Study On And Exploiture Of The Transcription Regulation Of Human Cytomegalovirus Major Immediate Early Enhancer/Promoter (HCMV MIEP)

The serious pathogenic effects associated with human cytomegalovirus (HCMV) infections, particularly in newborn and immunocompromised individuals, result from a complicate interplay of viral gene products and cellular factors. HCMV immediate early gene expression clearly plays a pivotal role in this scheme since the gene products are synthesized initially after infection primarily relying on the host factors. Therefore, the regulatory mechanisms governing the activity of the HCMV major immediate early enhancer/promoter (HCMV MIEP) have been receiving a universal attention. On the other side, the HCMV MIEP is known to exert a strong constitutive transcription activity in a broad spectrum of cells in vitro. However, the expression of extrinsic gene under this promoter in adult transgenic mice is often more restricted. Therefore, the knowledge on the regulatory mechanisms of MIEP is the prerequisite to reconstruct a stronger and more universal promoter used in transgenic mice. The HCMV MIEP, extending from -600 to +1 relative to the transcription start site, contains an array of transcriptional regulatory elements. Characteristic of HCMV MIEP are binding sites for eukaryotic transcription factors within reiterated sequences. For example, MIEP comprise 16-, 17-, 18-, 19-, 21-bp repeat elements. Among these repeats, the 18-bp and 19-bp repeats, each echoing four times, play an important role to the constitutive strength The of the MIEP. However, whether these elements repeat just for backing up or they really execute various indispensable functions, no one has given an answer. Here, we had found that these reiterated elements are different from each other functionally through deletion mutation, site-directed mutation, decoy perturbation, and EMSA. In addition, we also found that the CG-reverse in the first 19-bp repeat could enhance the MIEP strength in Hela cells, and this observation has been confirmed in transgenic mice. Meanwhile, the expression pattern of the extrinsic gene drove by the certain mutated MIEP is more ubiquitous. Furthermore, we also initially identified the functional communication among these 18-bp repeats and 19-bp repeats.