Human cytomegalovirus (HCMV) is a ubiquitous human pathogen. It is a major cause of infectious morbidity and mortality in immunocompromised individuals, especially in transplant recipients and AIDS patients. It is also a leading cause of congenital birth defects. My long-term research goal is to elucidate the interactions of HCMV with Its host cell at the molecular level, and evaluate the consequences of these interactions, especially the effects on viral replication and pathogenesis. In this proposal, I will focus on the significance of the induction of interferon-stimulated genes (ISGs) following HCMV infection.
My specific aims are as follows: I. Identify HCMV-response elements (HCRE)-binding proteins. Two elements, the interferon stimulated element (ISRE) and gamma-interferon activated site (GAS), have been identified responsible for HCMV-mediated ISG induction. Gel shift assays have shown that four protein complexes interact with these elements. Identification of the consensus sequence of HCRE and HCRE-binding proteins are critical for understanding molecular basis of this signal transduction pathway. I propose different experiments to approach these questions, including: (1) Test base-pair substitutions to identify the sequence motifs necessary for HCMV-mediated ISG induction. (2) Examine known HCRE-binding proteins in the interferon regulatory factor family for their roles in HCMV-mediated signal transduction pathway. (3) Purify HCRE-binding proteins by chromatography and identify these binding proteins by 2-dimensional electrophoresis and mass spectrometric analysis. (4) Screen HCRE-binding proteins using a yeast one-hybrid system. (5) Screen HCRE-binding proteins using retrovirus cDNA expression libraries. II. To investigate biological significance of the induction of ISGs by HCMV infection. HCMV envelope glycoprotein B (gB) has been identified for initiating this signal transduction pathway. Importance of initiating this signal pathway in HCMV replication will be studied using biochemical and genetic approaches, including: (1) The roles of HCMV activated proteins in the induction of ISGs and HCMV IE genes will be studied. (2) Mutate two GAS elements in the HCMV major IE promoter and examine their roles in viral gene expression and replication.
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