Author

Paul T. Wille

Date

November 2011

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Molecular Microbiology and Immunology

Institution

Oregon Health & Science University

Abstract

My studies focused on the role of human cytomegalovirus (HCMV) envelope glycoproteins in viral entry. My first set of studies questioned how a complex of HCMV glycoproteins, gH/gL/gO, functions to promote virus entry. Our laboratory has previously demonstrated that another complex of five HCMV proteins, gH/gL/UL128/UL130/UL131, is necessary for HCMV entry into epithelial and endothelial cells that requires endocytosis and low pH-dependent fusion with endosomal membranes. When I began my studies, it was believed that entry into fibroblasts was facilitated by HCMV gH/gL/gO and occurred at the plasma membrane. However, the exact role of gO in entry had not been thoroughly defined. I studied this genetically by constructing and characterizing a HCMV strain TR gO-null mutant. Together with biochemical characterization of gO, we discovered that in the HCMV strain TR, gO acts as a molecular chaperone to promote gH/gL ER export, then gO dissociates from gH/gL and is not found in the virion envelope. When gO was deleted, gH/gL incorporation into the virion envelope was significantly reduced, and these virus particles were unable to enter both fibroblasts and epithelial and endothelial cells. My second set of studies focused on HCMV glycoprotein gB. For other herpesviruses, gB is thought to be the fusion protein, causing the virion envelope to fuse with cellular membranes during virus entry. Triggering of gB is thought to involve interactions between gB and gH/gL complexes. However, little is known about how HCMV gB promotes fusion or even if HCMV gB is the fusion protein. I found that HCMV gB could be expressed in cellular membranes and would promote the entry of HCMV virus particles lacking gB. This process, denoted entry in trans, was novel in that no one has described an instance in which a viral fusion protein can be expressed in cells and mediate entry of virus particles lacking that fusion protein. By contrast, cells expressing HCMV gH/gL would not mediate entry of virus particles lacking gH/gL. In the entry in trans that I observed, gH/gL complexes are oriented toward the plasma membrane of cells and gB is oriented toward the virion envelope. Thus, these results support two important hypotheses: i) HCMV gH/gL complexes bind cellular ligands or receptors and can interact with gB across the space between the virion envelope and cell membranes and ii) gB is the fusion-inducing protein.

Identifier

doi:10.6083/M4PV6HC2

School

School of Medicine

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