Studies On Molecular Mechanism Of Baculovirus Infectivity

The Baculoviridae is a diverse family of arthropod-specific viruses with adouble-stranded genomic DNA of which most members infect Lepidoptera. It has beenwidely utilized for the expression of recombinant proteins and as a biopesticide.Baculoviruses are unique among virus families because they have two distinct phenotypesduring infection cycle: budded virus (BV) and occlusion derived virus (ODV). BV andODV use the different mechanisms for infection. It has been demonstrated that BV entriesthe insect cells via endocytosis and is released into the cytoplasm as a result of fusion withacidic late endosomes. However, our knowledge on oral infection is still limited. We areinteresting to understand the molecular mechanisms of the primary infection (ODV entire)and the second infection (BV) in host. We selected two genes vfgf, fibroblast growth factor(fgf) homology encoded by baculovirus which might involved in baculovirus pathogenesis,andper os infection factor 3 (pif-3) as the start point.In Chapter one, an overview briefly introduce baculovirus, especially on the infectionmechanism of BV and ODV. We also outline the research development and the prospect onFGF. The aims and singnifacnt of this thesis are presented at the end.Fibroblast growth factor (FGF) is a key regulator of developmental processe,highlighting its importance in a variety of multicellular biological processes such asmitogenesis, angiogenesis, cell proliferation, differentiation and cell migration. In chapter 2we characterized biochemical characteristics in detail and analyzed the functions duringHelicoverpa armigera nucleopolyhedrovirus (HearNPV) BV infection. HearNPV vfgftranscription and translation results indicated that vfgf is a late expression gene.Chemoattraction assay showed that HearNPV vFGF specially chemoattracted Hz-AM1, but not other insect cell lines such as Sf9 and Se-UCR and mammalian cells: 293 and HepG2.FGFs bind heparin-Sepharose results indicated that vHaFGF conserved the property ofbinding heparin strongly, which is critical for the function of all FGFs. At the same time,Western blot analysis showed that vFGF was secreted into the culture medium, suggestingthat it acts as an extracellular ligand. HearNPV vFGF had strong affinity to heparin, aproperty important for FGF signaling via an FGF receptor. It is most notable that HearNPVvFGF was also a envelope structural protein of budded virus but absent inocclusion-derived virus (ODV) by western blot and Immune colloidal gold technique,which coordinated to the chemotatic activity analysis and antiserum neutralized results,implying that it my have important function for BV infection. Then a recombinantHearNVP bacmid lacking a functional vFGF (Ha-vfgfKOeGFP) was constructed.Transfection-infection analysis indicated that vHa-vfgfKOeGFP could not set up thesecond infection. However, restoration of vfgf rescued the infectivity. Those resultsindicated that vFGF plays an essential role in HearNPV BV infection.In chapter 3, we characterized the pif-3 of Autographa californica NPV (AcMNPV)in more detail and studied the function of the N-terminal hydrophobic domain of AcMNPVPIF-3. The pif3 transcripts and Western blot analysis indicated that pif-3 is a late stageexpression gene. Western blot analysis also indicated that PIF-3 was a component ofocclusion derived virus but not of budded virus. The subcellular localization demonstratedthat PIF-3 hydrophobic domain riching in isoleucine, leucine and valine acts as nucleuslocation signal and is essential for protein trafficking to nucleus, but required other viralprotein(s) for efficient passage from the cytoplasm to the nucleus. To investigate thefunction of the SM domain of PIF-3, we constructed the recombinant viruses deletingPIF-3 or PIF-3 hydrophobic domain by red recombinant system. In vivo assay, we find thatdeletion of PIF-3 or PIF-3 N-terminal hydrophobic domain eliminates AcMNPV oralinfectivity but no affect on its BVs infectivity. One-step growth analysis also showed thatdeleting either PIF-3 or the SM domain of PIF-3 didn't affect the AcMNPV BV replication,assembly and budding.The paper first demonstrates that HearNPV vFGF is a new envelope structural proteinof BVs and plays an essential role in HearNPV BV infection, which provides the new way for studying on molecular mechanism of baculovirus infectivity. At the same time, we alsofind PIF-3 N-terminal hydrophobic domain acts as nucleus location signal and is essentialfor AcMNPV oral infectivity, which is a new idea for studying the mechanism of ODVinfectivity.