title: Conserved arginine residues in the carboxyl terminus of the equine arteritis virus E protein may play a role in heparin binding but may not affect viral infectivity in equine endothelial cells.
abstract: Equine arteritis virus (EAV), the causative agent of equine viral arteritis, has relatively broad cell tropism in vitro. In horses, EAV primarily replicates in macrophages and endothelial cells of small blood vessels. Until now, neither the cellular receptor(s) nor the mechanism(s) of virus attachment and entry have been determined for this virus. In this study, we investigated the effect of heparin on EAV infection in equine endothelial cells (EECs). Heparin, but not other glycosaminoglycans, could reduce EAV infection up to 93 %. Sequence analysis of the EAV E minor envelope protein revealed a conserved amino acid sequence (52 RSLVARCSRGARYR 65) at the carboxy terminus of the E protein, which was predicted to be the heparin-binding domain. The basic arginine (R) amino acid residues were subsequently mutated to glycine by site-directed mutagenesis of ORF2a in an E protein expression vector and an infectious cDNA clone of EAV. Two single mutations in E (R52G and R57G) did not affect the heparin-binding capability, whereas the E double mutation (R52,60G) completely eliminated the interaction between the E protein and heparin. Although the mutant R52,60G EAV did not bind heparin, the mutations did not completely abolish infectivity, indicating that heparin is not the only critical factor for EAV infection. This also suggested that other viral envelope protein(s) might be involved in attachment through heparin or other cell-surface molecules, and this warrants further investigation.
authors.affiliation: 108 Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, 40546, USA.|J. A. Baker Institute for Animal Health, College of Veterinary Medicine, 235 Hungerford Hill Road, Cornell University, Ithaca, NY, 14853, USA.
authors.forename: Zhengchun
authors.initials: Z
authors.lastname: Lu
authors.affiliation: 108 Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, 40546, USA.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Sarkar
authors.affiliation: 108 Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, 40546, USA.|Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, 1600 South 16th St, Ames, IA, 50011, USA.
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Zhang
authors.affiliation: 108 Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, 40546, USA. ubalasuriya@uky.edu.
authors.forename: Udeni B R
authors.initials: UB
authors.lastname: Balasuriya
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D001120:Arginine / Q000737:chemistry*; D003001:Cloning, Molecular; D042783:Endothelial Cells / Q000821:virology*; D006025:Glycosaminoglycans; D006493:Heparin / Q000378:metabolism*; D019763:Heparin Lyase; D006736:Horses; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; C120679:E protein, Equine arteritis virus; D006025:Glycosaminoglycans; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D006493:Heparin; D001120:Arginine; D019763:Heparin Lyase