Regulation And Expression Of Pseudorabies Virus IE180 And EP0 Genes

Pseudorabies virus(PRV) is a member of the family Herpersviridae,subfamily Alphaherpesvirinae.In addition to infection of its natural host swine,PRV infects many kinds of other domestic animals and even wild animals.The outbreak and spread of pseudorabies in swine populations cause great economic losses worldwide.Although several countries have been listed as pseudorabies-free,pseudorabies still occurs endemicly in many other countries and areas.So it is still an emphasis in the disease control of swine agriculture.Like almost all of the other herpesviruses,the viral genes of PRV are expressed in a coordinated cascade,characterized mainly by three phases: immediate-early(IE),early,and late.Also PRV undergoes a latent phase in its life cycle. Although most herpesviruses express several IE proteins and more early proteins,PRV encodes only one genuine IE gene(IE180),and only three early genes(EP0,UL54 and TK) have been reported.In spite of its noted homology to human alphaherpesviruses and its broad host range,PRV is insensitive to humans.Thus,PRV has been of interest to virologists as a simple model organism to study the coordinated gene regulation of herpesviruses.In this research,investigations were taken to elucidate the regulatory functions of IE 180 and EP0 involved in the early stage of PRV gene expression.The transactivating functions of IE180 on EP0 and TK genes were validated,and the autoregulation mechanism of IE180 and EP0 genes were tested.Additionally,it was found that the IE180 and TK gene promoters were markedly suppressed by the EP0 of the PRV strains Ea and Fa(EP0/Ea,EP0/Fa).Further mutational analysis of the two amino acids next to the RING finger domain(amino acid 85 and 86) showed that the variation of these two amino acids could affect the transregulatory function of EP0.The trans-suppression domain of EP0/Ea has been mapped to the region containing the RING finger domain. The main projects are:1.Isolation and functional identification of the IE180,EP0,and TK gene promoters of PRV strain EaA PCR-based approach was used to isolate the IE180,EP0,and TK gene promoters from the genome of PRV strain Ea.The nucleotide sequences were identified.To investigate the promoter activities of these regions,they were ligated into the EGFP and lacZ reporter gene plasmids constructed in this research(pcDNA-EGFP,pcDNA-lacZ), and the corresponding assays were taken by transient transfection.Results indicated that all of the three promoters could drive transcription of the reporter gene in the absence of any viral protein synthesis or infection. 2.Transactivation of the EP0 and TK gene promoters by IE180It has been reported that the gene expression from TK promoter was transactivated by IE180.No similar research on EP0 gene was reported.Cotransfection assays taken in this study showed that the IE180 significantly activated the TK gene promoter,which was in agreement with previous studies.As expected,IE180 also significantly activated the EP0 gene promoter.3.The negative auto-regulation of IE180 and EP0 genesPrevious studies indicated that expression of IE180 from PRV strain Ka decreased gene expression from its own promoter,reflecting negative auto-regulation. Cotransfection assays were taken using the expression plasmid of IE180 from PRV strain Ea and reporter plasmids promoted by the IE180 promoter.Results indicated that IE180 of PRV strain Ea reflected the same negative auto-regulation.In addition,the similar negative auto-regulation was found in EP0 gene in transfected cells.The expression of EP0 in transfected cells significantly suppressed the gene expression from its own promoter.This has not been reported previously.4.Cloning and sequence analysis of the EP0 gene of PRV strain FaThe complete coding sequence of the EP0 gene of PRV strain Fa(EP0/Fa) was amplified from the genomic DNA of PRV strain Fa,followed by DNA sequencing.The deduced amino acid sequence of EP0/Fa was compared.Sequence alignment indicated that,compared to EP0/YS-81,EP0/Ea and EP0/Fa both encoded 409 amino acids,had six same mutations and a same deletion.Beside this,the EP0/Ea has three other mutations than EP0/Fa.5.Suppression of the IE180 and TK gene promoters by EP0/Ea and EP0/FaIn the initial work,it was found that EP0/Ea significantly suppressed the IE180 and TK gene promoters in transient assays.To confirm it was the same in PRV strain Fa,the complete coding sequence of EP0 was cloned from the genomic DNA of PRV strain Fa. Following transfection assays showed that EP0/Fa also significantly suppressed the IE180 and TK gene promoters.6.Functional analysis of the substitution mutants from EP0/Ea and EP0/FaBased on the amino acid sequence analysis of EP0,the variation of the two-amino acid next to the RING finger domain may greatly affect the function of EP0.Reverse mutants were generated by replacing the KT of EP0/Ea and EP0/Fa with NA of EP0/YS-81.Following results revealed that,after changing the two amino acids,the suppression activity of EP0/Fa was significantly decreased,whereas that of EP0/Ea was not obviously affected.This indicated that this two-amino acid substitution did affect the regulatory function of EP0. 7.Mapping of the transregulatory domains of EP0 from PRV strain Ea and FaTo map the functional domains of EP0/Ea and Fa,a series of truncated mutants of EP0/Ea and EP0/Fa were obtained by PCR amplification and the corresponding expression plasmids were constructed.Then the resulting plasmids were cotransfected separately with reporter gene plasmids promoted by the IE180 or TK gene promoter. Analysis of regulatory activities of these truncated forms of EP0/Ea and EP0/Fa revealed that the region containing the RING finger domain were important for the trans-suppression.On the other hand,the mutant consisting of amino acids 1 to 113 exhibited a dominant-negative property.8.Screening of efficient siRNA target sites directed against the IE180 gene of PRV strain EaFive siRNAs were designed and synthesized according to the published sequence of IE180 gene from PRV strain TNL and identified sequence of IE180 gene from PRV Ea strain.Then the corresponding expression plasmids were constructed and cotransfected separately with the plasmid expressing IE180-EGFP fusion protein.Flow cytometric analysis revealed that all of these 5 siRNAs could not inhibit the IE180 expression effectively.One important reason could be that some sequences might be buried within secondary structures or within highly folded regions in target RNAs.Another reason is that the sequences identified are probably not accurate because of the difficulty in IE180 gene sequencing.