Complete Title of Thesis:
"Human Coronavirus-Receptor Interactions"
Prepared under the direction of: Kathryn V. Holmes, Ph.D.
Human coronaviruses are increasingly important respiratory pathogens. These viruses typically are difficult to grow in cell culture, thus little is known about their biology and molecular biology. The coronavirus attachment protein, the S glycoprotein, binds to receptor proteins on target cells to facilitate virus entry. Coronavirus S glycoproteins are therefore attractive targets for development of vaccines and therapeutic strategies. To determine the molecular characteristics of the interactions human coronavirus spike glycoproteins with their cellular receptors, I studied the human coronaviruses 229E and NL63.
Using purified, soluble cellular receptor for 229E (aminopeptidase N) and purified portions of the 229E S protein, I identified a receptor binding domain on the 229E S protein. I showed that soluble receptor neutralizes 229E virions at 37°C, but not at 4°C, suggesting a receptor-induced conformational change in the S glycoproteins on 229E virions.
Human coronavirus NL63 was discovered by two laboratories in the Netherlands in 2004. NL63 is closely related to human coronavirus 229E. I identified the cellular receptor for NL63 (angiotensin converting enzyme 2; ACE2) by inoculating cell lines that were transiently transfected with DNA encoding candidate receptor proteins with NL63 virus and then detecting viral antigen with a cross-reactive anti-229E polyclonal antibody. These results were particularly intriguing, as ACE2 also acts as a cellular receptor for the very distantly related Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). In addition, I identified potential target tissues for NL63 using primary human lung type II epithelial cells and alveolar macrophages, provided by Dr. Robert Mason at National Jewish Medical and Research Center. Both cell types are permissive for NL63 replication.
To characterize the molecular interactions of NL63 S with ACE2 cellular receptor, I developed a panel of anti-NL63 S monoclonal antibodies in collaboration with other members of the Holmes laboratory and Drs. Donna Ambrosino and Theresa Broering at the University of Massachusetts. Epitope-mapping experiments using purified, C-terminally truncated NL63 S proteins showed that NL63 S has at least three antigenic domains. Eight of eleven anti-NL63 monoclonal antibodies mapped to the N-terminal 200 amino acids of the S glycoprotein, indicating that this portion of the S protein is very immunogenic.
Neutralization tests with NL63 virions and purified monoclonal antibodies showed that each antigenic domain contains neutralizing epitopes. Antibodies mapping to the N-terminal 200 amino acids had the most potent neutralizing capacities. The S proteins of monoclonal antibody resistant variants revealed four amino acid substitutions relative to wild-type S proteins (P231S, S249F, L307V and Q999K), suggesting roles for these amino acids in antibody-mediated neutralization.
These results are significant in that they provide information about how human coronavirus S proteins engage their cellular receptors to achieve entry, how antibodies work to neutralize virus and how coronavirus evolution may affect cellular receptor usage. The data presented herein is therefore applicable to the development of sensitive diagnostic tools, immunogen selection for anti-coronavirus vaccines and prediction of receptor usage of human coronaviruses that may emerge in the future.