Complete Title of Thesis:
"Coronavirus Mediated Membrane Fusion"
Prepared under the direction of: Kathryn V. Holmes, Ph.D.
Coronaviruses infect cells by binding of the viral spike glycoprotein (S) to specific receptor proteins on the host cell membrane, followed by a series of conformational rearrangements in S that induce fusion of the viral envelope with the cellular membrane. To characterize domains of the coronavirus S glycoprotein that are essential for membrane fusion, and the series of conformational changes in S triggered by receptor binding, I studied the S protein of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). In collaboration with Drs Robert Hodges and Brian Tripet, I showed that the SARS-CoV S protein is a Class-I viral fusion protein by identifying and characterizing the HR-N and HR-C coiled-coil regions and the anti-parallel six-helix bundle they form in the post-fusion conformation of S.
I analyzed the highly conserved juxtamembrane domain (JMD) within the coronavirus S glycoprotein using pseudotyped viruses and cell-cell fusion assays to measure membrane fusion activities. I showed that specific tyrosine and tryptophan amino acids within the JMD were critical for virus-cell fusion, and that the phenyl groups in these amino acids were necessary for receptor-dependent cell-cell fusion. Interestingly, several alanine and phenylalanine substitutions in the JMD had different effects on virus-cell vs. cell-cell fusion. A peptide derived from the wild-type SARS-CoV S JMD destabilized membranes and the membrane-destabilizing activity of the JMD was required for both virus-cell and cell-cell fusion. However, amino acid substitutions in the JMD peptide that increased membrane destabilization but reduced virus-cell fusion when introduced into the S protein did not alter cell-cell fusion. Thus, fusion between virus and cell membranes was more sensitive than cell-cell fusion to substitutions within the JMD. The JMDs of coronavirus S proteins likely function during the final stages of membrane fusion, perhaps during hemi-fusion and/or the formation and widening of the fusion pore. Because of the high conservation between JMD of all coronavirus S proteins, drugs that target conformational changes in the JMD might inhibit several unrelated coronaviruses. The differences observed between virus-cell and cell-cell fusion offer a tantalizing glimpse into potential mechanistic differences between the contributions of the coronavirus JMD during fusion.