The Mechanism Of BmNPV Entry Into Host Cells And The Interactions Of The Envelope Protein GP64 With The Host Proteins

Bombyx mori nucleopolyhedrovirus(BmNPV)is the most important pathogen of Bombyx mori,and it belongs to the alphabaculovirus genus of the baculovirus family.During the viral infectious cycle,budded virus(BVs)and occlusion derived virus(ODVs)particles,which have identical genetic content but different phenotypes,are produced.ODVs infect the host midgut epithelial cells through oral transmission,triggering the primary infection.In contrast,BVs mediate the infection between cells and tissues within individuals,thereby causing systemic infections within their hosts.GP64 is a low pH triggered envelope fusion protein and it plays an indispensable role in BVs infection.In this study,we investigated the mechanism of BV entry,and also identified several novel host proteins interacting with viral GP64.Definitely,the determination of the mechanism of BV entry and the identification of host celluar proteins that interacting with GP64 will help us to well understand the mechanism of BmNPV invading the host cells.The main conclusions are listed as follows:1.The mechanism of BV entry into BmN cells.Endosome acidification inhibitor treatments were performed before or after BmNPV infection,and the viral yield appeared to be significantly reduced,indicating that BV internalized into BmN cells via a low-pH-dependent endocytosis pathway.Moreover,treatment of inhibitors of clathrin-mediated endocytosis and siRNA knockdown assays suggested that cell entry of BmNPV is dependent on clathrin-mediated endocytosis.By using the inhibitor of dynamin,viral reproduction was significantly inhibited in a dose-dependent manner,indicating that dynamin also participates in the internalization of BV.In addition,by using siRNA knockdown assay,we have shown that BmNPV infection of BmN cells requires rab5 and rab7,but not rabl 1,suggesting that vesicular trafficking to early endosomes and late endosomes are crucial in BmNPV infection cycle.2.Screening of host cell surface proteins binding to BV.To reveal the presence of viral receptor on the BmN cell surface,we conducted virus overlay protein binding assay.After incubated BV particles with BmN membrane,three distinct bands were detected by mouse anti-GP64 antibody,indicating the presence of viral receptor on the cell surface.Furthermore,a total of 158 proteins were identified from three positive separation strips by using mass spectrometry.The GO analysis classifies these proteins into three major categories:cellular components,molecular functions,and biological processes.According to the classification of molecular function,the binding active protein accounted for 45%,the translocating active protein accounted for 4%,and the catalytically active protein accounted for 32%.The above data provided us with a more comprehensive understanding of membrane proteins that bind to BV.3.Identification of host cellular proteins interacting with viral envelope protein GP64.The cDNA library of Bombyx mori cell line,BmN,was constructed and cellular proteins were expressed as GAL4 activation domain fusion proteins.As the bait,full-length envelope glycoprotein GP64 was expressed with a fusion to the GAL4 DNA binding domain.Yeast two hybrid(Y2H)assays were performed to screen the host cellular proteins that interact with GP64.After removal of false-positive-interaction proteins,eight proteins were finally identified as potential GP64-binding partners with a high confidence,including E3 ubiquitin-protein ligase SINA-like 10(SINAL10),uncharacterized protein LOC101738779,ran-binding protein 9,phosphoribosylaminoimidazole carboxylase,scaf49 2190774-2191531(-),cleavage and polyadenylation specific factor 5,protein abrupt-like and NADH-ubiquinone oxidoreductase.4.Roles of GP64 binding protein SINAL10 in BV infection.Further co-immunoprecipitation(Co-IP)analysis of the eight candidate proteins identified by the Y2H assay described above and validated the interaction between GP64 and SINAL10 in vitro.Meanwhile,SINAL10 was found to colocalize with GP64 in the cytoplasm near the cell membrane.Moreover,overexpression and siRNA knockdown assays suggested that SINAL10 is a positive regulator in BmNPV infection and crucial for viral life cycle.In conclusion,our results elucidated BV entry into BmN cells through clathrin-mediated endocytosis,which is dependent on dynamin and regulated by Rab5 and Rab7 proteins.In addition,cell membrane proteins that bind to BV particles were identified,and their functions were annotated and analyzed.Moreover,eight host cellular proteins that interacting with the viral envelope GP64 were identified,and the interaction between SINAL10 and GP64 was confirmed.Further research showed that SINAL10 promotes the proliferation of BV and revealed the mechanism of BV entry at the molecular level.