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Journal of Virology, March 2008, p. 2883-2894, Vol. 82, No. 6
0022-538X/08/$08.00+0     doi:10.1128/JVI.01805-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

Megan W. Howard,¹ Emily A. Travanty,¹ Scott A. Jeffers,¹ M. K. Smith,¹ Sonia T. Wennier,^2, Larissa B. Thackray,^1, and Kathryn V. Holmes^1*

Department of Microbiology,¹ Graduate Program in Molecular Biology, University of Colorado Denver School of Medicine, Aurora, Colorado 80045²

Received 16 August 2007/ Accepted 24 December 2007

The severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein (S) is a class I viral fusion protein that binds to its receptor glycoprotein, human angiotensin converting enzyme 2 (hACE2), and mediates virus entry and cell-cell fusion. The juxtamembrane domain (JMD) of S is an aromatic amino acid-rich region proximal to the transmembrane domain that is highly conserved in all coronaviruses. Alanine substitutions for one or two of the six aromatic residues in the JMD did not alter the surface expression of the SARS-CoV S proteins with a deletion of the C-terminal 19 amino acids (S 19) or reduce binding to soluble human ACE2 (hACE2). However, hACE2-dependent entry of trypsin-treated retrovirus pseudotyped viruses expressing JMD mutant S 19 proteins was greatly reduced. Single alanine substitutions for aromatic residues reduced entry to 10 to 60% of the wild-type level. The greatest reduction was caused by residues nearest the transmembrane domain. Four double alanine substitutions reduced entry to 5 to 10% of the wild-type level. Rapid hACE2-dependent S-mediated cell-cell fusion was reduced to 60 to 70% of the wild-type level for all single alanine substitutions and the Y1188A/Y1191A protein. S 19 proteins with other double alanine substitutions reduced cell-cell fusion further, from 40% to less than 20% of wild-type levels. The aromatic amino acids in the JMD of the SARS-CoV S glycoprotein play critical roles in receptor-dependent virus-cell and cell-cell fusion. Because the JMD is so highly conserved in all coronavirus S proteins, it is a potential target for development of drugs that may inhibit virus entry and/or cell-cell fusion mediated by S proteins of all coronaviruses.

Published ahead of print on 16 January 2008.

Present address: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610.

Present address: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110.