Study On The Infection Mechanism Of Heliothis Armigera Nucleopolyhedrosis Virus

Heliothis armigera nuclear polyhedrosis virus (HaNPV), due to its special pestiferous effect on Heliothis armigera and being innocent to human, livestock and environment, has been a commercial bioinsecticide widely in the world for years. HaNPV is the earliest commercial baculovirus insecticide in China and about 100,000 hectare cotton farmland had been used such insecticide to prevent Heliothis armigera by the end of 1999. Many aspects of HaNPV such as the isolation and identification of different virus strains, bioassay of their toxicity, infectivity and duplication in culture cells, genome sequencing and some functional genes have been researched. But there is little understanding of the virus-host interaction.Chariton C A &Volkman L Z researched the change of Actin in Sf21 cells during the AcMNPV infection process in 1991. They found that the original Actin can been induced to form the cable structure by the nucleocapsid just escaped from its envelope about 3hr p.i., even though the cellular protein synthesis was inhibited by aphidicolin. At the late-early phase of infection (3-6hr p.i.) and before the viral DNA replication, host cells became round and ventral aggregates of actin were observed. This precess were sensitive to cychoheximide but not to aphidicolin indicating that an early viral protein mediated this Actin rearrangement. Howerever, ventral aggregates itself did not result inthe rounding process, because uninfected cells becoming round pretreated with colchicine did not form ventral aggregates of Actin. During the viral infection login phase (6-12hr p.i.), viral nucleocapsid and filamentous Actin were found to coexisted in nucleoplasm. So nucleocapsid might induce Actin to form cable structure and this structure might associate with such viral infectious courses as the viral transportation in host cells, viral assembly in nucleus, host cells' liquefaction and viruses' release from the host cells.Because VP39 is the major component of AcMNPV nucleocapsid, it was speculated that VP39 might play an important role in the aggregation of host cell Actin caused by AcMNPV nucleocapsid. But the researchers hadn't revealed the molecular mechanism for the interaction of AcMNPV nucleocapsid and the host Actin, i.e., which component of the nuleocapsid actually interacted with the host actin and induced it to form cable structure? vp39 gene of AcMNPV was first identified by Miller et al. in 1989, which codes a 39kD nucleocapsid protein(VP39), the main component of AcMNPV's capsid. Almost all the newly synthesized VP39 were dispersed in the cytoplasm, but most of them were then transferred to the nucleus after about 12hr and distributed on the surface of nucleocapsid equably. Recent study has shown that VP39 is associated tightly with the infectious course of AcMNPV. Does the interaction of AcMNPV nucleocapsid and Actin also exist in other kinds of baculoviruses such as HaNPV? Little has been reported by far. So we explored the infection process of HaNPV deeply in this thesis.Using the Oligo(dT) cellulose column , 1.68mg mRNA was purified from total RNA that was isolated from 1 X 108Hz-AMl cells with TRIzol reagent. Then, cDNA of Hz-AMl cell (Ha-cDNA) was synthesized with "SuperScrip?choice system for the cDNA to synthesis kit": using Oligo(dT)12-18 as the primers, the first strains of cDNA were retrotranscripted from mRNA with SuperScripII RTwithout RNaseH activity, then using original mRNA as primers and the newly synthesized first strains of cDNA as templates, the second strain of cDNA was synthesized through the nick translation function of E.coli DNA polymerase I, at the same time E.coli RNaseH was added to digest the original mRNA and the newly synthesized DNA fragments were ligated by THE addition of E.coli DNA ligase. Finally, the terminal of the newly synthesized double strain cDNA was blunted by T4 DNA polymerase. Dephosphorylated EcoRI adapter was ligated to the newly blunted terminal of cDNA with T4 DNA ligase and then was phosphorylated by polynucleotide kinase. cDNA fragments that molecular...