Epstein-Barr Virus Encoded GPCR Protein BILF1Up-regulate ICAM-1and Its Molecular Mechanism
|Keywords||BILF1 GPCR ICAM-1 AP-1 NF-κB|
BackgroundG protein-coupled receptor (GPCR) is a large family of receptors coupled throughG-protein effector molecules to complete the transmembrane signal transduction, and avariety of physiological or pharmacological effects. In addition to eukaryotes, the viralproteins encoded by many viruses contained several hallmarks of GPCRs, including sevenhydrophobic transmembrane domains, conserved cysteines in the amino terminus and inthe extracellular loops, amino-terminal glycosylation sites, and intracellularphosphorylation sites. Many in vitro and in vivo studies suggest that the GPCR proteinencoded by the virus has an important regulatory role in viral replication and virus-induced pathology in the natural host physiological effects. For example, M33-deficientmurine CMV does not disseminate to the salivary glands of infected mice, R33-deficientrat CMV is less virulent for immunocompromised rats than wild-type virus, and in parallelto M33mutant murine CMV, R33mutant rat CMV does not replicate efficiently in thesalivary glands of infected rats. Furthermore, R78-deficient rat CMV is less virulent thanwild-type rat CMV, and M78deletion mutant murine CMV replicates and/or disseminatesto a lower level in the salivary glands, spleen, and liver in both immunocompetent andimmunocompromised mice. γ2-Herpesvirus-encoded GPCRs also have important functions.For example, ORF74is considered the key viral oncogene in Kaposi’s sarcomapathogenesis.Epstein-Barr virus-encoded BILF1is a GPCR protein, which is deemed to the only GPCR protein of Epstein-Barr virus. So far, only5literatures about BILF1are documentedby Pubmed. These studies have shown that BILF1belongs to a new type of Gprotein-coupled receptor subfamily, which is localized to the plasma membrane,constitutively activates the Gαi protein and passes biological signal through it; BILF1alsoplay a vital role in the process of viral immune evasion through down-regulating thesurface expression of MHC class I molecules in the host cell membrane. BILF1withGPCR activity is one of the Epstein-Barr virus immediate early genes. It is known thatBILF1involved in the regulation of ERK, NF-B signaling pathways and play a role in thelytic replication of the virus. However, more regulation of BILF1to the host cell remainsto be further explored.Epstein-Barr virus (EBV) is one of the most widespread viruses. Studies have shownthat EBV infection is found in>90~95%humans worldwide. An important feature of thevirus is that Epstein-Barr virus can complete several repeated latent-lytic conversion in thebody. As encountering specific stimulated signal, EBV latented in the peripheral bloodmemory B cells is reactivated, then gets released from the host cells and eventuallyexplosive replicates in oropharyngeal epithelial cells. EBV-positive B cells migrate in thecirculatory system, and finally settle and replicate in the effect site. The life cycle of EBVindicates that the virus can not survive in the body without the function of cell adhesionmolecules. A large numbers of clinical studies show that EBV transformes B cells andassociates with a variety of malignant diseases including Burkitt’s lymphoma,nasopharyngeal carcinoma, and gastric cancer and some cell adhesion molecules mediatethe malignant behavior of tumors.Intercellular adhesion molecules（ICAM-1）have gained more and more attentionamong the researchers due to its function of mediating heterogeneous adhesion. ICAM-1belongs to the immunoglobulin superfamily, is an important factor regulatingheterogeneous adhesion. It overexpresses in some malignant tumors including malignantmelanoma, gastric cancer, colon cancer and kidney cancer and is closely related with tumormetastasis and prognosis. We found in the study that BILF1can up-regulate the expression of ICAM-1molecule at the protein level. We designed experiments on this basis in order to furtheranalyze the expression regulation of BILF1to ICAM-1and its mechanisms.ObjectiveBased on the background above, we designe experiments and hope to analyze theeffect of EBV-encoded immediate early protein BILF1up-regulating intercellular adhesionmolecule ICAM-1and make a tentative research on its regulatory mechanism.MethodsFirst, we builded the cell model of TPA/SB-induced EBV activation in the human Blymphoma cell line Raji to analyze the expression pattern of BILF1during the process ofEBV activation. Then, we cloned the cDNA of BILF1and constructed its eukaryoticexpression vector and turned this exogenous BILF1into cells through electroporationtransfection technique to prepare a condition for further experiment. Western bolt,Real-time PCR, Dual-Luciferase reporter gene system and specific siRNA interferencewere employed to analyze the effect of BILF1on the expression of ICAM-1at the proteinlevel and mRNA level and its regulatory mechanism.ResultsThe results showed that BILF1is one of the Epstein-Barr virus-encoded immediateearly genes and its expression pattern is coincident with Zta（the immediate earlytranscription factor of EBV）in the cell model of TPA/SB-induced EBV activation. Theover-expression of BILF1up-regulates the expression of intercellular adhesion moleculeICAM-1at the mRNA level and protein level. BILF1activates the ICAM-1promoter,suggesting its regulation on the transcription of ICAM-1. Both NF-κB and AP-1areimportant response elements in the ICAM-1promoter region. Dual-Luciferase reportervector analysis showed that the over-expression of BILF1could increase the activity ofAP-1and NF-κB reporter vectors. Also, the activation of AP-1is more significant and theeffect are dosage-dependent. It suggests that it is via activating the promoters of NF-κBand AP-1that BILF up-regulates the promoter activity of ICAM-1, thus affecting its transcription and expression. Further analysis through WB experiment showed that whileup-regulating the expression of ICAM-1, the over-expression of BILF1actives p65byphosphorylation, up-regulates the expression of the NF-κB target genes Bcl-XL and IκBɑ,and induces the phosphorylation of c-Jun and increases the level of c-fos which both arethe members of AP-1family. Through blocking the activation of the ERK pathway withMEK1inhibitor, it is observed that only part of the effect that BILF1activates AP-1andup-regulates ICAM-1is affected. This phenomenon suggested that the up-regulation ofICAM-1by BILF1is dependent on other pathways. The GPCR activity of BILF1isdependent on its EKT motif, so we constructed the BILF1-K122A mutant with a defect inthe EKT motif. Dual-Luciferase reporter vector analysis and WB experiment showed thatthe activation of BILF1to AP-1is partially inhibited due to the functional defect of EKTmotif, suggesting that the activation of BILF1to AP-1is partly dependent on the EKTmotif. Finally, we confirmed that specifically knocking-down the expression of BILF1could inhibit the up-regulation of ICAM-1induced by the activation of BILF1throughsiRNA interference experiment.ConclusionsEpstein-Barr virus-encoded immediate early protein BILF1with GPCR activityup-regulates the expression of intercellular adhesion molecule ICAM-1at the protein leveland mRNA level. This effect is achieved through the AP-1signalling pathway which is notentirely dependent on the ERK molecule. Through up-regulating the expression level ofICAM-1, BILF1strengthens the adhesive power between different cells, mediates theadhesion between the infected cells and endothelial cells, promotes the process of piercingthe vascular endothelial cells, and then plays an important role in the tumor invasion andmetastasis. This result suggests that BILF1are likely to participate in the malignantevolution of EBV-associated tumors and play an important role in the tumor invasion andmetastasis.