Myristoylation of the arterivirus E protein: the fatty acid modification is not essential for membrane association but contributes significantly to virus infectivity

• Published in: Journal of general virology 2009-11, Vol.90 (11), p.2704-2712
• Main Authors: Thaa, Bastian, Kabatek, Aleksander, Zevenhoven-Dobbe, Jessika C, Snijder, Eric J, Herrmann, Andreas, Veit, Michael
• Format: Article
• Language: English
• Subjects: acylation; Animals; Biological and medical sciences; chemical reactions; CHO Cells; Cricetinae; Cricetulus; cytopathogenicity; E protein; Endoplasmic Reticulum - virology; Equartevirus - pathogenicity; Equine arteritis virus; Fatty Acids - metabolism; Fundamental and applied biological sciences. Psychology; glycine (amino acid); Golgi Apparatus - virology; host-pathogen relationships; Intracellular Membranes - virology; Microbiology; Miscellaneous; myristic acid; myristoylation; Protein Processing, Post-Translational; viral proteins; Viral Structural Proteins - metabolism; Virology; Virus Internalization
• ISSN: 0022-1317; 1465-2099
• DOI: 10.1099/vir.0.011957-0

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 log10. 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.