DNA Methylation Of Baculovirus And Its Role In Viral Replication

Epigenetic modification is involved in virus-host interactions of many virus infection, and is crucial to the infecting process and results. Most researches focused on latent and non-lytic persistent viruses while paid little attention to lytic virus. In this study we investigated the DNA methylation status of AcMNPV (Autographa californica multicapsid nucleopolyhedrosis, AcMNPV) during its lytic replication process in host cells Sf9.First, we used the DNA methyltransferase inhibitor DAC to study the effect of hypomethylated cell environment to AcMNPV’s repiication. When pre-treating the cell with 30nM DAC, DNA methyltransferase DNMT1 was found to be degraded, and viral DNA level was 8-fold to the control 8h post infection. DNA methylation sequencing showed that at this time point, the methylation of ieO promoter region was 40% lower than control, and iel promoter region was 50% lower than the control. DAC pre-treatment also improved the budded virus production at the first 24h post infection, while the difference reduced with time, until no difference was seen in final viral titer. These results suggested that decreased DNA methylation improves the viral DNA replication and budded virus production.In order to understand whether DAC improves virus replicaition through directly reducing the level of viral DNA methylation, or through modifying the cellular gene expression and indirectly affecting virus repliction, we prepared the hypomethlated virus stock by passaging the virus in Sf9 cells in the presence of 30nM DAC. Methylation sequencing indiciated that the methylation level of ieO and iel promoter decreased significantly with passages. When infecting cells, the hypomethylated virus produced 5 times more DNA DNA copies than the control the first 48h of infection on average. It also had increased transcription level of virus immediate early gene ie0, ie1, ie2 and late early gene Dnapol,lefl,lef2 at the first 24h post infection. When the hypomethylated virus was used to transduce the mammalian cell CHO, it showed improved foreign gene expression level by 3- fold.In addition, in vitro DNA methylation has been used to study the effect of methylation on the activity of viral promoters. We construct the reporter plasmids carrying different viral important promoter. They were treated with methyltrasferase in-vitro before being used to transfect Sf9 cells. The expression level of reporter gene was determined It was shown that some vrial promoters was suppressed by DNA methylatioin, while some others like p35、Dnapol、lefl、ief3、lef8 was slightly improved.In a word, DNA methyltransferase inhibitor DAC can improve the viral DNA replication and budded virus production, hypomethylated virus" has increased viral DNA level, as well as higher transcription level of virus immediate early gene ieO, iel, ie2 and late early gene Dnapol,lefl,lef2 at the first 24h post infection. In vitro DNA methylaiton experiment showed that the activity of some viral promoter are also influenced by DNA methylation. These results provided useful information to understand the effect of DNA methylation on lytic virus replication.