Molecular Mechanisms Of Inhibition Of MHC Ⅱ By Zta And A Preliminary Research On Cellular Proteins Interacting With Zta

Since the end of the nineteenth century when tobacco mosaic virus was found, many studies have confirmed that the virus lives with the host, against each other in the maintenance of a micro-ecological balance. On the one hand, immune system has developed a highly intricate and elaborate mechanism against the invision of microorganisms. On the other hand, pathogenic microorganisms have also adopted a variety of immune evasion mechanisms in evolution. For example, viruses may evade the recognition and attack of host's immune system by directly inhibiting the host's immune response or by antigen drifting as well as antigen switching.In the recent years, the critical roles of MHC class II molecules mediated immune recognition in anti-viral immunity have been noticed. MHC class II molecules are highly expressed on antigen-presenting cells, mainly macrophages, dendritic cells, Langerhans cells and B cells. Upregulation of MHC class II molecules has a physiological significance. It can promote the antigen presentation by APC to CD4+ T cells, thus enhancing the specific immune response. It has been confirmed that human immunodeficiency virus HIV-1, human Herpes Simplex virus HSP-1 can evade the attacks by host's immune system by affecting MHC class II-dependent immune recognition.Epstein-Barr virus (EBV) is one of the most widespread viruses. Studies have shown that EBV infection is found in >90 ~ 95% humans worldwide. A large numbers of clinical studies show that EBV associates with a variety of malignant diseases including Burkitt's lymphoma, nasopharyngeal carcinoma, and gastric cancer. In addition, post-transplant lymphoproliferative disease (PTLD) is also a serious problem due to EBV infection.EBV predominantly infects human B cells and epithelial cells. It exists in host cells in two different states, the latent and lytic states. In latent state, the virus does not express or only express a few viral proteins. In lytic infection, almost one hundred viral proteins are expressed. Zta, the product of EBV immediate-early gene bzlf1, is a member of basic leucine zipper (bZIP) transcription factor family, and plays a critical role in the life cycle of EBV. It is the first protein expressed in EBV reactivation. The expression of Zta switches EBV infection from the latent to lytic state, leading to the replication and dissemination of EBV. As EBV in absolute latency does not express any viral proteins, the immune system is unable to distinguish the infected cells from normal cells. So far, elimination of lytic EBV in the host cell is considered to be the key for control EBV infection. As the most critical protein during lytic infection of EBV, Zta has been an important target in anti-virus research.MHC II molecule restricted CD4+ T cell-mediated immune response is crucial in control of Herpes virus infection. It has been reported that the lytic cycle of EBV is associated with decreased expression of cell surface MHC class II molecules. It is noteworthy that the Herpes virus assembles viral particles in endosomes, where antigenic peptides bind to MHC II molecules. As B cells are not only the important host cells for EBV but also the antigen-presenting cells (APC), we speculate that EBV is likely to affect the expression of MHC II molecule in infected B cells and interfere with the presentation of viral antigens during lytic cycle. This study was designed to study the mechanisms by which EBV inhibits the expression of MHC class II molecules.In this study, B lymphoma cell line Raji was used. The reduced MHC II expression at transcription level was observed by inducing EBV reactivation in Raji cells. Downregulation of MHC II was confirmed to be associated with the expression of Zta. Further study demonstrated that Zta expression also resulted in the inhibition of MHC-II trans-activator (CIITA) at both transcriptional and protein levels. Sequence analysis revealed the presence of two potential binding sites of Zta (Zta response elements, ZRE) in the CIITA promoter. By luciferase assays, chromatin immunoprecipitation (ChIP) assay and oligonucleotide pull-down, binding of Zta to proximal ZRE221 in CIITA promoter was confirmed. The inhibition of CIITA and MHC II by Zta could be reversed by site-directed mutagenesis and Zta specific RNA interference, revealing a novel mechanism by which EBV inhibits MHC II molecules mediated antigen presentation and evades immune clearance.Zta not only regulates the transcription of cellular genes through its transcriptional activation domain, but also affects the functions of host proteins by directly interacting with host proteins through its coiled-coil domain. It has been reported that Zta can bind to a variety of host proteins, such as retinoic acid receptor, TATA-binding protein and p53, interfering with the functions of these proteins. The second part of this study was designed to screen the cellular proteins that interact with Zta by using immunoprecipitation and proteomic analysis to reveal the interactions between EBV and host cells and thus provide the information for antiviral strategies.For this purpose, we constructed a GST-Zta fusion protein. By GST pulldown and mass spectrometry analysis, we obtained 27 cellular proteins that may interact with Zta. One of these proteins, CENP-E, was verified as Zta binding protein by Western blot. It has been reported that CENP-E may be involved in spindle checkpoint control in the cell cycle. Downregulation of CENP-E expression can affect the sister chromatid alignment and block the mitosis in M phase, or result in formation of aneuploidy cells. Since CENP-E is involved in cell cycle regulation, we analyzed the DNA content in Raji cells induced by TPA and sodium butyrate by flow cytometry. A significant increase of the cell population (from 14.2% to 44.6%) at G2/M phase was detected, suggesting that the cell cycle arrest in lytic cycle of EBV may be related to the dysfunction of CENP-E in mitosis.In summary, the findings in this study include: 1. Zta specifically downregulates the expression of CIITA and MHC class II molecules in EBV reactivation. 2. A functional ZRE (ZRE221) was found in the proximal region of CIITA promoter and Zta inhibits CIITA transcription by binding to ZRE221 through its DNA binding domain. 3. 27 proteins potentially interacting with Zta were found from Raji cells induced by TPA/sodium butyrate and Zta-overexpressing Hela cells. CENP-E was verified as a Zta binding protein. 4. Induction of Raji cells by TPA/sodium butyrate resulted in G2/M arrest. It may attribute to the disrupted functions of CENP-E by Zta.