1887

Abstract

Porcine circovirus type 2 (PCV2) encodes two major proteins: the replication protein (Rep) and the capsid protein (Cap). Cap displays a conserved stretch of basic residues situated on the inside of the capsid, whose role is so far unknown. We used a reverse-genetics approach to investigate its function and found that mutations in these amino acids hindered Cap mRNA translation and hampered Cap/Rep co-localization, yielding unfit viruses. Intriguingly, co-transfection with a WT PCV2 of a different genotype partially rescued mutant Cap expression, showing the importance of this basic pattern for efficient translation of Cap mRNA into protein. Our results show that Cap and Rep are expressed independently of each other, and that this amino acid sequence of Cap is vital for virus propagation. This study provides a method for studying unfit PCV2 virions and offers new insights into the intracellular modus vivendi of PCV2.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000302
2015-12-01
2019-10-21
Loading full text...

Full text loading...

/deliver/fulltext/jgv/96/12/3566.html?itemId=/content/journal/jgv/10.1099/jgv.0.000302&mimeType=html&fmt=ahah

References

  1. Allan G. M. , McNeilly F. , Kennedy S. , Daft B. , Clarke E. G. , Ellis J. A. , Haines D. M. , Meehan B. M. , Adair B. M. . ( 1998;). Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. J Vet Diagn Invest 10: 3–10 [CrossRef] [PubMed].
    [Google Scholar]
  2. Costers S. , Lefebvre D. J. , Van Doorsselaere J. , Vanhee M. , Delputte P. L. , Nauwynck H. J. . ( 2010;). GP4 of porcine reproductive and respiratory syndrome virus contains a neutralizing epitope that is susceptible to immunoselection in vitro. Arch Virol 155: 371–378 [CrossRef] [PubMed].
    [Google Scholar]
  3. Crowther R. A. , Berriman J. A. , Curran W. L. , Allan G. M. , Todd D. . ( 2003;). Comparison of the structures of three circoviruses: chicken anemia virus, porcine circovirus type 2, and beak and feather disease virus. J Virol 77: 13036–13041 [CrossRef] [PubMed].
    [Google Scholar]
  4. Finsterbusch T. , Steinfeldt T. , Doberstein K. , Rödner C. , Mankertz A. . ( 2009;). Interaction of the replication proteins and the capsid protein of porcine circovirus type 1 and 2 with host proteins. Virology 386: 122–131 [CrossRef] [PubMed].
    [Google Scholar]
  5. Firth C. , Charleston M. A. , Duffy S. , Shapiro B. , Holmes E. C. . ( 2009;). Insights into the evolutionary history of an emerging livestock pathogen: porcine circovirus 2. J Virol 83: 12813–12821 [CrossRef] [PubMed].
    [Google Scholar]
  6. Guo L. , Lu Y. , Huang L. , Wei Y. , Liu C. . ( 2011;). Identification of a new antigen epitope in the nuclear localization signal region of porcine circovirus type 2 capsid protein. Intervirology 54: 156–163 [CrossRef] [PubMed].
    [Google Scholar]
  7. He J. , Cao J. , Zhou N. , Jin Y. , Wu J. , Zhou J. . ( 2013;). Identification and functional analysis of the novel ORF4 protein encoded by porcine circovirus type 2. J Virol 87: 1420–1429 [CrossRef] [PubMed].
    [Google Scholar]
  8. Heath L. , Williamson A. L. , Rybicki E. P. . ( 2006;). The capsid protein of beak and feather disease virus binds to the viral DNA and is responsible for transporting the replication-associated protein into the nucleus. J Virol 80: 7219–7225 [CrossRef] [PubMed].
    [Google Scholar]
  9. Hickman A. B. , Dyda F. . ( 2005;). Binding and unwinding: SF3 viral helicases. Curr Opin Struct Biol 15: 77–85 [CrossRef] [PubMed].
    [Google Scholar]
  10. Huang L. , Lu Y. , Wei Y. , Guo L. , Liu C. . ( 2011a;). Development of a blocking ELISA for detection of serum neutralizing antibodies against porcine circovirus type 2. J Virol Methods 171: 26–33 [CrossRef] [PubMed].
    [Google Scholar]
  11. Huang L. P. , Lu Y. H. , Wei Y. W. , Guo L. J. , Liu C. M. . ( 2011b;). Identification of one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of porcine circovirus type 2. BMC Microbiol 11: 188 [CrossRef] [PubMed].
    [Google Scholar]
  12. Khayat R. , Brunn N. , Speir J. A. , Hardham J. M. , Ankenbauer R. G. , Schneemann A. , Johnson J. E. . ( 2011;). The 2.3-angstrom structure of porcine circovirus 2. J Virol 85: 7856–7862 [CrossRef] [PubMed].
    [Google Scholar]
  13. Lefebvre D. J. , Costers S. , Van Doorsselaere J. , Misinzo G. , Delputte P. L. , Nauwynck H. J. . ( 2008;). Antigenic differences among porcine circovirus type 2 strains, as demonstrated by the use of monoclonal antibodies. J Gen Virol 89: 177–187 [CrossRef] [PubMed].
    [Google Scholar]
  14. Liu J. , Chen I. , Kwang J. . ( 2005;). Characterization of a previously unidentified viral protein in porcine circovirus type 2-infected cells and its role in virus-induced apoptosis. J Virol 79: 8262–8274 [CrossRef] [PubMed].
    [Google Scholar]
  15. Mankertz A. , Hillenbrand B. . ( 2001;). Replication of porcine circovirus type 1 requires two proteins encoded by the viral rep gene. Virology 279: 429–438 [CrossRef] [PubMed].
    [Google Scholar]
  16. Mankertz A. , Hillenbrand B. . ( 2002;). Analysis of transcription of Porcine circovirus type 1. J Gen Virol 83: 2743–2751 [CrossRef] [PubMed].
    [Google Scholar]
  17. Mankertz A. , Mankertz J. , Wolf K. , Buhk H. J. . ( 1998a;). Identification of a protein essential for replication of porcine circovirus. J Gen Virol 79: 381–384 [CrossRef] [PubMed].
    [Google Scholar]
  18. Mankertz J. , Buhk H. J. , Blaess G. , Mankertz A. . ( 1998b;). Transcription analysis of porcine circovirus (PCV). Virus Genes 16: 267–276 [CrossRef] [PubMed].
    [Google Scholar]
  19. McNeilly F. , McNair I. , Mackie D. P. , Meehan B. M. , Kennedy S. , Moffett D. , Ellis J. , Krakowka S. , Allan G. M. . ( 2001;). Production, characterisation and applications of monoclonal antibodies to porcine circovirus 2. Arch Virol 146: 909–922 [CrossRef] [PubMed].
    [Google Scholar]
  20. Meerts P. , Misinzo G. , McNeilly F. , Nauwynck H. J. . ( 2005;). Replication kinetics of different porcine circovirus 2 strains in PK-15 cells, fetal cardiomyocytes and macrophages. Arch Virol 150: 427–441.[CrossRef]
    [Google Scholar]
  21. Misinzo G. , Delputte P. L. , Meerts P. , Lefebvre D. J. , Nauwynck H. J. . ( 2006;). Porcine circovirus 2 uses heparan sulfate and chondroitin sulfate B glycosaminoglycans as receptors for its attachment to host cells. J Virol 80: 3487–3494 [CrossRef] [PubMed].
    [Google Scholar]
  22. Nauwynck H. J. , Pensaert M. B. . ( 1995;). Effect of specific antibodies on the cell-associated spread of pseudorabies virus in monolayers of different cell types. Arch Virol 140: 1137–1146 [CrossRef] [PubMed].
    [Google Scholar]
  23. Nawagitgul P. , Morozov I. , Bolin S. R. , Harms P. A. , Sorden S. D. , Paul P. S. . ( 2000;). Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein. J Gen Virol 81: 2281–2287 [CrossRef] [PubMed].
    [Google Scholar]
  24. Roux S. , Enault F. , Bronner G. , Vaulot D. , Forterre P. , Krupovic M. . ( 2013;). Chimeric viruses blur the borders between the major groups of eukaryotic single-stranded DNA viruses. Nat Commun 4: 2700 [CrossRef] [PubMed].
    [Google Scholar]
  25. Saha D. , Lefebvre D. J. , Van Doorsselaere J. , Atanasova K. , Barbé F. , Geldhof M. , Karniychuk U. U. , Nauwynck H. J. . ( 2010;). Pathologic and virologic findings in mid-gestational porcine foetuses after experimental inoculation with PCV2a or PCV2b. Vet Microbiol 145: 62–68 [CrossRef] [PubMed].
    [Google Scholar]
  26. Saha D. , Lefebvre D. J. , Ducatelle R. , Doorsselaere J. V. , Nauwynck H. J. . ( 2011;). Outcome of experimental porcine circovirus type 1 infections in mid-gestational porcine foetuses. BMC Vet Res 7: 64 [CrossRef] [PubMed].
    [Google Scholar]
  27. Saha D. , Huang L. , Bussalleu E. , Lefebvre D. J. , Fort M. , Van Doorsselaere J. , Nauwynck H. J. . ( 2012a;). Antigenic subtyping and epitopes' competition analysis of porcine circovirus type 2 using monoclonal antibodies. Vet Microbiol 157: 13–22 [CrossRef] [PubMed].
    [Google Scholar]
  28. Saha D. , Lefebvre D. J. , Ooms K. , Huang L. , Delputte P. L. , Van Doorsselaere J. , Nauwynck H. J. . ( 2012b;). Single amino acid mutations in the capsid switch the neutralization phenotype of porcine circovirus 2. J Gen Virol 93: 1548–1555 [CrossRef] [PubMed].
    [Google Scholar]
  29. Segalés J. . ( 2012;). Porcine circovirus type 2 (PCV2) infections: clinical signs, pathology and laboratory diagnosis. Virus Res 164: 10–19 [CrossRef] [PubMed].
    [Google Scholar]
  30. Steinfeldt T. , Finsterbusch T. , Mankertz A. . ( 2001;). Rep and Rep′ protein of porcine circovirus type 1 bind to the origin of replication in vitro. Virology 291: 152–160 [CrossRef] [PubMed].
    [Google Scholar]
  31. Tischer I. , Gelderblom H. , Vettermann W. , Koch M. A. . ( 1982;). A very small porcine virus with circular single-stranded DNA. Nature 295: 64–66 [CrossRef] [PubMed].
    [Google Scholar]
  32. Xiao C. T. , Halbur P. G. , Opriessnig T. . ( 2015;). Global molecular genetic analysis of porcine circovirus type 2 (PCV2) sequences confirms the presence of four main PCV2 genotypes and reveals a rapid increase of PCV2d. J Gen Virol 96: 1830–1841 [CrossRef] [PubMed].
    [Google Scholar]
  33. Yu S. , Vincent A. , Opriessnig T. , Carpenter S. , Kitikoon P. , Halbur P. G. , Thacker E. . ( 2007;). Quantification of PCV2 capsid transcript in peripheral blood mononuclear cells (PBMCs) in vitro. Vet Microbiol 123: 34–42 [CrossRef] [PubMed].
    [Google Scholar]
  34. Zinchuk V. , Zinchuk O. . ( 2008;). Quantitative colocalization analysis of confocal fluorescence microscopy images. Curr Protoc Cell Biol 39: 4191–41916 [CrossRef].
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000302
Loading
/content/journal/jgv/10.1099/jgv.0.000302
Loading

Data & Media loading...

Most Cited This Month

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