Date of Award

2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Abstract

Viruses have evolved complex ways to penetrate host barriers and cause disease. One of the most important barriers the virus has to cross is the cellular membrane. Enveloped viruses accomplish this task by viral glycoprotein-mediated binding to host cells and fusion of virus and host cell membranes. For the coronaviruses, viral spike (S) proteins execute these cell entry functions. In my dissertation research I focused on understanding the coronavirus spike proteins as well as other cofactors required for S-mediated entry into cells.

The S proteins are set apart from other viral and cellular membrane fusion proteins by their extensively palmitoylated membrane-associated tails. In our experiments, substitution of alanines for the cysteines that are subject to palmitoylation had effects on both S incorporation into virions and S- mediated membrane fusions. In specifically dissecting the effects of endodomain mutations on the fusion process, we used antiviral peptides that bind only to folding intermediates in the S-mediated fusion process, and found that mutants lacking three palmitoylated cysteines remained in transitional folding states nearly ten times longer than native S proteins. This slower refolding was also reflected in the paucity of post-fusion six-helix bundle configurations amongst the mutant S proteins. Viruses with fewer palmitoylated S protein cysteines entered cells slowly and had reduced specific infectivities. These findings indicate that lipid adducts anchoring S proteins into virus membranes are necessary for the rapid, productive S protein refolding events that culminate in membrane fusions.

The membrane fusion process also requires an S protein conformational flexibility that is facilitated by proteolytic cleavages. The severe acute respiratory syndrome (SARS) coronavirus S proteins rely on host cell proteases for fusion activation. I identified the human lung transmembrane serine protease, TMPRSS2, as an important factor for SARS coronavirus entry. TMPRSS2 co-localized on cell surfaces with the virus receptor ACE2, and enhanced the cell entry of both SARS S - pseudotyped HIV and authentic SARS-CoV. Enhanced entry correlated with TMPRSS2-mediated proteolysis of both S and ACE2. These findings indicate that a cell-surface complex comprising a primary receptor and a separate endoprotease operate as portals for activation of SARS coronavirus cell entry.

Creative Commons License

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

Included in

Virology Commons

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