The broad and long-term objectives of this application focus on the role of viral and cellular transcription factors in mediating the pathogenesis associated with human cytomegalovirus (HCMV) infection. HCMV is a clinically relevant, ubiquitous pathogen that is often the cause of death in AIDS, cancer and transplant patients. Levels of the major IE regulatory proteins are proposed to determine the activated or latent state of the virus. Therefore, the control of transcription by the HCMV major immediate-early promoter (MIEP) is an important event in determining the replicative state of the virus.
The specific aims are to elucidate the molecular interactions between host-cell and viral transcription factors in governing HCMV MIEP function. In this proposal, I will identify and characterize cis and trans-acting elements associated with a newly discovered regulatory domain of MIEP located in the 5' untranslated leader region. This regulatory domain will be characterized by analyzing a series of mutations with in vivo and in vitro transcription assays. DNA-binding factors will be analyzed using mobility shift, DNasel protection, methylation interference and UV-crosslinking experiments. Furthermore, in vitro transcription oligonucleotide competition experiments will be used to specify the interaction of transcription factors. I will investigate the biological significance of this domain as well as the modulator and enhancer domains in uninfected/infected human foreskin fibroblasts and undifferentiated/differentiated NT2 cells as models for the regulation of MIEP during a permissive infection and latency/reactivation, respectively. Changes in sequence specific transcription factors associated with the infection or latency/activation will be identified using DNA-binding experiments described above. The functional characterization of these factors will be determined by utilizing mutations of MIEP in in vivo and in vitro transcription assays established from the model cell systems. Finally, I will characterize feedback mechanisms associated with the 55 kDa major IE protein in activating the MIEP by using IE cDNA effector plasmids in dose-response cotransfection experiments with mutant MIEP reporter plasmids. These experiments will identify the responsive cis-acting target for the 55kDa in the MIEP. I will explore the molecular basis of action by addressing whether the 55 kDa protein functions via a direct or indirect mechanism or whether it involves accessory factors associated with the activation. The identification and characterization of the cellular and viral factors controlling the expression of MIEP will be essential in defining events regulating latency and reactivation of HCMV.
