1887

Abstract

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) contains four glycoproteins, GP, GP, GP and GP, the functions of which are still largely unresolved. In this study, the significance of the -glycosylation of the GP and GP proteins of PRRSV strain LV was investigated. Both glycoproteins contain two predicted -glycosylation sites that are highly conserved between North American-type and European-type PRRSV. Using site-directed mutagenesis, single and double mutant full-length PRRSV cDNA clones were generated. After analysing the expression of the mutant proteins and the actual use of the four putative glycosylation sites in the wild-type proteins, the production of mutant virus particles and their infectivities were investigated. The results showed that the -linked glycans normally present on the GP protein are not essential for particle formation, as is the oligosaccharide attached to N53 of the GP protein. In contrast, the oligosaccharide linked to N46 of the GP protein is strongly required for virus particle production. The specific infectivities of the mutant viruses were investigated by comparing their infectivity-per-particle ratios with that of wild-type virus. The results showed that the lack of either one or both of the -linked oligosaccharides on GP or of the oligosaccharide attached to N53 of GP did not significantly affect the infectivities of the viruses. In contrast, the two recombinant viruses lacking the oligosaccharide bound to N46 exhibited a significantly reduced specific infectivity compared with the wild-type virus. The implications of the differential requirements of the modifications of GP and GP for PRRSV assembly and infectivity are discussed.

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2004-12-01
2019-11-22
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References

  1. Back, N. K. T., Smit, L., De Jong, J.-J., Keulen, W., Schutten, M., Goudsmit, J. & Tersmette, M. ( 1994; ). An N-glycan within the human immunodeficiency virus type 1 gp120 V3 loop affects virus neutralization. Virology 199, 431–438.[CrossRef]
    [Google Scholar]
  2. Benfield, D. A., Nelson, E., Collins, J. E. & 7 other authors ( 1992; ). Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332). J Vet Diagn Invest 4, 127–133.[CrossRef]
    [Google Scholar]
  3. Cavanagh, D. ( 1997; ). Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Arch Virol 142, 629–633.
    [Google Scholar]
  4. Chen, Z., Li, K. & Plagemann, P. G. ( 2000; ). Neuropathogenicity and sensitivity to antibody neutralization of lactate dehydrogenase-elevating virus are determined by polylactosaminoglycan chains on the primary envelope glycoprotein. Virology 266, 88–98.[CrossRef]
    [Google Scholar]
  5. Cowley, J. A., Dimmock, C. M., Spann, K. M. & Walker, P. J. ( 2000; ). Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses. J Gen Virol 81, 1473–1484.
    [Google Scholar]
  6. Daniels, R., Kurowski, B., Johnson, A. E. & Hebert, D. N. ( 2003; ). N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin. Mol Cell 11, 79–90.[CrossRef]
    [Google Scholar]
  7. Dea, S., Gagnon, C. A., Mardassi, H., Pirzadeh, B. & Rogan, D. ( 2000; ). Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (PRRS) virus: comparison of the North American and European isolates. Arch Virol 145, 659–688.[CrossRef]
    [Google Scholar]
  8. Dekker, A., Leendertse, C. H., van Poelwijk, F., Rebel, J. M. & Moormann, R. J. ( 2000; ). Chimeric swine vesicular disease viruses produced by fusion PCR: a new method for epitope mapping. J Virol Methods 86, 131–141.[CrossRef]
    [Google Scholar]
  9. Delputte, P. L., Vanderheijden, N., Nauwynck, H. J. & Pensaert, M. B. ( 2002; ). Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages. J Virol 76, 4312–4320.[CrossRef]
    [Google Scholar]
  10. Dobbe, J. C., van der Meer, Y., Spaan, W. J. & Snijder, E. J. ( 2001; ). Construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus tropism in cell culture. Virology 288, 283–294.[CrossRef]
    [Google Scholar]
  11. Doms, R. W., Lamb, R. A., Rose, J. K. & Helenius, A. ( 1993; ). Folding and assembly of viral membrane proteins. Virology 193, 545–562.[CrossRef]
    [Google Scholar]
  12. Faaberg, K. S. & Plagemann, P. G. ( 1995; ). The envelope proteins of lactate dehydrogenase-elevating virus and their membrane topography. Virology 212, 512–525.[CrossRef]
    [Google Scholar]
  13. Gonin, P., Mardassi, H., Gagnon, C. A., Massie, B. & Dea, S. ( 1998; ). A nonstructural and antigenic glycoprotein is encoded by ORF3 of the IAF-Klop strain of porcine reproductive and respiratory syndrome virus. Arch Virol 143, 1927–1940.[CrossRef]
    [Google Scholar]
  14. Hebert, D. N., Zhang, J. X., Chen, W., Foellmer, B. & Helenius, A. ( 1997; ). The number and location of glycans on influenza hemagglutinin determine folding and association with calnexin and calreticulin. J Cell Biol 139, 613–623.[CrossRef]
    [Google Scholar]
  15. Helenius, A. & Aebi, M. ( 2001; ). Intracellular functions of N-linked glycans. Science 291, 2364–2369.[CrossRef]
    [Google Scholar]
  16. Huso, D. L., Narayan, O. & Hart, G. W. ( 1988; ). Sialic acids on the surface of caprine arthritis-encephalitis virus define the biological properties of the virus. J Virol 62, 1974–1980.
    [Google Scholar]
  17. Jacobs, C. E., Hakze-van der Honing, R. W. & Steverink, P. J. G. M. ( 2000; ). Detection of porcine reproductive and respiratory syndrome virus in semen by real-time RT-PCR. In Proceedings of the Fifth International Congress of the European Society for Veterinary Virology (ESVV), Brescia, Italy, pp. 246–247.
  18. Johnson, W. E., Sanford, H., Schwall, L., Burton, D. R., Parren, P. W. H. I., Robinson, J. E. & Desrosiers, R. C. ( 2003; ). Assorted mutations in the envelope gene of simian immunodeficiency virus lead to loss of neutralization resistance against antibodies representing a broad spectrum of specificities. J Virol 77, 9993–10003.[CrossRef]
    [Google Scholar]
  19. Liljestrom, P. & Garoff, H. ( 1993; ). Expression of proteins using Semliki Forest virus vectors. In Current Protocols in Molecular Biology, pp. 16.20.1–16.20.16. Edited by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. Seidman & K. Struhl. New York: Greene Publishing Associates and Wiley Interscience.
  20. Magnusson, P., Hyllseth, B. & Marusyk, H. ( 1970; ). Morphological studies on equine arteritis virus. Arch Gesamte Virusforsch 30, 105–112.[CrossRef]
    [Google Scholar]
  21. Mardassi, H., Massie, B. & Dea, S. ( 1996; ). Intracellular synthesis, processing, and transport of proteins encoded by ORFs 5 to 7 of porcine reproductive and respiratory syndrome virus. Virology 221, 98–112.[CrossRef]
    [Google Scholar]
  22. Mardassi, H., Gonin, P., Gagnon, C. A., Massie, B. & Dea, S. ( 1998; ). A subset of porcine reproductive and respiratory syndrome virus GP3 glycoprotein is released into the culture medium of cells as a non-virion-associated and membrane-free (soluble) form. J Virol 72, 6298–6306.
    [Google Scholar]
  23. Mateu, E., Martín, M. & Vidal, D. ( 2003; ). Genetic diversity and phylogenetic analysis of glycoprotein 5 of European-type porcine reproductive and respiratory virus strains in Spain. J Gen Virol 84, 529–534.[CrossRef]
    [Google Scholar]
  24. Meng, X. J. ( 2000; ). Heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development. Vet Microbiol 74, 309–329.[CrossRef]
    [Google Scholar]
  25. Meulenberg, J. J. M. & Petersen-den Besten, A. ( 1996; ). Identification and characterization of a sixth structural protein of Lelystad virus: the glycoprotein GP2 encoded by ORF2 is incorporated in virus particles. Virology 225, 44–51.[CrossRef]
    [Google Scholar]
  26. Meulenberg, J. J., Petersen-den Besten, A., De Kluyver, E. P., Moormann, R. J., Schaaper, W. M. & Wensvoort, G. ( 1995; ). Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus. Virology 206, 155–163.[CrossRef]
    [Google Scholar]
  27. Meulenberg, J. J., Bos-de Ruijter, J. N., Wensvoort, G. & Moormann, R. J. ( 1998; ). An infectious cDNA clone of porcine reproductive and respiratory syndrome virus. Adv Exp Med Biol 440, 199–206.
    [Google Scholar]
  28. Ohuchi, M., Ohuchi, R., Feldmann, A. & Klenk, H. D. ( 1997; ). Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety. J Virol 71, 8377–8384.
    [Google Scholar]
  29. Oleksiewicz, M. B., Botner, A., Madsen, K. G. & Storgaard, T. ( 1998; ). Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes. Vet Microbiol 64, 7–22.[CrossRef]
    [Google Scholar]
  30. Pol, J. M. & Wagenaar, F. ( 1992; ). Morphogenesis of Lelystad virus in porcine lung alveolar macrophages. Am Assoc Swine Pract Newsl 4, 29.
    [Google Scholar]
  31. Snijder, E. J., Dobbe, J. C. & Spaan, W. J. M. ( 2003; ). Heterodimerization of the two major envelope proteins is essential for arterivirus infectivity. J Virol 77, 97–104.[CrossRef]
    [Google Scholar]
  32. Stadejek, T., Stankevicius, A., Storgaard, T., Oleksiewicz, M. B., Belák, S., Drew, T. W. & Pejsak, Z. ( 2002; ). Identification of radically different variants of porcine reproductive and respiratory syndrome virus in Eastern Europe: towards a common ancestor for European and American viruses. J Gen Virol 83, 1861–1873.
    [Google Scholar]
  33. van Nieuwstadt, A. P., Meulenberg, J. J., van Essen-Zandbergen, A., Petersen-den Besten, A., Bende, R. J., Moormann, R. J. & Wensvoort, G. ( 1996; ). Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion. J Virol 70, 4767–4772.
    [Google Scholar]
  34. Verheije, M. H. ( 2002; ). In Genetic engineering of the porcine reproductive and respiratory syndrome virus: exploration of avenues towards a vaccine, pp. 95–104. PhD thesis, Utrecht University, The Netherlands.
  35. Verheije, M. H., Welting, T. J., Jansen, H. T., Rottier, P. J. & Meulenberg, J. J. ( 2002; ). Chimeric arteriviruses generated by swapping of the M protein ectodomain rule out a role of this domain in viral targeting. Virology 303, 364–373.[CrossRef]
    [Google Scholar]
  36. Wensvoort, G., Terpstra, C., Boonstra, J., Bloemraad, M. & Van Zaane, D. ( 1986; ). Production of monoclonal antibodies against swine fever virus and their use in laboratory diagnosis. Vet Microbiol 12, 101–108.[CrossRef]
    [Google Scholar]
  37. Wensvoort, G., Terpstra, C., Pol, J. M. A. & 19 other authors ( 1991; ). Mystery swine disease in the Netherlands: the isolation of Lelystad virus. Vet Q 13, 121–130.[CrossRef]
    [Google Scholar]
  38. Wieringa, R. ( 2003; ). The envelope proteins of equine arteritis virus, pp. 67–85. PhD thesis, Utrecht University, The Netherlands.
  39. Wieringa, R., De Vries, A. A. & Rottier, P. J. ( 2003; ). Formation of disulfide-linked complexes between the three minor envelope glycoproteins (GP2b, GP3, and GP4) of equine arteritis virus. J Virol 77, 6216–6226.[CrossRef]
    [Google Scholar]
  40. Wissink, E. H. J., van Wijk, H. A. R., Kroese, M. V., Weiland, E., Meulenberg, J. J. M., Rottier, P. J. M. & van Rijn, P. A. ( 2003; ). The major envelope protein, GP5, of a European porcine reproductive and respiratory syndrome virus contains a neutralization epitope in its N-terminal ectodomain. J Gen Virol 84, 1535–1543.[CrossRef]
    [Google Scholar]
  41. Wu, W. H., Fang, Y., Farwell, R., Steffen-Bien, M., Rowland, R. R., Christopher-Hennings, J. & Nelson, E. A. ( 2001; ). A 10-kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b. Virology 287, 183–191.[CrossRef]
    [Google Scholar]
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