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
2021-02-28
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 Most Cited RSS feed

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