Identification Of The Cleavage Site Of AcMNPV Per Os Infectivity Factor P74

Baculoviruses are a family of enveloped double-stranded circular DNA virus that produce two viral phenotypes——budded virus(BV)and occlusion-derived virus(ODV)in their life cycle.ODVs mediate virus primary oral infection to host midguts,while BVs mediate the further systemic infection among other tissues.So far,ten ODV envelope proteins,termed as per os infectivity factors(PIFs),have been reported to be essential for primary infection.Among those,P74 was first reported among all the PIFs and thereafter named as PIF0.P74 undergoes two sequential cleavage when ODVs infect midguts: the first cleavage occurs when ODVs released from lysed occlusion body(OB);while the second cleavage happens during the contact between ODVs with host midgut epithelial cells.Till now,the specific first cleavage site and impact on the oral infection are unknown.The thesis focuses on the cleavage of P74,also explores interactions between PIFs on the ODV envelope to investigate the mechanism of baculoviruses oral infection.Chapter 1: Introduction of the research background,including the life cycle of baculoviruses,the detailed process of oral infection and summary of the state of the art of PIFs,especially P74.Chapter 2: Research on cleavage of Autographa californica multiple nucleopolyhedrovirus(AcMNPV)P74.We use infected cell sample and ODV sample from larvae to conduct Western blot analysis.Results indicated that P74 could be cleaved in ODVs,but not in infected cells.Then,we use soybean trypsin inhibitor(SBTI)which inhibits trypsin activity specially to perform OB dissolve time-course experiment.The result suggested the enzyme that cleaved P74 might be a trypsin like one.According to the molecular weight of cleaved protein and bioinformatic analysis,we proposed that four arginines might be the candidate cleavage sites.We have constructed p74 knock-out,repaired and other seven arginine site mutated recombinant viruses by Bac-to-Bac system.One step growth curve and transmission electron microscope(TEM)and scanning electron microscope(SEM)showed that the biological characteristics of all the recombinant viruses are similar to the wild type control viruses.We purified ODVs of recombinant virus and conducted Western blotting analysis.The results showed that the R325/R334 Q mutant could partially inhibit P74 cleavage,while the four-site mutant R319Q/R323Q/R325Q/R334 Q could completely block the cleavage of P74.Therefore,the first cleavage site of AcMNPV P74 is R319/R323/R325/R334.Chapter 3: Investigation on interactions of ten PIFs.We used yeast two-hybrid assay(Y2H)to detect interactions between each PIF and find eight direct interactions including P74-PIF4,PIF1-PIF2,PIF1-PIF3,PIF1-PIF4,PIF2-PIF3,PIF2-PIF4,PIF3-PIF4,PIF4-Ac110.PIF5 and VP91 were excluded from the experiment because of autoactivation property.The result indicates extensive interactions between PIFs,supporting the hypothesis that PIFs facilitate oral infection by forming a complex(PIF complex).The data provided us valuable information for further exploring the structure of the PIF complex.Chapter 4: Summary and discussion for the thesis.