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Journal of Virology, May 2004, p. 4508-4516, Vol. 78, No. 9
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.9.4508-4516.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Chimeric Protein of Simian Immunodeficiency Virus Envelope Glycoprotein gp140 and Escherichia coli Aspartate Transcarbamoylase

Bing Chen,1 Yifan Cheng,2 Lesley Calder,3 Stephen C. Harrison,1,4* Ellis L. Reinherz,5 John J. Skehel,3 and Don C. Wiley1,4,{dagger}

Laboratory of Molecular Medicine, The Children's Hospital,1 Howard Hughes Medical Institute,4 Department of Cell Biology, Harvard Medical School,2 Dana-Farber Cancer Institute, Boston, Massachusetts 02115,5 National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom3

Received 27 October 2003/ Accepted 31 December 2003

The envelope glycoproteins of the human immunodeficiency virus and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing viral and target cell membranes. We have reported expression, purification, and characterization of gp140 (also called gp160e), the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp160 (B. Chen et al., J. Biol. Chem. 275:34946-34953, 2000). We have now expressed and purified chimeric proteins of SIV gp140 and its variants with the catalytic subunit (C) of Escherichia coli aspartate transcarbamoylase (ATCase). The fusion proteins (SIV gp140-ATC) bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp140. The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains. Thus, the fusion protein is trimeric. When ATCase regulatory subunit dimers (R2) are added, the fusion protein assembles into dimers of trimers as expected from the structure of C6R6 ATCase. Negative-stain electron microscopy reveals spikey features of both SIV gp140 and SIV gp140-ATC. The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein either by electron cryomicroscopy or X-ray crystallography.

* Corresponding author. Mailing address: Laboratory of Molecular Medicine, The Children's Hospital, and Howard Hughes Medical Institute, 320 Longwood Ave., Boston, MA 02115. Phone: (617) 355-7372. Fax: (617) 730-1967. E-mail: harrison{at}crystal.harvard.edu.

{dagger} Deceased.

Journal of Virology, May 2004, p. 4508-4516, Vol. 78, No. 9
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.9.4508-4516.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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