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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

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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.

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Acknowledgments

This study was supported by the Kentucky Agricultural Experiment Station, College of Agriculture, University of Kentucky, and the Agriculture and Food Research Initiative competitive grant number 2013-68004-20360 from the United States Department of Agriculture National Institute of Food and Agriculture (USDA-NIFA). The authors would like to thank Ms. Jennifer Strange and Mr. Greg Bauman, Flow Cytometry Core Facility, Department of Microbiology, Immunology and Molecular Genetics, College of Medicine, University of Kentucky (UK), for their assistance with flow cytometry. Dr. Zhengchun Lu was the recipient of a Geoffrey C. Hughes Foundation graduate fellowship from the Maxwell H. Gluck Equine Research Center. The authors would like to thank Dr. Hans W. Heidner and Jacqueline C. Williams, Division of Life Science, University of Texas at San Antonio, San Antonio, TX 78249, USA, for providing the pVR21 plasmid for this study.

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  1. Zhengchun Lu

    Present address: J. A. Baker Institute for Animal Health, College of Veterinary Medicine, 235 Hungerford Hill Road, Cornell University, Ithaca, NY, 14853, USA

  2. Jianqiang Zhang

    Present address: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, 1600 South 16th St, Ames, IA, 50011, USA

Authors and Affiliations

  1. 108 Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, 40546, USA

    Zhengchun Lu, Sanjay Sarkar, Jianqiang Zhang & Udeni B. R. Balasuriya

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Correspondence to Udeni B. R. Balasuriya.

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Lu, Z., Sarkar, S., Zhang, J. et al. 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. Arch Virol 161, 873–886 (2016). https://doi.org/10.1007/s00705-015-2733-3

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