Regulation of Human Cytomegalovirus Major Immediate-Early Gene Promoter by Novel Viral Genes

• Main Authors: Shang-Kwei Wang, 王雙桂
• Other Authors: Tai-Tsung Chang
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/99043510386040825681

博士 === 高雄醫學大學 === 醫學研究所 === 89 === Human cytomegalovirus (HCMV) major immediate early gene (MIE, UL122-123) is known to play an essential role in viral replicative lytic cycle, latent infection as well as viral reactivation from latency. Major immediate-early promoter/enhancer region (MIEP, －1139 to +52) is regulated by cell-type specific transcriptional factors and its own MIEs proteins (IE2p40, IE1p55, IE1p72 and IE2p86) which manifest an autoregulatory mechanism. Three HCMV proteins, pUL69 (tegument protein, encoded by UL69), pUL82 (pp71, upper matrix protein, encoded by UL82) and pUL84 (encoded by UL84), have been demonstrated to be involved in the regulation of MIEP. To investigat whether that HCMV MIEP is modulated by additional viral genes the HCMV (AD169) genomic sublibraries were constructed and transient co-transfection assays were performed to assess the ability of these sublibraries to modulate MIEP expression. In this study, sublibraries, enhancing the production of reporter enzymes chloramphenicol acetyl transferase (CAT) or luciferase (Luc), were selected. Three sublibraries SL-F, SL-M and SL-Q, each contributed one essential clone, pCL77, pCL196 or pCL1066, for augmenting the CAT activity. To identify the loci responsible for the stimulatory effect, we further subcloned the inserting fragments and identified three distinct loci, UL76, UL87 and UL112-113, modulate MIEP expression in the absence of other viral gene products. To generate a UL76-specific antibody for immunodetection, the UL76 ORF was constructed as a histidine-tagged fusion protein that was produced in prokaryotic cells. A polyclonal antibody raised against the UL76 fusion protein immunoreacts with a protein of 38 kDa (pUL76) in UL76 ORF-transfected cells. Additionally, pUL76 is present in HCMV-infected cells at the immediate-early to late stage of the reproductive cycle. UL76 is a highly basic protein (predicted pI 11.6). pUL76, tagged with a green fluorescent protein was found to localize exclusively at the nucleus. Taken together pUL76 is characterized as a novel regulatory protein that mediates both activation and repression of gene expression, depending on the promoter context and the concentration of the protein.