease of bovine viral diarrhea virus: characterization of active site residues, NS4A cofactor domain, and protease–cofactor interactions. Virology 273:351–363 [View Article][PubMed]
    [Google Scholar]
  34. Terpstra C., de Smit A. J. 2000; The 1997/1998 epizootic of swine fever in the Netherlands: control strategies under a non-vaccination regimen. Vet Microbiol 77:3–15 [View Article][PubMed]
    [Google Scholar]
  35. Uttenthal A., Storgaard T., Oleksiewicz M. B., de Stricker K. 2003; Experimental infection with the Paderborn isolate of classical swine fever virus in 10-week-old pigs: determination of viral replication kinetics by quantitative RT-PCR, virus isolation and antigen ELISA. Vet Microbiol 92:197–212 [View Article][PubMed]
    [Google Scholar]
  36. Wang P., Wang Y., Zhao Y., Zhu Z., Yu J., Wan L., Chen J., Xiao M. 2010; Classical swine fever virus NS3 enhances RNA-dependent RNA polymerase activity by binding to NS5B. Virus Res 148:17–23 [View Article][PubMed]
    [Google Scholar]
  37. Wei D., Li M., Zhang X., Xing L. 2004; An improvement of the site-directed mutagenesis method by combination of megaprimer, one-side PCR and DpnI treatment. Anal Biochem 331:401–403 [View Article][PubMed]
    [Google Scholar]
  38. Weiland E., Stark R., Haas B., Rümenapf T., Meyers G., Thiel H. J. 1990; Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer. J Virol 64:3563–3569[PubMed]
    [Google Scholar]
  39. Widjojoatmodjo M. N., van Gennip H. G., de Smit A. J., Moormann R. J. 1999; Comparative sequence analysis of classical swine fever virus isolates from the epizootic in the Netherlands in 1997–1998. Vet Microbiol 66:291–299 [View Article][PubMed]
    [Google Scholar]
  40. Xu J., Mendez E., Caron P. R., Lin C., Murcko M. A., Collett M. S., Rice C. M. 1997; Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication. J Virol 71:5312–5322[PubMed]
    [Google Scholar]
  41. Yu H., Grassmann C. W., Behrens S. E. 1999; Sequence and structural elements at the 3′ terminus of bovine viral diarrhea virus genomic RNA: functional role during RNA replication. J Virol 73:3638–3648[PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.052688-0
Loading
/content/journal/jgv/10.1099/vir.0.052688-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed