Research On The Machnism Of Baculovirus Envelope Protein P74 In Primary Infection To Host

Baculoviruses are unique among virus families because of their two distinct infectious phenotypes: BV and ODV. The latter is contained within matrix crystalline structures termed as occlusion bodies (OBs), which is used for stabilizing the virus in the environment. When consumed by insect, the OBs are degraded under the alkaline conditions of the larval midgut, releasing ODV, which is able to establish the primary infection. P74 of AcMNPV ,one of the structural peptides, is the first ODV envelope protein demonstrated to have a functional significance in primary infection .In order to study the function of envelope protein P74, two kinds of p74 -null recombinant baculoviruses, rAc-gfpAp74 and rHa-gfpAp74, were constructed by inserting gfp driven by polyhedrin promoter into the p74 locus of Autographa californica multiple nucleocapsid nucleopolyhedroviras (AcMNPV) genome and that of Heliothis armigera single nucleocapsid nucleopolyhedroviras (HaSPNV) genome, respectively. Plaque purification was carried out to isolate putative recombinants with GFP inserts. Expression of GFP, PCR amplification and DNA sequencing were used to confirm the authenticity of the recombinant viruses.On the other hind, two kinds of p74 over-expression and polyhedra-positive recombinant viruses, rAc-p74++polh+ and rHa-p74++polh+, were produced by usingBac-to-Bac system. OBs were formed in Sf9 cells and Hz-AMI cells when infected with rAc-p74++polh+ and rHa-p74++polh+ respectively. Two specific bands appeared on the SDS-PAGE gel of Sf9 cells infected with rAc-p74++polh+ and the Hz-AMI cells infected with rHa-p74++polh+ r, one is the 30kD polyhedrin protein band, the other is the 74 kD protein band. And this 74kD protein was detected by anti-His-tag mAb in western blot. Ant it proved that the p74 over-expression and polyhedra- positive recombinant viruses were constructed successfully.The one- step growth curves of three kinds of virus(p74-null virus including rAc-gfp A p74 and rHa-gfp A p74, p74++ virus including rAc-p74++polh+ and rHa-p74++polh+, and p74+ virus including wtAcMNPV and wtHaSNPV) were performed by Plaque-assay to investigate whether the inactivation of p74 or over-expression of p74 influence the duplication and propagation of BVs. Infectious virus production fromp74-null virus-infected cells was similar to that from wild type virus-infected cells or P74 over-expression virus-infected cells, indicating that the deletion/inactivation and over-expression of p74 gene did not have a significant effect on either the production or propagation of infectious budded virus.Both the productivity and the characters of the cell culture-derived OBs of p74-null virus, wild type virus and P74 over-expression virus were investigated. The three kinds of virus OBs showed no remarkable difference in productivity together with shape and size, suggesting that p74 gene did not influence the assembling of OBs in cells.However, the bioassay analysis results revealed that all larvae infected with p74-null viruses did not appear any infection symptom, which was similar to the control group. Neither polyhedra nor GFP was observed in the hemolymph cells of the host insects fed with p74-null virus per os. OBs of rAc-gfpAp74 failing to infect S. exigua larvae showed that the function of AcMNPV p74 gene is similar or conserved to its susceptive host.Similarly, the other p74-null\ virus rHa -gfp p74 OBs also lost its virulence to H.armigra larvae, which showed HaSNPV p74 gene played a similar role like AcMNPV p74.It proved that the p74 gene of HaSNPV was essential to the oral infectivity of Group II baculovirus OBs, too. And it illuminated that the functional of baculovirus p74 is conserved, though the amino acid homology is only 51%.However, LD50 values of P74 over-expression viruses (rAc-p74++polh+ and rHa-p74++polh+) were not significant different from that of wild type viruses correspondingly ( wtAcMNPV and wtHaSNPV), showing that the high expression of P74 protein did not influence the virulence of OBsThe deletion/inactivation of p74...