Human cytomegalovirus (HCMV) is a ubiquitous pathogen that is a major cause of disease in immunocompromised individuals, such as AIDS and bone marrow transplantation patients. HCMV is also the most common congenital viral infection and is the leading cause of infectious CNS maldevelopment in children. Despite the clinical importance of this viral pathogen, little is known regarding the molecular and cellular basis of HCMV assembly in infected cells. Current models of HCMV suggest that following transport of viral capsids to the cytoplasm, they are enveloped at the trans-Golgi Network (TGN) and associated endosomal compartments by membranes studded with envelope glycoproteins. Correct localization and processing of the envelope glycoproteins is required for the production of infectious virus. Key to understanding HCMV biogenesis is a determination of the cellular and molecular mechanisms that localize the mature glycoproteins to the compartment(s) of viral assembly. Recent studies of the cell biology of the major HCMV envelope glycoprotein gB, an essential herpes virus glycoprotein, show that: 1) HCMV gB is localized to the TGN and sorts to the apical surface in polarized cells (the surface of HCMV release), 2) TGN localization is mediated by interaction of gB with the cellular connector, PACS- 1, and 3) inhibition of furin-dependent cleavage of gB causes missorting of the unprocessed glycoprotein and is coupled with a dramatic block in the production of infectious virus. The goal of this application is to determine the role of gB in HCMV assembly. To achieve this goal, we will address four specific aims: 1) identify the cis-acting sorting signals that direct binding of gB to PACS-l and the gB motifs and domains necessary and sufficient to direct the TGN localization and sorting, 2) determine the mechanism(s) of PACS-1-mediated localization of gB to the TGN, and the requirement of this interaction for the production of infectious HCMV, 3) investigate the role of the furin-dependent processing of gB in both TGN localization of the viral glycoprotein and in virus assembly, 4) determine the role of gB TGN localization, endosome sorting, and furin-dependent proteolysis in virus assembly by analysis of HCMV recombinants expressing gB genes defective in these processes. Together, these studies will increase our knowledge of the cell biology of HCMV gB and provide insight into the molecular mechanisms of viral assembly that will enable development of novel therapeutics to treat viral disease.
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