A Preliminary Functional Study Of Baculovirus Per Os Infectivity Factor 5 (PIF5)

Baculoviridae is a family of arthropod-specific large double stranded DNA viruses that infect insect larvae.Baculoviruses form two different virion phenotypes: budded viruses(BVs)and occlusion-derived viruses(ODVs).The BV virion type is responsible for systemic infection within the larvae.ODVs are responsible for the oral infection.Oral infection was medicated by per os infectivity factors(PIFs)which locate on ODV envelope.Now there are 10 PIFs in baculovirus namely PIF0~PIF9.Some of PIFs can form a stable complex which facilitates ODVs infection.But PIF5 is not a component of the PIF complex and the level of pif5 transcript is highest among all pif transcripts.The role of PIF5 in oral infection process is still unclear.In order to study the impacts of PIF5 on virions production and infection,the impacts of AcMNPV PIF5 on the formation of BV and ODV were studied.The transcription and expression time course of pif5 showed that pif5 is a late gene.PIF5 is an ODV envelope specific protein localized on the nucleus microvesicles and ODV envelope during viral infection.Recombination viruses were constructed by homolog recombination and transposition.Knocking out pif5 had no impacts on BV production and infection,ODV formation and embedding,OB morphology and the embed ODVs in an OB.But pif5 delection could decrease the infectivity of ODV in Sf9 cells and oral infection.In order to study the role of PIF5 in oral infection,truncated PIF5 was expressed and purified.The soluble PIF5-P-T1 protein was obtained by expressing truncated PIF5 with a prokaryotic expression system.Protein purification revealed that PIF5-P-T1 protein had various forms: monomer and polymer.In order to obtain more monomer,the purification method was optimized,and finally the monomer proportion of PIF5-PT1 was increased.The eukaryotic expression system was used to express different forms of truncated PIF5 proteins,and a S2 cell line stably expressing PIF5-E-T1 protein was selected.At the same time,far-western blot was conducted using exogenously expressed PIF5-P-T1 and PIF5-E-T1 protein with proteins from the brush broder membrane vesicle(BBMV)of host insect.Pull-down were also performed to identify proteins interacting with PIF5-P-T1 and PIF5-E-T1.The exogenously expressed PIF5-P-T1 protein was cleaved after co-incubation with BBMV,suggesting that BBMV may promote PIF5 cleavage.In this thesis,the impacts of PIF5 on BV production and infection,ODV embedding and OB morphology were studied.And truncated PIF5 was expressed and purified for searching interating host proteins to understand the mechanism of oral infection mediated by PIFs.