Study On The Function Of Pseudorabies Virus Tugement Protein UL14 And The Construction Of TK～-/gG～- Strain Infectious Clone

Pseudorabies virus (PRV) is the causative agent of Pseudorabies (Aujeszky's disease)of many domestic and wild animals. Especially, porcine Pseudorabies has become one ofthe most economically important diseases in worldwide swine industry. PRV genome is adouble-stranded DNA of approximately 150 kb and encodes 70-100 proteins. Like otheralpha herpesvirus, PRV has some typical characteristics of alpha herpesvirus such aslatent infection and neurotropism. Although cloning and sequencing of some importantfunctional genes of PRV were carried out in the middle of 1980s, full-length sequence ofPRV genome has not been reported because of high content of G+C of PRV genome (themean content of G+C is 74%). Functions of some genes still remain unclear, such as UL4,UL7, UL14, UL24 and US2. It is significant to study and analyze unknown genes, whichcontributes to clarify entirely the structure and function of PRV genome.For a long time, the main method studying on the functional genes of herpesviruswas to construct genetic-deleting mutants. However, it was tedious to producerecombinant mutants using homologous recombinant in infected cells. In 1997, Messerleet al., German researchers, firstly constructed infectious clone of MCMV based onbacterial artificial chromosomes (BACs). This technique actualized that the genomes ofinfectious virus could be stored, propagated or modificated by BAC-plasmid pattern in E.coli, and it was convenient and rapid to manipulate the technique. This technique lead toadvancement in studying on gene functions of herpesvirus, and provided new ways todevelope high-performance herpesviral-vetor system.Based on the above mentioned researchs background, the present study investigatedthe functions of PRV tegument protein UL14, and constructed PRV TK^-/gG^- straininfectious clone. The main projects are as following:1. Cloning and sequence analysis of PRV Ea strain BamHI-3 fragmentThe BamHI-3 fragment was isolated from PRV genome DNA digested with BamHI,and cloned into pBR322 to generate the recombinant plasmid pBRB₃. A 7.5kb unknownfragment was isolated from the plsmid pBRB₃ digested with BamHI and KpnI to generatethe plasmid pUCB₃. Sequences analysis showed that the UL13-UL17 fragment containedcomplete coding-region of UL13, UL14, UL16 and UL17 and partial coding-region ofUL15. UL14 gene with 480bp encodes 159 amino acid residues. Compared with aminoacid residues of UL14 gene of other Alphaherpesviruses, it was found that N-terminalamino acid residues sequence of UL14 of PRV is highly conservative and there was 62%,62%, 72% homology with HSV-1, HSV-2, BHV-1, respectively, but the homology ofC-terminal is low. 2. Cloning and expression of PRV UL14 gene in E.coli and IBRS-2 cellsPRV UL14 gene was amplified by PCR and cloned into pMD18-T vector to obtainthe recombinant plasmid pT-UL14. The UL14 gene was removed from pT-UL14 andcloned into prokaryotic expression vector pGEX-KG resulting in the recombinant plasmidpGEX-UL14, which expressed GST-UL14 fused protein. After induction by IPTG, therecombinant fused protein with about 44kDa was highly expressed in E.coliBL21(DE3)plysS and expressing mainly in the form of inclusion body. The GST-UL14fused protein was used to immunize rabbits to generate polyclonal antibodies againstUL14. UL14 gene was inserted into vector pcDNA3.1(+) resulting in the eukaryoticexpression plasmid pcDNA-UL14. After transfection, UL14-expressing IBRS-2 cell linewas obtained by G418 screening. Western blotting analysis demonstrated that this IBRS-2cell line could stably express UL14.3. Construction of PRV UL14 mutants and analysis of function domainsUL14 gene was inserted into vector pEGFP-C1 resulting in the eukaryoticexpressing plasmid pC1-UL14 expressing EGFP-UL14 fused protein, pC1-UL14 wastransfected into Hela cells, and the fluorescence mainly distributed in cytoplasm at 24 hposttransfection, but as time went on, the fluorescence imaging occured to transfer andaggregated in nucleus at 48h posttransfection. In order to analyze UL14 function domains,a series of C- (1-146aa, 1-124aa, 1-88aa, 1-77aa, 1-65aa) or N-(14-159aa, 37-159aa,63-159aa, 85-159aa, 106-159aa, 121-159aa) terminal truncated mutants were obtained,and EGFP-fused expressing plasmids were constructed. When transfected into Hela cells,different fluorescence imaging in cells transfected with pC1-UL14 at various timesposttransfection were observed. The deletion of the C-terminal 94aa or N-terminal 37aasequences did not affect the intracellular localization of UL14, whereas, 62aa-deletion atthe N-terminal sequences resulted in the diffusedly cytoplasmic distribution. The resultssuggested that the 37aa to 65aa sequence of UL14 played an important role onintracellular localization.4. Expression analysis of UL14 after infecting cells with PRVCells were collected at various time after PRV-Ea infection, RT-PCR was performedto amplify cDNA of UL14 and late gene gD of PRV. Like gD gene, the transcriptionproduct of UL14 was observed at 9 h postinfection. Meanwhile, Western blottingdemonstrated that UL14 protein was expressed at 12 h postinfection. The resultsconfirmed that PRV UL14 protein was expressed with late kinetics.5. Construction of PRV UL14 mutant strainEGFP expression cassette drived by CMV promoter was amplified by PCR, andinserted into StuI site of UL14 gene of plasmid pUC3.0 containing entire UL14, UL16 gene and partial UL13, UL15 gene to generate transfer plasmid pUC3.0-14/EGFP. Thenthis plasmid was co-transfected into IBRS-2 cells with PRV-Ea genome. After cytopathiceffect occurred in cells, recombinant virus PRV-UL14D expressing EGFP was screenedfrom the cytopathic cells by EGFP fluorescence marker and plaque purification. Therecombinant virus was verified by PCR, Western blotting and Flow Cytometry (FCM),and can stably express EGFP protein.6. Function analysis of PRV UL14The penetration kinetics, plaque size, one step growth curves, and morphogonesis ofPRV-UL14D mutant were investigated and compared with the parent virus, PRV Ea ininfected cells. The results showed that the penetration and release of PRV wereindependent of UL41, but the UL41 deleted mutant exhibited a delayed growth andsignificantly small plaque size. Meanwhile, electron microscopic examination indicatedthat the nonenveloped particles obviourly increased in the PRV-UL14D-infected cells.The results illuminated that UL 14-deletion affected the virus's enveloping, and furtherlyinfluence the mature of PRV particles. But these defects above in vitro can be completelyrecovered in UL14-expressing IBRS-2 cells, which indicated that the UL 14-deletion maybe responsible for these changes. In further researches, we found that the average deathtime of BALB/c mice inoculated with PRV-UL14D was obviously delayed, comparedwith the same dose of PRV-Ea. The results suggested that the UL14-deletion also affectedthe replication of PRV in vivo. In additional, anti-apoptotic activity of UL14 wasinvestigated by cell apoptosis detection and electron microscopic examination. The resultsindicated that UL14 expressing cell lines could not be resistant to apoptosis induced bysorbitol treatment. Electron microscopy showed that there were no obviously differencesof apoptotic phenomenon between cells infected with PRV-Ea and PRV-UL14D, whichsuggested that UL14 protein did not share anti-apoptotic function, contrast to HSV-1,HSV-2 and BHV-1 UL14 as reported previously.7. Construction of PRV TK^-/gG^- infectious cloneBAC vector sequence containing F-factor was cloned into plasmid pgG-Uni togenerate transfer plasmid pSB301. And then, this plasmid was co-transfected into IBRS-2cells with PRV TK^-/gG^-/LacZ⁺ genome. When eytopathie effect occurred in cells,recombinant virus PRV TK^-/gG^-/BAC was screened from the cytopathic cells by plaquepurification. Replieative intermediate (covalently closed circular) viral DNA extractedfrom cells infected with the recombinant virus was electroporated into E.coli DH10B cells.The BAC plasmid DNA isolated from E.coli harboring PRV full-length BAC DNA wastransfected into PK-15 cells, and cytopathic effect was visible, suggested that PRV BACDNA possess infection. Furthermore, the results of one step growth curves of PRV TK^-/gG^- infectious clone showed that the insertion of BAC vector sequence had noinfluence on the propagation of recombinant viruses in cells.8. Immunogenicity of PRV TK^-/IgG^- BAC plasmid DNA in miceImmunogenicity of PRV BAC plasmid DNA was detected in mice, and comparedwith transfer plasmid pSB301, recombinant PRV TK^-/gG^-/BAC and parent strain PRVTK^-/gG^-/LacZ⁺. The results showed that PRV neutralization antibodies induced by PRVBAC plasmid DNA corresponded with that induced by TK^-/gG^-/BAC, TK^-/gG^-/LacZ⁺.Moreover, mice immunized with BAC plasmid DNA could resist the challenge of lethalPRV and displayed the same protective efficacy with that vaccinated with TK^-/gG^-/BACor TK^-/gG^-/LacZ⁺. The results indicated that PRV TK^-/gG^- infectious clone had favourableimmunogenicity, and lay foundation for further research on immunology and thedevelopment of perfect viral-vector system of PRV.