Molecular Function Of Bombyx Mori Cytoplasmic Polyhedrosis Virus Genome Segment7

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the mainpathogens, which can infect midgut cells of silkworm larvae and cause midgutpolyhedrosis. Considerable economic loss was frequently occurred in sericulturebecause of midgut polyhedrosis. BmCPV genome consists of10dsRNA segments andsegment7(S7) encoded structural protein VP7. In this study, subcellular localization,cleavage and expression phase of VP7were detected by Western blotting analysis andimmunohistochemistry with VP7antibody. In addition, the effect of silencing vp7geneon BmCPV proliferation was explored. The interacting host cell proteins of VP7werescreened by Co-IP and the candidates were identified by mass spectrometry. Theseresults can help us clearly understand the molecular functions of VP7and provide newclues for the study of interactions between BmCPV and the host.1. Prokaryotic expression VP7of BmCPV and preparation of polyclonalantibodyThe recombinant expression vector pET-28a-S7was constructed by cloning vp7gene of BmCPV into the prokaryotic expression vector pET-28a(+). The recombinantplasmids were transformed into E.coli strain BL21and inducible expression. The resultsof SDS-PAGE and Western blotting showed that the molecular mass of the recombinantprotein VP7was approximately55kDa. The mice were immunized with VP7recombinant protein and polyclonal antibody was preparaed. Western blotting analysisshowed that polyclonal antibody can be used for the detection of VP7because of itsspecificity.2. Cleavage anlysis of VP7In order to analyze whether VP7could be cleavaged, the recombinant expressionvector pIZT/V5-His-S7was constructed by cloning vp7gene into pIZT/V5-His vector.The BmN cells were trasfected with recombinant plasmid pIZT/V5-His-S7and screenedby the antibiotic Zeocin to obtain transformed cells that could stably express VP7.Western blotting analysis with His6antibody showed that the molecular mass of the recombinant protein VP7was approximately65kDa in transformed cells. However, themolecular mass is slightly larger than the theoretical molecular weight. Except for the65kDa protein could be detected, but two35kDa proteins (V35a, V35b) also could bedetected by Western blotting analysis with VP7antibody, indicating that VP7could becleavaged into smaller proteins in transformed cells.Western blotting analysis with VP7antibody was conducted in BmN cells by thechallenge of BmCPV on the6th day post-infection and in posterior midgut tissues bythe challenge of BmCPV on the8th day post-infection. The55kDa protein could bedetected in two conditions. Besides that, one20kDa protein could be detected ininfected BmN cells and25,20kDa proteins could be detected in infected midgut tissues.These results demonstated that VP7could be cleavaged in BmCPV-infected cells, butthere were differences existed in the virus-infected cultured cells and midguts or in thevirus-infected cells and non-infected cells.Western blotting analysis with VP7antibody was also performed in BmCPVparticle. The results showed that four proteins with the molecular mass of55,38,25and10kDa could be detected, suggesting that VP7could be cleavaged in differentfragments in BmCPV particle.3. Subcellular localization of VP7and the changes of VP7protein levelsduring BmCPV infectionThe expression of VP7in posterior midgut tissues on the8th day post-infected byBmCPV was detected by immunofluorescence. Fluorescence observation showed thatgreen fluorescence appeared in the cytoplasm, indicating VP7was located at cytoplasm.Western blotting analysis was performed after quantifing the proteins in posteriormidgut tissues on the1st to12th day post-infection with BmCPV. One20kDa proteincould be detected in7th day post-infected tissues. The expression of VP7was increasedand55,25and20kDa proteins were detected in8th day post-infected tissues. Theexpression of VP7in9th day post-infected tissues reached its peak and four proteinswith the molecular mass of55,28,25,20and18kDa could be detected. The expressionof VP7in10th,11th and12th day post-infected tissues were similar to that in9th day.However, there was a difference: the extra22kDa protein was expressed in10th,11thand12th day post-infected tissues. So we speculated that there may be subtledifferences existed in the cleavage patterns of VP7during virus proliferation at differenttimes. 4. Effect of inhibition vp7gene expression on the virus proliferationTo further investigate the effect of vp7gene expression on the virus proliferation,3th instar silkworms were injected with VP7-siRNA (1μg/each) and infected byBmCPV after24h, and then the RNA levels of vp7and vp1were detected on the48hpost-infection. The relative expression levels of vp7and vp1were reduced by5.2and15.4times respectively. BmN cultured cells were transfected with3VP7-siRNAs andinfected by BmCPV after24h, and then the RNA levels of vp7and vp1were detected onthe48h post-infection. The relative expression levels of vp7were decreased by17.5,9.7and4.7times respectively. The relative expression levels of vp1were decreased by13.0,6.3and3.0times respectively. These results indicated that inhibition vp7geneexpression can suppress the proliferation of BmCPV in silkworm.5. Screening and identification of the host proteins interacting with VP7In order to study the interactions between VP7and the host proteins, the proteinsof posterior midgut tissues infected by BmCPV were identified by experiment ofco-immunoprecipitation. By analysis of the mass spectrometry we found that18proteins entirely encoded by silkworm genes could be interacted with VP7. Informationanalysis showed that these proteins are involved in glucose metabolism, cholesterolmetabolism, nucleotide degradation, anion transportation between cytoplasm andmitochondria, peritrophic membrane formation, immune response, transcriptionalregulation, JH binding, protein degradation, cell cycle and apoptosis and other aspects.