Function Of VP22 And UL13 Protein Of Marek's Disease Virus Serotype 1 CVI988 Vaccine Strain

The UL49 and UL13 homolog gene of Herpes simplex virus (HSV) in Marek's disease virus serotype 1 (MDV-1) encodes the major tegument protein VP22 and UL13 of 27.6kDa and 57.1kDa respectively. VP22 possess a remarkable property of protein transduction independent of any other viral proteins and can be used in protein delivery technology to overcome the limitations in gene therapy and DNA vaccine. UL13 plays important roles in virus replication, virion assembly, regulation of host cells'transcription and translation as a viral serine/threonine protein kinase. VP22 is a major substrate of UL13 in HSV, and phosphorylation of VP22 may influence the localization of this protein. However, phosphorylation of VP22 by UL13 in MDV has not been demonstrated so far.Currently, most researches about VP22 and UL13 are focus on HSV. However, whether can MDV VP22 be utilized as a transporter for protein delivery? If Yes, what kinds of proteins can be transported by MDV VP22? Can MDV VP22 enhance the immune response induced by these proteins? What is the transduction characterization of MDV VP22? Does UL13 phosphorylate VP22 in MDV? Does the modification of phosphorylation influence the biological activity of VP22? All these questions remain unclear. We aimed to resolve these questions in this study for the further application of MDV VP22.1. MDV-1 VP22: a transporter that can selectively deliver proteins into cellsMDV VP22 shows a remarkable property of protein transduction independent of any other viral proteins. To confirm whether it can be utilized as a transporter for protein delivery, five heterogenes, encodes GFP, AIV-NP, BoIFN-γ, IBDV-VP2 and NDV-F were respectively fused to MDV UL49 gene. Transient expression were carried out by transfecting these fusion genes into COS-1 cells. The results suggested that GFP, AIV-NP,BoIFN-γbe highly transported by VP22 while NDV-F be transported at low efficiency. However IBDV-VP2 could not be deliveried at all. Our data suggests that the VP22 of MDV-1 selectively transport heterogenous proteins into cells and the original localization of cargo proteins may be changed after transporting by MDV-1 VP22.2. Evaluation of MDV VP22 enhances the immune response by DNA vaccinationMDV VP22 possesses the property of protein delivery and GFP, AIV-NP, BoIFN-γ, IBDV-VP2 and NDV-F can be deliveried into cells by VP22. To further evaluate the immune response enhancements induced by VP22, balb/c mice were immunized with recombinant plasmids expressing NP-VP22, BoIFN-γ-VP22 and F-VP22 respectively. Antiserum and spleen cells were isolated from the mice after 4 times immunization. ELISA, ELISPOT and FACS analysis was carried out to evaluate the immune response of each group. The results indicated that the cellular immunity level of BoIFN-γwas significantly enhanced by VP22 while the humoral immunity was not. In contrast, humoral immunity level of NP was significantly enhanced and a slight enhancement of cellular immunity. However, the immune response induced by F-VP22 and F alone has no difference. It implied that the immunogenicity of F can not be enhanced by MDV VP22. Taken together, MDV-1 VP22 selectively increases the immunity of antigens, but the type of immune response may be influenced by the way in which VP22 transported.3. Mechanism of VP22 protein transductionVP22 shows a remarkable property of protein transduction independent of any other viral proteins, and can be used for protein delivery. However, the subcellular localization mechanism of this protein remains unclear. In this study, VP22 was expressed with recombinant adenovirus. Lysates of recombinant virus infected 293 cells were added to normal MDBK cells. The results revealed that infected 293 cells expressed VP22 protein can almost enter all the monolayers, suggests that adenovirus expressed VP22 remains its transduction property. Subsequent study showed that in recombinant adenovirus infected 293 cells, VP22 first gather round the nucleus membrane, and then concentrated in particles in cytoplasm, which differs from the nuclei localization pattern of VP22 in MDV infected CEF and transient expressed VP22 in 293 cells. We also characterized the protein transduction property of Bac-to-Bac Baculovirus Expression System expressed VP22, and examined the transduction ability of transient expressed VP22 in different cell lines. The results suggested that VP22 expressed by Bac-to-Bac System remained its protein transduction property and the subcellular localization of the protein may be influenced by temperature; transient expressed VP22 remained its transduction property in different cells and was localized in the nucleolus of all these cell types.4. Sequence analysis of MDV-1 UL13 and phosphorylation sites prediction of VP22The protein kinases comprise one of the largest superfamilies of homologous proteins containing hundreds of members. Although there is a rich diversity of structures, regulation modes, and substrate specificities among the protein kinases, there are also common motifs and structural features in their functional domains. In this study, amino acid sequence analysis was carried out between the homologous gene of UL13 in different strains of MDV and in different Herpesviridae subfamilies, analysis by MegAlin function of DNAstar software. The results indicted that UL13 share almost 100% homology between different strains of MDV, and was highly conserved in kinase domains while the sequences contained a rich diversity in different Herpesviridae subfamilies. Taxonomy analysis of UL13 catalytic center using CDTree3.1 software provided by NCBI implied that UL13 is more homologous to Drosophila melanogaster pelle protein. The conserved domain of UL13 was analyzed with protein blast and Cn3D 4.1 online software of NCBI, the results suggest that 152-297 residue is kinase catalytic center of UL13. However conserved glycine in kinase subdomainⅦfor most protein kinase was replaced by serine in UL13 and proline in kinase subdomainⅧreplaced by cysteine. Phosphorylation sites prediction of VP22 was carried out with NetPhos 2.0 online Server. Numerous phosphorylation sites were found, most of which were enriched in the N and C terminus of VP22, implying that VP22 may be a kinase substrate of UL13 in MDV.5. Expression of UL13 by prokaryotic and eukaryotic system and antiserum preparationAs a viral serine/threonine protein kinase, the kinase activity of UL13 is similar to the eukaryotic protein kinases, and is conserved in Herpesviridae subfamily. To further investigate the function of UL13 kinase, UL13 gene was amplified by polymerase chain reaction (PCR) from MDV-1 CVI988/Rispens strain, followed by introducing to the pFastTMBac1 vector. The recombinants were transformed into DH10Bac in which translocation was occurred. Recombinant genome of baculovirus was identified by PCR and then transfected into sf9 cells to obtain the recombinant baculovirus expressing UL13, named as rBac-UL13. The codon bias and antigenicity of UL13 in E.coli was analyzed by online service GENEART ( www.gcua.de ) and DNAstar software respectively. The UL13 truncated fragments were expressed in E.coli as GST fusion protein, and mice were immunized with the expressed GST fusion protein to obtain antiserum against UL13. Sf9 cells infected with rBac-UL13 were reacted with the antiserum. The result suggested that the antiserum contains antibodies specific to UL13.