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Epigenetic Regulation Of Baculovirus Replication And Gene Expression

Posted on:2011-01-30Degree:DoctorType:Dissertation
Country:ChinaCandidate:X WangFull Text:PDF
GTID:1110360305997598Subject:Microbiology
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In the last three decades, epigentics has been one of the fast developing areas in life science. Many great results were reported. However, virologists seem to be spectators in this field. Until recently, only a few reports about viral epigenetic regulation could be found. Compared with viruses with latent infection phase, such as herpes simplex virus (HSV), hepatitis B virus (HBV), Epstein-Barr virus (EBV) and human papillomavirus (HPV), less concern was given to the epigenetic regulation in the replication of lytic viruses. In this dissertation, we focused on epigentics regulation of baculovirus AcMNPV(Autographa californica multicapsid nucleopolyhedrovirus) during its lytic infection in insect cells. We studied the roles of viral minichromosome structure, histone acetylation and DNA methylation in viral gene expression and virus replication.We first studied the roles of viral minichromosome structure and histone acetylation in viral gene expression. We found that chickenβ-globin 5'-HS4 insulator (HS4), which was placed downstream of the polyhedrin promoter-directed foreign gene expression cassette in AcMNPV, markedly increased the expression of reporter genes. When enhanced green fluorescence protein gene (egfp) was used as the reporter gene, cells infected by the recombinant virus with HS4 (AcEGFP-HS4) showed 3.0 and 2.1-fold stronger fluorescence than that by the control virus without HS4 (AcEGFP) at 72 and 96 h post infection, respectively. The level of egfp mRNA was also much higher in cells infected by AcEGFP-HS4 than that by AcEGFP. An increase in gene expression was also seen when firefly luciferase gene or secreted alkaline phosphatase gene was used as a reporter. The insertion of HS4 in the polyhedrin locus has no significant effect on virus replication. The effect of HS4 was orientation-dependent, and sensitive to inhibitors of histone acetyltransferase. In DNase I sensitivity assay, HS4 significantly increased the sensitivity of neighbouring DNA to nuclease, but had little effect on DNA of a distal locus. These results suggested that HS4 insulator might affect baculovirus gene expression by modifying the structure of neighbouring chromatin in the virus minichromosome. These results provided a new method to improve baculovirus-insect cell expression system, and clearly suggested viral minichromosome and histone acetylation play an important role in virus replication and viral gene expression.To analyse other epigenetic regulation mechanisms in baculovirus replication, we then investigated DNA methylation during virus replication. We for the first time showed that the expression of viral immediate-early gene ieO was affected by the DNA methylation in the promoter area. IEO was an important viral regulator that was expressed at relatively higher level at the early phase of infection, and then down-regulated at late stage. The down-regulation of ieO was known to be imporant for normal virus replication. We found that ieO promoter was hypermethylated Oh p.i., hypomethylated 8h p.i., and back to hypermethylated state again after 18h p.i. In vitro DNA methylation test showed that DNA methylation inhibited ieO expression. Treatment with 50nM DAC, a kind of DNA methyltransferase inhibitor (DNMTi) maintained the hypomethylation of ieO promoter and increased ieO expression. These results indicated that DNA meythlation in the ieO promoter contributed to the down-regulation in ieO expression at the late phase of viral infection, and showed that DNA methylation played a role in the regulation of gene expression in lytic virus replication.To understand the general relationship between DNA methylation and virus replication and viral gene expression, we also studied the effect of DNA methylation inhibitor, DAC on the virus replication and viral gene expression. Presence of 50nM DAC decreased viral titer to only 1/30 and delayed viral DNA replication significantly. Treatment with DAC has different effect on immediate early gene expression in different phases of infection. DAC increased the expression of immediate early gene iel and ie2 2h p.i., and inhibited their expression later. Meanwhile, DAC increased the expression of another immediate early gene, ie0, both early and late in the infection. The effect of DAC on the expression of viral delayed early genes was not uniformed, but it inhibited the expression of all late genes tested. DAC had more significant effect on the expression of viral very late genes, resulted in almost complete elimination of the protein production. The mechanism of DAC to inhibit very late gene expression was further studied. It was found that additional expression of IE1 by plasmid relieved the inhibitory effect of DAC on very late promoter. These data, in combination with results above, and known function of IEO and IE1 proteins, implied that DAC inhibited very late promoter by decreasing the expression iel, while while preventing the down-regulation of ieO expression. This will resulted in the improper ratio of IEO and IE1 in infected cells, which was known to be essential for the activation of viral very late promoter late in infection.These results suggested that viral DNA methylation might affect virus replication and viral gene expression by modifying the expression of a few important viral regulatory genes.Apart from infecting insect cells, baculovirus could also enter many mammalian cells, too. In recent years, baculovirus has attracted more and more attentions as a gene delivery vector for mammalian cells. However, the silencing of target gene mediated by baculovirus limited its application. Although histone deacetylation inhibitors were known to improve baculovirus-mediated gene expression in mammalian cells for long time, another group of chemicals that induces epigenetic changes, DNMTi, was reported to have no such effect. In this dissertation, we for the first time found that DNMTi AZA or DAC improved baculovirus-mediated gene expression by fourfold or more in all four mammalian cell lines tested when they were added prior to virus inoculation. This improvement of gene expression was less significant or even abolished when drugs were added after virus inoculation. The further results indicated that DNMTi improved baculovirus-midated gene expression by inhibiting viral DNA degradation in transduced mammalian cells. This research provided a new method to improve the application of baculovirus vector in mammalian cells, and offered new insights to understand the relationship between baculovirus vector and mammalian cells, and the response to non-specific invasion by mammalian cells.
Keywords/Search Tags:baculovirus, epigentics, histone modification, DNA methylation
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