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Journal of Virology, June 2007, p. 5893-5901, Vol. 81, No. 11
0022-538X/07/$08.00+0     doi:10.1128/JVI.02022-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Hendra Virus G Glycoprotein Residues That Are Critical for Receptor Binding{triangledown}

Kimberly A. Bishop,1 Tzanko S. Stantchev,1 Andrew C. Hickey,1 Dimple Khetawat,1 Katharine N. Bossart,2 Valery Krasnoperov,3 Parkash Gill,3 Yan Ru Feng,1 Lemin Wang,1 Bryan T. Eaton,2 Lin-Fa Wang,2 and Christopher C. Broder1*

Department of Microbiology and Immunology, Uniformed Services University, Bethesda, Maryland 20814,1 CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Victoria 3220, Australia,2 VasGene Therapeutics, Inc., 1929 Zonal Ave, Los Angeles, California 900333

Received 15 September 2006/ Accepted 9 March 2007

Hendra virus (HeV) is an emerging paramyxovirus capable of infecting and causing disease in a variety of mammalian species, including humans. The virus infects its host cells through the coordinated functions of its fusion (F) and attachment (G) glycoproteins, the latter of which is responsible for binding the virus receptors ephrinB2 and ephrinB3. In order to identify the receptor binding site, a panel of G glycoprotein constructs containing mutations was generated using an alanine-scanning mutagenesis strategy. Based on a predicted G structure, charged amino acids residing in regions that could be homologous to those in the measles virus H attachment glycoprotein known to be involved in its protein receptor interaction were targeted. Using a coprecipitation-based assay, seven single-amino-acid substitutions in HeV G were identified as having significantly impaired binding to both the ephrinB2 and ephrinB3 viral receptors: D257A, D260A, G439A, K443A, G449A, K465A, and D468A. The impairment of receptor interaction conferred a concomitant diminution in their abilities to promote membrane fusion when coexpressed with F. The G glycoprotein mutants were also recognized by three or more conformation-dependent monoclonal antibodies of a panel of five, were expressed on the cell surface, and retained their abilities to bind and coprecipitate F. Interestingly, some of these mutant G glycoproteins coprecipitated with F more efficiently than wild-type G. Taken together, these data provide strong biochemical and functional evidence that some of these residues could be part of a conformation-dependent, discontinuous, and overlapping ephrinB2 and -B3 binding domain within the HeV G glycoprotein.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Uniformed Services University, Bethesda, MD 20814. Phone: (301) 295-3401. Fax: (301) 295-1545. E-mail: cbroder{at}usuhs.mil

{triangledown} Published ahead of print on 21 March 2007.

Journal of Virology, June 2007, p. 5893-5901, Vol. 81, No. 11
0022-538X/07/$08.00+0     doi:10.1128/JVI.02022-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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