Interaction Of Bombyx Mori Cytoplasmic Polyhedrosis Virus Structural Protein VP7 And Polyhedrin

Bombyx mori cytoplasmic polyhedrosis virus(BmCPV)is a representative member of the genus cypovirus of Reoviridae.BmCPV virus particles are icosahedral with a single capsid and protected by polyhedra structure.A polyhedron contains several to tens of thousands of virus particles.It is known that the genome of BmCPV is composed of 10 dsRNAs of different sizes,which are S1-S10 from large to small.Among them,S1-S4,S6 and S7 encod structural proteins VP1,VP2,VP3,VP4,VP6 and VP7 respectively,and S5,S8,S9 and S10 encode nonstructural proteins NSP5,NSP8,NSP9 and polyhedrin(Polh)respectively.Studying the structure and function of virus encoded proteins and their interaction is an indispensable part of virus research,which will help to study the assembly process of virus particles and provide new ideas for the development and utilization of virus and its components.In this study,the interaction between BmCPV VP7 and Polh was proved by yeast two hybrid system.Then,the insect cell expression plasmids pIZT-V5/His-S7 and pIZT-S10 expressing VP7 and Polh were constructed respectively.The results of immunofluorescence showed that VP7 and Polh colocalized in the cytoplasm of BmN cells.Furthermore,the 168 amino acid sequence of N-terminal of DsRed was fused with Myc tag,and the remaining C-terminal amino acid sequence was fused with HA tag,and colned into insect expression vector pIZT-V5/His respectively to construct pIZT-Myc-DsRedN168 and Pizt-HA-DsRed △N168.VP7 and Polh genes were further cloned into the above two plasmids to construct expression plasmids of VP7/Polh fused with truncated DsRed.After transfection,the fluorescence observation showed that VP7 and Polh were close to each other in cells,resulting in two DsRed segments close to each other and emitting red fluorescence.Then,in order to identify the key region of interaction between Polh and VP7,we predicted the secondary structure of VP7 and Polh,designed a series of truncated proteins according to the predicted results,and locked the key region of VP7 interacting with Polh at the N-terminal 331-360 amino acid(VP7-N331-360)by yeast two hybrid system.When Polh was truncated,it lost the interaction with VP7 in yeast system.The structural analysis of VP7-N331-360 shows that its main body is a helix.The results of yeast two hybrid showed that when the histidine at position 342 of VP7 was mutated to glycine,the interaction between VP7 and Polh was lost.The cytoplasmic polyhedron has a good protection effect on the virus particles embedded in it in the natural environment,and is a good choice for the development of protein drug carriers.In order to confirm that VP7 can use its its interaction with Polh to guide the foreign protein into polyhedra,EGFP gene and bFGF gene were fused at the 5’end of VP7-N331-360 sequence respectively,and then cloned into P10 promoter downstream of pFastBacTM Dual vector containing Polh gene expression cassette to construct EGFP/bFGF vector expressing Polh and VP7-N331-360 at the same time.Furthermore,the recombinant viruses BmNPV-EGFP-VP7-Polh and BmNPV-bFGF-VP7-Polh were obtained by Bac-to-Bac system.Western blotting results showed that EGFP and bFGF fused with VP7-N331-360 were successfully embedded in the polyhedra,and the embedding efficiency of EGFP and bFGF was 6%and 51%respectively.SDS-PAGE was used to detect the protective effect of polyhedra on the embedded recombinant protein under different storage conditions.It was found that when polyhedra were stored at room temperature or-20℃ without lysis,the embedded recombinant protein did not degrade significantly.When polyhedra were stored at room temperature for 2 weeks after lysis,the embedded protein basically degraded completely.It is confirmed that polyhedra have a good protective effect on foreign protein.Transwell experiment showed that the polyhedra embedded with bFGF could promote the migration of L-929 cells without contacting with L-929 cells.It is speculated that bFGF embedded in polyhedra can be released into the culture medium under the condition of cell culture.In conclusion,the N331-360 region of VP7 is the key region for the interaction with Polh.VP7 N331-360 can guide the fusion protein into polyhedra and protect the encapsulated protein from degradation.The results provide a new clue for understanding the mechanism of virus particles embedded in polyhedra and a new method for the development of polyhedra embedded protein/drug microcrystals.