1887

Abstract

The envelope of equine arteritis virus (EAV) contains two glycoprotein complexes (GP2b/GP3/GP4 and GP5/M) and the small, non-glycosylated E protein. As E is essential for the production of infectious progeny but dispensable for assembly and release of virus-like particles, it probably mediates virus entry into cells, putatively in concert with the GP2b/GP3/GP4 complex. The E protein contains a central hydrophobic domain and a conserved potential site for N-terminal myristoylation, a hydrophobic modification usually pivotal for membrane targeting of the modified protein. Here, it was shown by radiolabelling that E is myristoylated at glycine-2, both in transfected cells as a fusion protein with yellow fluorescent protein (YFP) and in virus particles. Biochemical fractionation revealed that E–YFP with an inactivated acylation site was still completely membrane-bound, indicating that the putative transmembrane domain of E mediates membrane targeting. Confocal microscopy showed that both myristoylated and non-myristoylated E–YFP were localized to the endoplasmic reticulum and Golgi complex, the membranes from which EAV buds. The presence of a myristoylation inhibitor during replication of EAV, whilst completely blocking E acylation, reduced virus titres by 1.5 log. Similarly, a mutant EAV with non-myristoylatable E grew to a titre five- to sevenfold lower than that of the wild-type virus and exhibited a reduced plaque size. Western blotting of cell-culture supernatants showed that N and M, the major structural proteins of EAV, are released in similar amounts by cells transfected with wild-type and mutant genomes. Thus, E myristoylation is not required for budding of particles and probably has a function during virus entry.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.011957-0
2009-11-01
2019-11-13
Loading full text...

Full text loading...

/deliver/fulltext/jgv/90/11/2704.html?itemId=/content/journal/jgv/10.1099/vir.0.011957-0&mimeType=html&fmt=ahah

References

  1. Balasuriya, U. B. & MacLachlan, N. J. ( 2004; ). The immune response to equine arteritis virus: potential lessons for other arteriviruses. Vet Immunol Immunopathol 102, 107–129.[CrossRef]
    [Google Scholar]
  2. Barrera, A., Guerra, B., Notvall, L. & Lanford, R. E. ( 2005; ). Mapping of the hepatitis B virus pre-S1 domain involved in receptor recognition. J Virol 79, 9786–9798.[CrossRef]
    [Google Scholar]
  3. Bruss, V., Hagelstein, J., Gerhardt, E. & Galle, P. R. ( 1996; ). Myristylation of the large surface protein is required for hepatitis B virus in vitro infectivity. Virology 218, 396–399.[CrossRef]
    [Google Scholar]
  4. Chow, M., Newman, J. F., Filman, D., Hogle, J. M., Rowlands, D. J. & Brown, F. ( 1987; ). Myristylation of picornavirus capsid protein VP4 and its structural significance. Nature 327, 482–486.[CrossRef]
    [Google Scholar]
  5. Cordo, S. M., Candurra, N. A. & Damonte, E. B. ( 1999; ). Myristic acid analogs are inhibitors of Junin virus replication. Microbes Infect 1, 609–614.[CrossRef]
    [Google Scholar]
  6. De Falco, S., Ruvo, M., Verdoliva, A., Scarallo, A., Raimondo, D., Raucci, A. & Fassina, G. ( 2001; ). N-terminal myristylation of HBV preS1 domain enhances receptor recognition. J Pept Res 57, 390–400.[CrossRef]
    [Google Scholar]
  7. Del Piero, F. ( 2000; ). Equine viral arteritis. Vet Pathol 37, 287–296.[CrossRef]
    [Google Scholar]
  8. de Vries, A. A., Chirnside, E. D., Horzinek, M. C. & Rottier, P. J. ( 1992; ). Structural proteins of equine arteritis virus. J Virol 66, 6294–6303.
    [Google Scholar]
  9. Farazi, T. A., Waksman, G. & Gordon, J. I. ( 2001; ). The biology and enzymology of protein N-myristoylation. J Biol Chem 276, 39501–39504.[CrossRef]
    [Google Scholar]
  10. Gordon, J. I., Duronio, R. J., Rudnick, D. A., Adams, S. P. & Gokel, G. W. ( 1991; ). Protein N-myristoylation. J Biol Chem 266, 8647–8650.
    [Google Scholar]
  11. Gripon, P., Le Seyec, J., Rumin, S. & Guguen-Guillouzo, C. ( 1995; ). Myristylation of the hepatitis B virus large surface protein is essential for viral infectivity. Virology 213, 292–299.[CrossRef]
    [Google Scholar]
  12. Harper, D. R., Gilbert, R. L., Blunt, C. & McIlhinney, R. A. ( 1993; ). Inhibition of varicella-zoster virus replication by an inhibitor of protein myristoylation. J Gen Virol 74, 1181–1184.[CrossRef]
    [Google Scholar]
  13. Hearps, A. C. & Jans, D. A. ( 2007; ). Regulating the functions of the HIV-1 matrix protein. AIDS Res Hum Retroviruses 23, 341–346.[CrossRef]
    [Google Scholar]
  14. Krausslich, H. G., Holscher, C., Reuer, Q., Harber, J. & Wimmer, E. ( 1990; ). Myristoylation of the poliovirus polyprotein is required for proteolytic processing of the capsid and for viral infectivity. J Virol 64, 2433–2436.
    [Google Scholar]
  15. Krauzewicz, N., Streuli, C. H., Stuart-Smith, N., Jones, M. D., Wallace, S. & Griffin, B. E. ( 1990; ). Myristylated polyomavirus VP2: role in the life cycle of the virus. J Virol 64, 4414–4420.
    [Google Scholar]
  16. Lee, C. & Yoo, D. ( 2005; ). Cysteine residues of the porcine reproductive and respiratory syndrome virus small envelope protein are non-essential for virus infectivity. J Gen Virol 86, 3091–3096.[CrossRef]
    [Google Scholar]
  17. Lee, C. & Yoo, D. ( 2006; ). The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties. Virology 355, 30–43.[CrossRef]
    [Google Scholar]
  18. MacLachlan, N. J., Balasuriya, U. B., Hedges, J. F., Schweidler, T. M., McCollum, W. H., Timoney, P. J., Hullinger, P. J. & Patton, J. F. ( 1998; ). Serologic response of horses to the structural proteins of equine arteritis virus. J Vet Diagn Invest 10, 229–236.[CrossRef]
    [Google Scholar]
  19. Marc, D., Masson, G., Girard, M. & van der Werf, S. ( 1990; ). Lack of myristoylation of poliovirus capsid polypeptide VP0 prevents the formation of virions or results in the assembly of noninfectious virus particles. J Virol 64, 4099–4107.
    [Google Scholar]
  20. Matrosovich, M., Matrosovich, T., Garten, W. & Klenk, H. D. ( 2006; ). New low-viscosity overlay medium for viral plaque assays. Virol J 3, 63 [CrossRef]
    [Google Scholar]
  21. Maurer-Stroh, S. & Eisenhaber, F. ( 2004; ). Myristoylation of viral and bacterial proteins. Trends Microbiol 12, 178–185.[CrossRef]
    [Google Scholar]
  22. Maurer-Stroh, S., Gouda, M., Novatchkova, M., Schleiffer, A., Schneider, G., Sirota, F. L., Wildpaner, M., Hayashi, N. & Eisenhaber, F. ( 2004; ). MYRbase: analysis of genome-wide glycine myristoylation enlarges the functional spectrum of eukaryotic myristoylated proteins. Genome Biol 5, R21 [CrossRef]
    [Google Scholar]
  23. Nitschke, M., Korte, T., Tielesch, C., Ter-Avetisyan, G., Tunnemann, G., Cardoso, M. C., Veit, M. & Herrmann, A. ( 2008; ). Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis. Virology 377, 248–254.[CrossRef]
    [Google Scholar]
  24. Paige, L. A., Zheng, G. Q., DeFrees, S. A., Cassady, J. M. & Geahlen, R. L. ( 1990; ). Metabolic activation of 2-substituted derivatives of myristic acid to form potent inhibitors of myristoyl-CoA : protein N-myristoyltransferase. Biochemistry 29, 10566–10573.[CrossRef]
    [Google Scholar]
  25. Peitzsch, R. M. & McLaughlin, S. ( 1993; ). Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins. Biochemistry 32, 10436–10443.[CrossRef]
    [Google Scholar]
  26. Perez, M., Greenwald, D. L. & de la Torre, J. C. ( 2004; ). Myristoylation of the RING finger Z protein is essential for arenavirus budding. J Virol 78, 11443–11448.[CrossRef]
    [Google Scholar]
  27. Resh, M. D. ( 2006; ). Trafficking and signaling by fatty-acylated and prenylated proteins. Nat Chem Biol 2, 584–590.[CrossRef]
    [Google Scholar]
  28. Saad, J. S., Miller, J., Tai, J., Kim, A., Ghanam, R. H. & Summers, M. F. ( 2006; ). Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly. Proc Natl Acad Sci U S A 103, 11364–11369.[CrossRef]
    [Google Scholar]
  29. Snijder, E. J. & Meulenberg, J. J. ( 1998; ). The molecular biology of arteriviruses. J Gen Virol 79, 961–979.
    [Google Scholar]
  30. Snijder, E. J., van Tol, H., Pedersen, K. W., Raamsman, M. J. & de Vries, A. A. ( 1999; ). Identification of a novel structural protein of arteriviruses. J Virol 73, 6335–6345.
    [Google Scholar]
  31. Snijder, E. J., Dobbe, J. C. & Spaan, W. J. ( 2003; ). Heterodimerization of the two major envelope proteins is essential for arterivirus infectivity. J Virol 77, 97–104.[CrossRef]
    [Google Scholar]
  32. Strecker, T., Maisa, A., Daffis, S., Eichler, R., Lenz, O. & Garten, W. ( 2006; ). The role of myristoylation in the membrane association of the Lassa virus matrix protein Z. Virol J 3, 93 [CrossRef]
    [Google Scholar]
  33. Thaa, B., Herrmann, A. & Veit, M. ( 2009; ). The polybasic region is not essential for membrane binding of the matrix protein M1 of influenza virus. Virology 383, 150–155.[CrossRef]
    [Google Scholar]
  34. van den Born, E., Posthuma, C. C., Gultyaev, A. P. & Snijder, E. J. ( 2005; ). Discontinuous subgenomic RNA synthesis in arteriviruses is guided by an RNA hairpin structure located in the genomic leader region. J Virol 79, 6312–6324.[CrossRef]
    [Google Scholar]
  35. van Dinten, L. C., den Boon, J. A., Wassenaar, A. L., Spaan, W. J. & Snijder, E. J. ( 1997; ). An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription. Proc Natl Acad Sci U S A 94, 991–996.[CrossRef]
    [Google Scholar]
  36. Veit, M., Kabatek, A., Tielesch, C. & Hermann, A. ( 2008a; ). Characterization of equine arteritis virus particles and demonstration of their hemolytic activity. Arch Virol 153, 351–356.[CrossRef]
    [Google Scholar]
  37. Veit, M., Ponimaskin, E. & Schmidt, M. F. ( 2008b; ). Analysis of S-acylation of proteins. Methods Mol Biol 446, 163–182.
    [Google Scholar]
  38. Wieringa, R., de Vries, A. A., Raamsman, M. J. & Rottier, P. J. ( 2002; ). Characterization of two new structural glycoproteins, GP3 and GP4, of equine arteritis virus. J Virol 76, 10829–10840.[CrossRef]
    [Google Scholar]
  39. Wieringa, R., De Vries, A. A., Post, S. M. & Rottier, P. J. ( 2003a; ). Intra- and intermolecular disulfide bonds of the GP2b glycoprotein of equine arteritis virus: relevance for virus assembly and infectivity. J Virol 77, 12996–13004.[CrossRef]
    [Google Scholar]
  40. Wieringa, R., de Vries, A. A. & Rottier, P. J. ( 2003b; ). 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]
  41. Wieringa, R., de Vries, A. A., van der Meulen, J., Godeke, G. J., Onderwater, J. J., van Tol, H., Koerten, H. K., Mommaas, A. M., Snijder, E. J. & Rottier, P. J. ( 2004; ). Structural protein requirements in equine arteritis virus assembly. J Virol 78, 13019–13027.[CrossRef]
    [Google Scholar]
  42. Zevenhoven-Dobbe, J. C., Greve, S., van Tol, H., Spaan, W. J. & Snijder, E. J. ( 2004; ). Rescue of disabled infectious single-cycle (DISC) equine arteritis virus by using complementing cell lines that express minor structural glycoproteins. J Gen Virol 85, 3709–3714.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.011957-0
Loading
/content/journal/jgv/10.1099/vir.0.011957-0
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