The Roles Of Adenovirus Type 12 E1B 55-kDa Oncoprotein In Its Interaction With Host Proteins

Adenovirus is an important tool for detecting fundamental theory of cell biology. Cells can be transformed to neplastic cancer cell by continued expression of the ad E1B and E1A proteins. Nevertheless as we know that Sin3A as a transcriptional corepressor locates in the nucleus, it's unclear if Sin3A express in the cytoplasm. It has been documented that E1B-p53 interaction results in the inactivation of p53 mediated transactivation function and facilitates Ad-mediated cell transformation [1,2], Our data demonstrate Ad 12 E1B 55-kDa cann't suppresses p53 mediated transcription, and it can suppress p53 mediated transcription with corepressor Ad12 E1A. Abstractly, adenovirus leaves a lot to tell us about basic cell active.E1B 55-kDa is a substrate of the ubiqutin ligase; it conjugates p53 or other subunits of the MRN complex for target proteosomal degradation. Although E1B-55kDa can surpresses p53 gene mediated function as a transcriptional repressor by conjugating a corepressor to it. So EIB 55-kDa requires an unclear cellular corepressor for completed it. These issues need us that in spite of Ads have taught us too much, but they still have left a lot to tell us.There are nearly 50 distinct serotypes of the human adenoviruses. They share similar proteinaceous capsid morphology and linear double-stranded DNA genomes. However, important functional differences have been observed among the Ad serotypes. For examples, intravenous injection of Ad5 viral particles results in significant liver sequestration of the virus. It was reported recently that Ad5 transduces liver cells via the coagulation factor X (FX), whereas many other Ad serotypes do not share this property. It was shown that the hypervariable regions of the Ad5 coat protein hexon mediate hexon-FX interaction. Hypervariable regions also exist in many other Ad proteins. They conceivably also confer distinct functions. From now on, there are three generations of adenovirus vector, and most of them made from Ad2 or Ad5. Though the third generation of Ads transfer vector still require helper virus for its function in the cells. Ad 12 DNA conjugates with cell chromosomes after virus infection[5], Ad12 DNA is firmly at selective chromosomal sites[6]. Ad12 DNA with the terminal protein still covalently attached (Ad12 DNA-TP) is more efficiently taken up by mammalian cells[7]. Understanding differences among these Ad serotypes may allow for the design of better gene transfer vectors for therapeutic purposes with improved efficacy and reduced toxicity.Under the leading and direction of Professor Shengquan Zou, we have studied on the biological of malignant tumor, especially on its early diagnosis, early treatment. I have been assined to Dr. Daiqing Liao's lab, Shands cancer center, University od Florida, to further study the mechanism of cell malignant transformation and properly adenoviral genetic vector for treating cancer as a joint Doctor training candidate sponsored by China Scholarship Council. We have studied on the biological function of p53, especially on its interaction with adenorirus oncopreins E1B and E1A in the cancer cells. The present study is based on the previous results, concentrating on the mechanism of adenovirus oncopreins E1B and E1A interaction with infected host celluar corepressor, trying to provide an experimental basis for further study and excpted to refer some new approachs for cancer early diagnosis and treatment. Objects:To investigate adenovirus type 12 E1B 55-kDa protein in regulating its interactions with host proteins Sin3 and intracellular location.Methods:By using molecular biology method to construct various constructs spanning different regions of Sin3A and the modification of E1B, then transfected them into Saos2 cells and determined their subcellular localization using IF.Results:1. The aa439-443 (KKKPK) of Sin3A is critcal for its location in nuclear. Sin3A constructs containing PAH1 in the absence of the NLS was found together with E1B in cytoplasmic bodies but they failed to colocalize in the nucleus. when the NLS is included, constructs lacking C-terminal sequence localized exclusively in the nucleus and colocalized with E1B in nuclear dots.2. Phosphorylation at Ser476 and Ser477 of Ad12 E1B also appears to have an important role in regulating its interaction with Sin3A.3. N terminal of Adl2 E1B 55-kDa stalled is important for its entrance into nuclearConclusions:1. The NLS of Sin3A regulates its interaction of E1B-Sin3A and its intracellular location.2. Phosphorylation of C terminal of Ad12 E1B regulates its intracellular location.3. N terminal of Ad 12 E1B 55-kDa regulates its traffic from nuclear. Objects:To investigate the SUMO modification of Ad12 E1B 55-kDa in regulating its intracullar location and its interaction with the host proteins Sin3A and Sin3B.Methods:By using molecular biology method to construct and mutate various E1B and design a system that allowed for inducible attachment of SUMO1 to E1B is that two copies of rapamycin-binding domain FKBP were fused to the N-terminus of Ad12 E1B to make 2xFKBP-E1B and the rapamycin-binding domain of the mTOR kinase, FRB, was fused to the C-terminus of SUMO1 to produce HA SUMO1-FRB, then expression in Saos2 cells. By using immunfluoresence and western blotting to detect the location and associated proteins of E1B complex.Results:1. Lys88 and perhaps an additional lysine within the N terminal domain of Ad 12 E1B are not sites of SUM03 modification but SUMO1.2. Exclusion of SUMO1 modified Ad12 E1B from the nucleus to cytoplasm.3. SUMO1-modification of Ad12 E1B prevents its interaction with Sin3 corepressor proteins.Conclusions:1. SUMO1 modification of Ad12 E1B 55-kDa sequesters into cytoplasm.2. SUMO1 modification of Ad12 E1B 55-kDa block its interaction with Sin3A and Sin3B. Objects:To investigate Ad12 E1B, E1A and SUMO1 modification of adenovirus type 12 E1B 55-kDa proteins in regulating viral DNA synthesis.Methods:The expression of constructs E1A266, wtE1B and SUMO1-E1B 55-kDa in LN229 cells by Ad3875 virus infection. We detect the amount of virus DNA replication by viral replication assay.Results:1. Ad12 E1B 55-kDa or Ad12 E1A is able to stimulate viral DNA replication.2. Ad E1B 55-kDa is significant able to stimulate viral DNA replication in cooperation with E1A.3. SUMO1-E1B, despite exclusive localization in the cytoplasm, can still support viral DNA replication in cooperation with Ad12 E1A.Conclusions:1. The critical roles of Ad12 E1B 55-kDa and E1A in regulating viral replication.2. The important roles of SUMO modification of Ad12 E1B 55-kDa in regulating viral replication with Ad 12 E1A. Objects:To investigate the mechanism in Ad12 E1B 55-kDa and E1A represses p53-mediated transcription.Methods:We used anti-p53 and anti-ElB 55-kDa antibodies for ChIP in G401 and G401-CC3 cell lines, then PCR amplified two regions of the p21 and Mdm2 promoter to detect whether E1B only associates with the p53-binding sites of the p21 and Mdm2 promoter; p53 and its target geneorteins were analyzed in Western blotting analysis and quantitative real-time PCR analysis of p21 mRNA levels in G401 and G401-CC3 cells, the mRNA levels were normalized against that of beta-actin; HCT116 cells were transfected with firefly luciferase reporter under the control of the Mdm2 promoter and the control Renilla luciferase reporter alone (Reporter) or together with other indicated expression plasmids (P53, E1A, E1B, E1A, E1B, P53, P300), Firefly luciferase activities were normalized against the Renilla luciferase activities, Shown are average values of two independent experiments with standard deviations. G401 cells were stably transduced with lentiviral vector expressing GFP, GFP-E1B, GFP-E1B and E1A, or E1A and treated with TSA (1μM) or etoposide (50μM) for 24h for Western blotting assays; we also have made a number of E1A mutant constructs to test whether these mutants could cooperate with E1B to repress the Mdm2 promoter in reporter gene assays.Results:1. Ad12 E1B 55-kDa associates with p53-binding sites of the p21 and mdm2 promoter in cells.2. Ad12 E1B 55-kDa alone could stable the expression of p53. 3. E1A can cooperate with the E1B 55-kDa for suppressing p53-mediated transcription.4. Coexpression of E1A and E1B 55-kDa completely reversed p300-mediated coactivation.5. The CBP/p300, Rb and CtBP-binding motif are not required for cooperation with E1B 55-kDa in repressing p53.Conclusions:1. Ad12 E1B 55-kDa binds to p53 in chromatin in cells and active the expression of p53.2. Ad12 E1B 55-kDa or E1A alone could not suppress p53-mediated transcription, but coexpression of E1A along with E1B 55-kDa resulted in repression of p53 target genes.3. The binding sites for p300, Rb and CtBP in E1A may not be necessary for cooperation with E1B 55-kDa.