Interactions Between The Major Capsid Proteins Of Pseudorabies Virus (PRV)

Pseudorabies virus(PRV) is the causative agent of pseudorabies(Aujeszky's disease), a serious infectious disease of several domestic and wild animals.PRV genome is a double-stranded DNA of approximately 150 kb and encodes 70-100 proteins.Like other alpha herpesvirus,PRV has some typical characteristics of alpha herpesvirus such as latent infection and neurotropism.Membership in the Herpesviridae family was based historically on their unique virion architecture and PRV virions resemble others in the family.The mature virion,or infectious viral particle,consists of four morphologically distinct structural components:the central core contains the linear double-stranded DNA genome of the virus;the DNA is enclosed within a protective icosahedral capsid to form a nucleocapsid;the capsid is embedded in a protein matrix known as the tegument;finally, the tegument is surrounded by the envelope,a lipid membrane containing several viral glycoproteins.Nearly half of all the PRV gene products are structural components of the mature virion.Most of what is known about the PRV capsid is inferred from detailed studies of the prototypical HSV-1 virion.The PRV capsid is icosahedron(150 hexons and 12 pentons), The hexons,composed of six molecules of UL19(VP5) and six molecules of UL35 (VP26),form the capsid edges and faces,while the 12 pentons comprise the vertices. Eleven of the pentons are pentamers of UL19(VP5),while the twelfth vertex is a unique cylindrical portal made of 12 molecules of UL6.The portal's ring-like channel allows one copy of the viral DNA genome to be packaged into the preformed capsid.Thus,each mature capsid contains 955 molecules of UL19(VP5),900 molecules of UL35(VP26), 640 molecules of UL18(VP23),320 molecules of UL38(VP19C),and 12 molecules of UL6(portal protein).So the PRV capsid needs six mature capsid proteins(UL38,UL35,UL25,UL19,UL18,UL6)and two scaffold proteins(UL26,UL26.5)for assembling in the cell nucleus,But the Scaffold proteins isn't in the virion of PRV.Therefore,in this study,we explored the formative mechanism of the PRV virion capsid,and its packing,maturation and stabilization.Furthermore,we study the interactions between capsid proteins.The detailed research was as following:1.Cloning and sequence analysis UL6,UL18,UL19,UL25,UL26,UL26.5,UL35,UL38 genes of PRV Strain EaThe UL6,UL18,UL25,UL26,UL26.5,UL35,UL38 genes of PRV Strain Ea were amplified by PCR,then were separately inserted into pMD18-T vector and sequenced.The length of UL6,UL18,UL25,UL26,UL26.5,UL35,UL38 genes of PRV'Strain Ea were 1938bp,891bp,1605bp,1599bp,861bp,312bp,1107bp, respectively.Compared with the sequence of PRV Strain Ka,the homology of nucleotide is corresponding 98%,98%,99%,97%,95%,99%and 98%.Besides,the homology of amino acid is corresponding 97%,98%,99%,97%,95.8%,99%,and 99%.And the genes encoded amino acid residues were 646aa,297aa,535aa,533aa,287aa,104aa and 369aa.The UL19 gene of PRV strain Ea was also cloned by several fragments.The upstream and downstream of UL19 was amplified by PCR.Sequence analysis showed that the upstream is 368bp in length and the downstream is 600bp in length.Then the BamHI-fourth fragment was isolated from PRV genome DNA digested with BamHI, resulting in the recombination expression plasmid pShuttle-UL19 finally.2.Expression of the capsid protein of PRV strain Ea in E.coli and HeLa cellThe UL6 gene of pseudorabies virus(PRV) Strain Ea was further subcloned into the prokaryotic expression vector pET-28a,resulting in the prokaryotic expression plasmid pET-28a-UL6.After transfection into E.coli,the expression of a fusion protein (6×His-UL6) with 70kDa molecular mass was observed in E.coli under induction with IPTG.The result of SDS-Page and Western blot demonstrated that the fusion protein can be recognized.Correspondingly,the fusion protein 6×His-UL18 is 33KDa;fusion protein GST-UL26.5) is 56kDa;fusion protein 6×His-UL38 is 40kDa.Then UL6 gene was inserted into eurokaryotic expression vector pEGFP-C2, resulting in the expression plasmid pEGFP-UL6.Then the expression plasmids pEGFP-UL18,pEGFP-UL19,pEGFP-UL25,pEGFP-UL26,pEGFP-UL26.5,pEGFP-UL35 and pEGFP-UL38 were obtained through the same method.After these expression plasmids and pEGFP-C2 transfected into HeLa cells,the fluorescence was observed 48h post transfection.The results showed that the fluorescence of EGFP-UL6 mainly located in cytoplasm.The fluorescence of EGFP-UL18,EGFP-UL26.5 and EGFP-UL38 mainly located in cell nucleolus 48h post transfection.However,the fluorescence of EFGP-UL19,EGFP-UL25,EGFP-UL26,EGFP-UL35 and pEGFP-C2 was uniformly distributed throughout the cytoplasm of cells.3.The interactions beween capsid proteins of PRV strain Ea as measured by Mammalian two-hybrid systemThe plasmid pMD-UL18 was digested with BamHI and KpnI,and subcloned into the same restriction sites of mammalian two-hybrid expression vector pACT and pBIND, resulting in the mammalian two-hybrid expression plasmids pACT-UL18 and pBIND-UL18.In the same method,the other fourteen mammalian two-hybrid expression plasmids will be gained,such as pACT-UL19.The pACT vetor were separately transfected with pBIND,pBIND-UL6,pBIND-UL18,pBIND-UL19,pBIND-UL25,pBIND-UL26,pBIND-UL26.5,pBIND-UL35,pBIND-UL38 into CHO cells.So the pBIND vetor were separately transfected with pACT-UL6,pACT-UL18,pACT-UL19,pACT-UL25,pACT-UL26,pACT-UL26.5,pACT-UL35,pACT-UL38 into CHO cells.The self-activation was detected by Dual-Luciferase reporter assay after 48h post transfection.The results showed that pACT and pBIND-UL19,pACT and pBIND-UL25, pBIND and pACT-UL26.5 were existent self-activation.According the result of self-activation,two of the mammalian two-hybrid expression plasmids were transfected into CHO cells.It was tested the interaction of PRV Ea capsid proteins using Dual-Luciferase reporter assay system after 48h.Then,three interactions have been identified:interactions between UL19 and UL26.5,molecular interactions between UL18 and UL38,and interactions between UL26 andUL26.5.4.The interactions beween eapsid proteins of PRV strain Ea as verified by GST Pull-Down and co-immunoprecipitationUsing GST Pull-Down assay,biochemical evidence of the interaction between UL19 and UL26.5 was obtained.With Expression product of UL26.5 in E.coli and cell Lysis buffer of recombinant baculovirus rBacUL19 infected sf9 cells after 48 h,The result demonstrated that the fusion protein with 140kD molecular mass can be recognized, which is the Expression product of UL19 in recombinant baculovirus.The interactions between UL19 and UL26.5 were demonstrated.Mammalian expression plasmids pCMV-HA-UL18,pCMV-Myc-UL18, pCMV-HA-UL38,pCMV-Myc-UL38 were constructed.Then mammalian expression plasmids pCMV-HA-UL18 and pCMV-Myc-UL38,pCMV-HA-UL38 and pCMV-Myc-UL18 were transfected into HeLa cell.After 48h post transfection,the cell was lysised by RIPA buffer.The protein which was precipitated by anti-HA polyclonal antibody was analyzed by immunoblotting using anti-Myc monoclonal,the fusion protein Myc-UL38 was recognized in the lysate of HeLa cell transfection with HA-UL18 and Myc-UL38.Besides,the fusion protein Myc-UL18 was recognized in the lysate of HeLa cell transfection with HA-UL38 and Myc-UL18.Molecular interactions between UL18 and UL38 were verified by Co-immunoprecipitation.