Effects Of Different Regions Of Insulator HS4 On Baculovirus Gene Expression

Epigentics has become one of the hot spots in recent years. In the field of virology, studies on the effect of epigenetic modifications in the infection and replication of viruses have mostly been focused on viruses with latent infection phase, such as Epstein-Barr virus (EBV), herpes simplex virus (HSV) or hepatitis B virus (HBV). Yet there are fewer researches about the epigenetic regulation in the replication of lytic viruses. Previous research in our lab has shown that chicken β-globin hypersensitive site4(HS4), when was placed downstream of the polyhedrin promoter-directed foreign gene expression cassette in baculovirus AcMNPV (Autographa californica multicapsid nucleopolyhedrovirus), could remarkedly increased the expression of reporter gene EGFP. In this dissertation, we further investigated the effect of different regions of HS4on baculovirus-mediated gene expression in insect Sf9cells.HS4is a sequence located20kb upstream of chicken β-globin locus, which provides enhancer blocking activity and reduces position effects. Previous research has identified the proximal250bp of HS4as the core. The CTCF motif in the core is primarily responsible for enhancer blocking, while the USF-1/2motif could recruit histone modifying enzymes to increase active chromatin markers. However, other work has also shown that unique insulator activity resided in the3’distal400bp of HS4sequences, which when combined with the core, restored full insulator activity.In this study we obtained different regions of HS4through PCR, and insert them into baculovirus vectors downstream of the expression cassette of repoter gene egfp. Six different recombinant viruses were obtained:vBGc (containing the5’core region of HS4), vBG550(containing the HS4fragment without the5’core and the3’distal400bp region), vBG650(containing the combined5’core and3’distal region of HS4), vBG950(containing the HS4fragment without the core region), vBG’(with the full-length HS4), as well as a control virus without insertion of any HS4fragment, vGFP. It was found that the difference between viruses in the expression of reporter gene was most significant36h post infection.In this experiment, through inserting different regions of HS4into baculovirus vectors we obtained results similar to that of in the mammal cells. The results further proved that HS4could affect the expression of reporter gene, and that epigenetic regulation play an important role in viral gene expression during baculovirus lytic infection.