Date of Award

2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Abstract

Coronaviruses (CoVs) represent some of the greatest modern threats to global health. CoVs are zoonotic viruses that generally cause respiratory or enteric infections. The ability of CoVs to move between species and into human populations ensures that CoVs will remain important and dangerous pathogens. Therefore, understanding how CoVs infect different hosts is vital to human health.

CoVs are enveloped and must undergo fusion of viral and host membranes to initiate infection. Viral glycoproteins, called Spike (S) proteins, are responsible for host cell binding and carrying out the membrane fusion reaction. S proteins store energy in their folded, pre-fusion conformation that is released upon interaction with host cell factors. These host cell factors include cellular receptors, which bind to S proteins and change their conformation, and host proteases, which cleave S proteins which releases the stored energy and catalyzes fusion. Thus, to successfully infect target cells, CoV S proteins must encounter cellular receptors and proteases at the same time and place.

Our goal was to determine where on the cell surface, CoVs encounter receptors and proteases. Using biochemical isolation of cellular membranes, we showed that these entry factors are concentrated in small, ordered regions of the membrane that are also rich in scaffolding proteins called tetraspanins. These isolated membrane fractions were capable of cleaving CoV S proteins in a manner similar to trypsin, indicating the presence of active proteases within these regions. Furthermore, we used CoV infection and pseudovirus transduction to determine that CoVs reside in or near these tetraspanin-

enriched membrane microdomains (TEMs) following receptor binding and that antibody crosslinking of tetraspanins inhibits CoV entry.

While our results suggested that TEMs function as CoV entry portals, the identity of tetraspanin interactions with CoV entry factors was not clear. We used CRISPR/Cas9 knockout of the tetraspanins CD9 and CD81 and determined the effect on CoV entry. We found that CD9-deficient cells were resistant to infection by MERS and 229E CoVs but were still susceptible to infection by SARS and MHV CoVs. The receptors for MERS and 229E, but not SARS or MHV, were absent from isolated TEM membrane fractions in CD9 deficient cells. These results indicated that CD9 acts as the specific tetraspanin partner for MERS and 229E receptors. The presence of CD9 in target cells also influenced the route of entry that MERS viruses utilized. Without CD9, MERS infection was dependent on endocytosis and cathepsin activation of S proteins. In cells replete with CD9, MERS entry was more rapid and more dependent on host cell transmembrane proteases.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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