The Expression Profiling Of PUL43 Protein From Marek's Disease Virus And Its Effect On The Stability Of Cell Surface MHC Class ?

Marek's disease(MD)is a highly contagious disease with neoplasm caused by the definitive etiological agent,Marek's disease virus(MDV).MD-susceptible chicken flocks are largely predisposed to develop severe immunosuppression and proliferation of T cell lymphoma.Although vaccination is widely practiced as the most effective means to prevent and control MD,the increasing emergence of very virulent plus(vv+)MDV has prompted a worldwide concern that breaks in the protection conferred by the best available vaccines will become more frequent in years to come.Therefore,more research efforts are needed to uncover the biology of MDV infection.A particular emphasis on decoding the viral gene products that potentially dampen the host immune responses will be of great significance for the optimization of the current MD vaccines or even for the development of a novel vaccine.MDV belongs to the alphaherpesviridae subfamily in the herpesviridae.In the alphaherpesvirus subfamily,the UL43 gene homologues are revolutionarily conserved.Regarding their expression profiles and biological functions,only the UL43 gene products from herpesviruses infected mammalian have been studied.Chicken is the major natural host for MDV,but until now the study on the MDV p UL43 protein,which is encoded by the UL43 gene,has yet to be undertaken.In order to characterize the p UL43 protein,a polyclonal antibody(p Ab)was generated by immunization with a polypeptide formulation;meanwhile,an expression vector to abundantly produce the p UL43 protein was constructed by homologous recombination to verify the p Ab specificity.Indirect immunofluorescence assay(IFA)and Western blotting were performed to detect the presence of the p UL43 protein in the transfected HEK293 T cells.The results showed that the over-expressed p UL43 protein was trapped into puncta or vesicles surrounding the nucleus.The apparent molecular weight of the p UL43 protein was in agreement with that of the predicted one.The properties of the p UL43 protein were analyzed using bioinformatics tools,which included the transmembrane topology,hydrophobicity,hydrophilicity and antigenicity.The p UL43 protein was predicted to consist of 9 transmembrane(TM)domains at both ends,while a distinct hydrophilic stretch resided in the middle(222-281 aa).There were 6 and 3 spanning TM domains clustered within the N-and C-terminal,respectively.Despite the extremely hydrophobic nature of the p UL43 protein,two antigenic segments were identified within the positions 1-55 aa and 259-276 aa.Given the higher yield and purity,the second segment was preferred for the peptide synthesis.Following 5 inoculations with the adjuvant-coated peptide,the anti-sera were harvested from the experimental rabbits and subjected to affinity chromatography,resulting in the purification and concentration of the p Ab.Dot blotting was performed to test the titer of the anti-sera,.The results showed that the titer of the anti-sera against the peptide was greater than 1:100 000.In the transfected or MDV-infected cells,specific binding of the p Ab with the p UL43 protein was observed.Next,the focus of this study was directed to the sub-cellular localization of the p UL43 protein and its expression characteristics during MDV infection.To determine whether the multiple TM domains at the N-terminal(1-222 aa)or Cterminal(281-419 aa)affect the p UL43 intracellular distribution,the EGFP gene that allows for green fluorescent protein expression was fused to the 5' terminal of the sequences corresponding to the full-length protein and TM-deletion mutants.At 24 h after transfection,the p UL43 protein and its truncated mutants were examined under a confocal microscope in combination with the staining of the endoplasmic reticulum(ER),Golgi network and plasma membrane.The imaging results showed that the p UL43 protein and its truncated mutants were predominantly co-localized with the Golgi marker 58 K and ER,implying that some motifs within the long hydrophilic structure might play a critical role in stabilizing the proper p UL43 localization inside the cell.To reveal the relationship between the MDV virulence and the expression of UL43 gene products,CEF cells were individually infected with the 814 vaccine strain and the field isolate GX20NNM1.At 12,24,48,72,96,120 hours post-infection(hpi),real-time quantitative PCR(RT-q PCR)was performed to detect the expression levels of UL43 m RNA.In parallel,the changes in p UL43 protein were examined by Western blot assay,accompanied by the treatment with phosphonoacetic acid(PAA)that blocks viral DNA synthesis.Our results showed that the expression of UL43 m RNA and p UL43 protein began to be detectable at 12 hpi,and their levels were increased steadily throughout the course of infection.The PAA treatment significantly inhibited the expression of p UL43 protein.Regardless of infection with different MDV strains,the expression patterns of the p UL43 protein remained identical,and its apparent molecular weight ranged between 45-50 k Da.These results indicated that variation of MDV virulence did not essentially alter the expression of the UL43 gene products,and the p UL43 protein belonged to the category of late proteins synthesized after MDV infection.Finally,the effect of p UL43 protein on the stability of the cell surface major histocompatibility complex class I(MHC-I)was investigated.The vectors expressing full length p UL43 or its truncated mutants were transfected separately into DF1 or HEK293 T cells.At 48 h after transfection,Flow Cytometry was performed to analyze the MHC-I levels on the cell surface.Compared to the control DF1 cells only with EGFP expression,both the full length p UL43 and its mutant with the N-terminal or C-terminal deletion were able to decrease the surface MHC-I expression level to 37.16%,20.74% and 31.47%,respectively(p<0.05).On the contrary,the p UL43 protein failed to inhibit the presentation of MHC-I on the human HEK293 T cell surface,which in turn suggested that there might be specific host factors in the chicken cell line that worked with the p UL43 protein to prevent the MHC-I antigen presentation process.Additionally,treatments with various chemical inhibitors that block intracellular transportation pathways demonstrated that the expression levels of MHC-I in transfected cells were significantly recovered in the presence of Chloroquine or MG132,indicating that the p UL43 protein might trigger the downregulation of the surface MHC-I molecules by manipulating the cellular pathways associated with autophagy and proteasome.In conclusion,the present study primarily focused on the generation of a polyclonal antibody specific to the p UL43 protein and characterized its expression profile in vitro.Importantly,the p UL43-mediated MHC-I reduction at cell surface occurred independently of other viral proteins.Moreover,the involvement of either the autophagy or the proteasome pathway might be necessary for the MHCI downregulation caused by the pUL43 expression.