Study On DNA Methylation Of Baculovirus And Regulation Of Its Viral Gene Expression

DNA methylation has been reported in many kinds of virus genomes, and was proposed to play an important role in virus gene expression regulation and virus-host interaction. Most epigenetic research in virus focused on latent and non-lytic viruses, such as Epstein-Barr virus (EBV), human papillomavirus (HPV), and hepatitis B virus (HBV), while the status and effect of DNA methylation in lytic virus, such as baculovirus, were rarely studied. In the current thesis, we studied the DNA methylation status of specific genes in the genome of AcMNPV (Autographa californica multicapsid nuclear polyhedrosis virus), its dynamics during infection, and the effect of methylation on the regulation of ieO and iel expression.After software prediction and MBD (methyl-CpG binding domain) enrichmen, we did bisulfite sequencing for CpG islands of selected regions in AcMNPV genome. The results showed that the level of DNA methylation in AcMNPV was generally low, and there were great differences between genomic regions. DNA methylation levels in ieO and p95were relatively high, which made them hot spots for methylation, while there were low or no DNA methylation in the CpG rich regions in iel promoter, gp64, gp41, hr3. Since ieO and iel are very important immediate-early gene of the virus, their difference in DNA methylation might have implications in virus replication.We then did bisulfite sequencing of selected regions in viral genome using DNA isolated at different time during infection. The resuts showed that the DNA methylation status of ieO region changed during infection. DNA methylation was detectatable at4h postinfection (p.i.), and the level of methylation decreased at8h p.i., and began to increase afterwards, reaching highest in virion. Examining the p95region showed the same trend. These results showed that the DNA methylation level in the hot spots in virus genome was not constant during infection, it changed during infection. Although the mechanism was not clear, it may be related with the temporal regulation of gene expression during infection.To study the effect of DNA methylation on the regulation of promoters of ieO and iel, plasmids p402-ie1and p402-ie0M-A, which can express IE1and IEO protein respectively, and reporter plasmids pie0p-luc and pie1p-luc, which express luciferase gene by ieO and ie1promoter, respectively, were reconstructed. Using in vitro DNA methylation and transient luciferase gene expression in transfected Sf9cells, the effect of DNA methylation on ie0/ie1expression and regulation was studied. The results confirmed that IE0activated ie0p whereas IE1inhibited it. DNA methylation reduced the activity of ieO and iel promoter greatly. Both IEO and IE1increased ielp activity, either non-methylated or methylated. Different ratio of p402-iel to p402-ie0M-A were cotransfected with reporter plasmid into Sf9cells, luciferase activity assay showed that DNA methylation could reverse the regulation on ieOp by IE0/IE1when IEO>IE1. These results indicate that DNA methylation might be able to affect the regulation of baculovirus gene expression.In conclusion, baculovirus AcMNPV genome were methylated, and there were hot spots of methylation in the genome, like ieO and p95. The methylation level in the hot spots changed during infection, which was decreased at8h p.i. DNA methylation in ieOp affected its gene expression regulation. These results suggested that DNA methylation may be related with temporal regulation of gene expression, and would be helpful for further study on baculovirus replication and the epigenetic regulation in virus infection.