Molecular Biology Of BmNPV ODV-E56/BmP95and Occlusion Of Foreign Protein Into Polyhedra

The family Baculoviridae encompasses a group of arthropod-specific viruses found ubiquitously in the environment. During the typical biphasic infection cycle, two structurally and functionally distinct enveloped virion phenotypes are produced, budded virus (BV) and occlusion-derived virus (ODV). Both virions have a common nucleocapsid structure and carry identical genetic information, but differ in the source and the composition of their envelopes. The Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most studied baculoviruses which specifically infects the domestic silkworm. BmNPV produces large amount of polyhedrin at late stage and the polyhedrin is subsequently assembled to form supermolecular, crystal polyhedra, in which lots of ODV embeded. The polyhedron can effectively protect the incorporated virus from desiccation, high temperature, even acid cauterization, and allow the virus to survive for several years in soil. In this study, in order to make clear the molecular mechanism for the ODV entry into the polyhedron, two ODV envelope proteins, ODV-E56and BmP95(ORF69) which conserved in baculoviruses are characterized. In addition, the occlusion mechanism for the foreign proteins encapsulating into the polyhedron of BmNPV are also discussed. The results are summarized as following:1. The characterization and biological function of BmNPV odv-e56The odv-e56is located at118,837-119,964nt, contains1,128bp, and encodes a375amino-acid peptide with predicted molecular weight of41kDa. The C-terminus region of ODV-E56contains a highly hydrophobic transmembrane domain. The RT-PCR analyses indicated that the1,128bp specific transcripts are detectable from12hpi until to96hpi.5’RACE analysis showed that the odv-e56mRNA transcript initiated at the second A of ATAAG motif, indicating that the odv-e56is a late gene.To determine its role in the BmNPV life cycle, a recombinant virus that delete the odv-e56was constructed through homologous recombination. The results showed that in tissue culture, odv-e56is not required for viral DNA replication, BV production, ODV envelopment and polyhedra formation. Larvae bioassays demonstrated that injection of mutant BV into the hemocoel can killed Bombyx mori larvae as efficiently as wild-type viruses; however, it is unable to infect the Bombyx mori larvae when inoculated per os. Thus, these results indicated that odv-e56encodes a new per os infectivity factor (PIF-5).2. Functional analysis of BmNPV BmP95The BmP95is located at62,249-64,768nt in the genome of BmNPV. It contains2,520bp and is predicted to encode a839amino-acid peptide. The BmP95is a core gene, homologous of Ac83of AcMNPV, and is a conserved gene present in all of the sequenced baculoviruses. A conserved highly hydrophobic transmembrane domain is located in the N-terminus region of the BmP95, and is predicted to contain two functional domain:Pfam:Baculo VP91N and ChtBD2.To investigate the role of BmP95in virus infection, a BmP95deletion virus (vBmP95-De) was generated by use of the λ-Red homologous recombination system in Escherichia coli. Fluorescence and light microscopy and titration analysis indicated that the BmP95deletion bacmid led to a defect in production of BV. However, deletion of BmP95did not affect viral DNA replication. Electron microscopy showed that masses of aberrant tubular structures were presented in the cells transfected with the BmP95deletion bacmid, indicating that deletion of BmP95affected the assembly of nucleocapsid and subsequently the BV production, the formation of ODVs, as well as functional polyhedra. This defect could be rescued by insertion of full length of BmP95into the polyhedrin locus of the Bm.P95-knockout bacmid, but not the N-terminal domain of BmP95. In combination, full length of BmP95is essential for the BV production and is required for the nucleocapsid assembly.3. Occlusion of foreign protein into polyhedra of BmNPVThe BmNPV polyhedra can occlude ODV virion particles and protect these virions from hostile environmental conditions. In order to make clear whether the ODV envelope protein can function as a polyhedrin recognition signal leading to the incorporation of foreign proteins into polyhedra, two pivotal envelope protein genes odv-e25and odv-e56were cloned and ligated with egfp, and using BmNPV Polh Bac-to-Bac expression system to construct two recombinant baculoviruses which can co-express the polyhedrin and fused protein. Fluorescence microscopy and Western blotting indicated that the fusion proteins can be detected in the BmNPV polyhedra. Two recombinant viruses which can express the polyhedrin and foreign protein (EGFP/LacZ) simultaneously were also generated by the BmNPV Polh Bac-to-Bac expression system. The results indicated that the foreign protein can be incorporated into the polyhedra and be protected from the harsh environment. The recombinant virus which can express EGFP but not contain the polyhedrin was used to infect BmN cells jointly with wild-type BmNPV, Fluorescence microscopy showed that EGFP and polyhedrin can be expressed in the same BmN cell simultaneously, and the foreign proteins were presented in the polyhedra. In conclusion, a nonspecific recognition mechanism exists in the embedment of foreign proteins into BmNPV polyhedra.