A Preliminary Study On Mechanism Of BmNPV Envelope Fusion Protein GP64

Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Baculoviruses are obviously distinct among virus families, because they have biphasic replication cycle during which they produce two different phenotypes of virions: budded virus(BV) and occlusion derived virus(ODV). These two virions use different mechanisms to conduct infection. It has been demonstrated that ODV enters host cells via oral infection and that BV uses endocytosis to infect insect cells. BmNPV(Bombyx mori nucleopolyhedrovirus, BmNPV) is an important representative member of the baculovirus branch NPV genus. Although a great number of researches have been done on the mechanisms of BV. But there are still many details remaining obscure.This paper will do some research on the mechanisms of BmNPV BV entering host cells, which mediated by GP64 and cholesterol. Meanwhile, we also clarify the characteristics of BmNPV GP64 signal peptide which wo?l d not be cutted after guiding GP64 for transmembrane. It has been demonstrated that the CRACs(cholesterol recognition amino acid consensus) domain in GP64 play a key role in the infection of the insect cells. The interactions between GP64 and cholesterol need to be expounded explain the mechanisms for BV infection. This study will enrich the theory of molecular mechanisms of baculovirus infection.This paper will be elaborated from several sections as followed: sequence analysis of BmNPV GP64 and the characteristic analysis of its signal peptide, construction of virus BmNPV GP64 deletion, analysis of BmNPV GP64 CRACs domain and its study of interaction with cholesterol. These studies will provide effective reference for the research of the mechanisms of BmNPV BV entering insect cells.1) Sequence analysis of BmNPV GP64 and characteristic analysis of its signal peptide: Total length of BmNPV GP64 is 1593 bp, encoding 530 amino acids, and its N-terminal signal peptide reaches 38 amino acid residues. By sequence alignment, we discover that homology of BmNPV GP64 and AcMNPV GP64 is 93%. For the following research, a c-Myc-Tag has been added in the middle of BmNPV GP64 signal peptide. Then, recombinant bacmid is transfected into BmN cells to get budded virus(BV). After getting purified BV, we use western blotting to identify whether BmNPV GP64 signal peptide has been cutted when it finishes its function for GP64 transmembrane.2) Construction of virus BmNPV GP64 deletion: using homologous recombination methods to transformate targeting DNA molec?l es into the Red recombination system by electroporation, selecting positive colonies via two kinds of antibiotics, designing identifying primer and internal primer for PCR identification, finally, construction of new Bac-to-Bac system.3) Analysis of BmNPV GP64 CRACs domain and its study of interaction with cholesterol: According to some article reports and sequence alignment, we find Ac MNPV gp64 has three CRACs, however, BmNPV GP64 has four CRACs, one of which is just in the signal peptide. To clarify which CRAC domain in BmNPV GP64 has binding activity with cholesterol, we design a series of recombinant bacmids to transfect insect cells for infection identification.Through these studies, we successfully constructed virus of BmNPV GP64 deletion; For the first time, we revealed the BmNPV GP64 protein signal peptide sequence is not cutted in post-processing; At last, we speculate that BmNPV GP64 CRAC2 may play a key role in the cell invasion of BmNPV BV.