Human cytomegalovirus (HCMV), a betaherpesvirus, can cause congenital defects in utero and pneumonitis, hepatitis, retinitis and gastrointestinal disease in immunocompromised adults. HCMV characteristically causes latent and persistent infections. Latent infections are associated with various undifferentiated cells of the blood and endothelial cells of various organs. Productive infection is associated with terminally differentiated macrophages and epithelial cells. The cell type and the stage of differentiation effect the level of HCMV immediate early (IE) gene transcription. The IE genes code for regulatory proteins that effect both cellular and viral gene transcription and determine whether the infection is productive.
In Specific Aim I, we propose to identify and characterize cellular protein(s) that binds to a 5'TRTCGC3' motif repeated in the silencer elements upstream of the viral IE1/IE2 and IEUS3 genes and represses transcription. In higher eucaryotes, little is known about silencer elements, DNA binding-dependent repressor proteins, and the mechanism of transcriptional repression.
In Specific Aim II, we propose to isolate recombinant HCMVs with the silencer element upstream of the IEUS3 gene deleted or substituted with all or parts of the silencer-modulator from the UL component of the viral genome. The viral IEUS3 gene, which is dispensable for replication in human foreskin fibroblast cells, will be replaced with the indicator gene chloramphenicol acetyl transferase (CAT). The effect of these mutations on transcription of the CAT gene in recombinant virus infected nonpermissive, undifferentiated myelomonocytic THP-1 cells or permissive, differentiated THP-1 cells will be determined. These cells will be used to evaluate the effect of silencer elements and repressor proteins on IE transcription in cells similar to monocytes and macrophages.
In Specific Aim III, we propose to characterize repression of transcription from a HCMV representative early promoter and activation of transcription by the viral IE2 DNA binding protein. The early viral promoter has upstream binding sites for cellular protein(s) and the region represses downstream transcription. IE2 protein homodimer competes for binding to the same region and negates the repressive effect. The IE2 protein is a member of a small group of DNA binding proteins that interact with A-T rich sequences within the minor groove of the DNA molecule. The effect of wild type and mutated DNA binding sites on downstream transcription during transient transfection will be determined. In addition, recombinant viruses with wild type and mutated DNA binding sites will be characterized. We are interested in the effect of IE2 protein template binding on repressor protein activity. We are also interested in the cellular repressor protein.
