Aromatic amino acids in the juxtamembrane domain of SARS coronavirus spike glycoprotein are important for receptor-dependent virus entry and cell-cell fusion

Department of Microbiology, and Graduate Program in Molecular Biology, University of Colorado Denver School of Medicine, Aurora, CO, 80045 (USA)

^* To whom correspondence should be addressed. Email: kathryn.holmes{at}uchsc.edu.

	Abstract

The 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 19 proteins or reduce binding to soluble human ACE2. 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 10-60% of wild-type. The greatest reduction was caused by residues nearest the transmembrane domain (TM). Four double alanine substitutions reduced entry to 5-10% of wild-type. Rapid hACE2-dependent S-mediated cell-cell fusion was reduced to 60-70% of wild-type 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 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.