The Role Of Bombyx Mori Niemann-pick Disease Type C (BmNPC1) In Baculovirus Infect Host Cell

Niemann-Pick disease?Niemann-Pick type C,NPC?is a fatal autosomal recessive hereditary disease,and characterized by lysosomal storage of cholesterol and gangliosides,which results from defects in intracellular lipid trafficking.Mutations in the NPC1 gene cause 95% of the cases,the rest being caused by NPC2 mutations.As an important cholesterol trafficking protein,NPC1 is also a critical filovirus receptor.In addition,the infection of HIV-1?family: Retroviridae?,Chikungunya virus?family:Togaviridae?,Zika,West Nile,and Dengue viruses?family: flaviviridae?are also closely related to the NPC1,it is suggested that NPC1 not only a host cholesterol trafficking receptor,but also an important virus infection factor.As a typical double-stranded DNA enveloped virus,baculoviruses are known to infect invertebrates,with over 600 host species described.Due to a strong species-specific tropism for arthropods,baculovirus has been widely used as a biopesticide.Concurrently,baculovirus can transduce a broad range of vertebrate cells,including human,bovine,fish,avian,and even primitive cells such as embryonic stem cells,warranting its application as a biological tool for gene delivery.Roles for a number of host molecules including heparin sulfate and phospholipids have been identified in BV attachment or binding;however,the exact identity of host receptors for baculovirus still remains elusive.It has been reported previously that Bombyx mori promoting protein?BmPP?,a member of NPC2 family proteins,was able to enhance BmNPV infection in silkworm Bo Mo cells.However,the detailed molecular mechanism has not yet been fully elucidated.The recent findings of NPC1 protein as an intracellular receptor for filovirus,coupled with prior evidence supporting the involvement of BmPP protein in BmNPV infection,prompted us to investigate thepotential role of NPC1 homologs in BmNPV infection in insect cells.Here,we analyze the sequences of Bmnpc1 on the baisis of genomic datas,the glycosylation sites and protein domain were analyzed according to the protein sequence.And we also expressed the recombinant protein of BmNPC1 and the polyclonal antibodies were prepared.Furthermore,indirect immunofluorescence assay?IFA?was performed to determine the localization of BmNPC1.Subsequently,pretreatment of Bombyx mori embryonic cells?BmE?with NPC1 antagonists and down-regulation of NPC1 expression resulted in a significant reduction in baculovirus BmNPV?Bombyx mori nuclear polyhedrosis virus?infectivity.Furthermore,we showed that the major glycoprotein GP64 of BmNPV,responsible for both receptor binding and fusion,was able to interact predominantly with the BmNPC1 C domain,with an enhanced binding capacity at low p H conditions,indicating that NPC1 most likely plays a role during viral fusion in endosomal compartments.The main results showed as follows.1.The bioinformatic characteristics of Bmnpc1 of B.mori.To identify Bombyx mori homologs of the npc1 gene,the human NPC1?h NPC1?gene sequence?gi|255652944?was used to conduct a BLAST search in the NCBI database.Results revealed that the gene?XP₀12544312.1?encoding a 1334 amino acid-protein shares the highest sequence similarity of 43% with h NPC1,indicating this gene is a potential homolog of a vertebrate NPC1 family member;it is named BmNPC1 in our study.Bmnpc1 was predicted to have a signal peptide,and structure prediction revealed that the putative BmNPC1 protein contains 13 transmembrane-spanning helices and 3 large luminal loops,described here as BmNPC1-A,BmNPC1-C and BmNPC1-I.Subcellular localization analysis show BmNPC1 localized in the plasma membrane,vesicle lumen and membranes.Multiple glycosylation modification sites and phosphorylation modification sites were predicted,and revealed that BmNPC1 is a glycoprotein.Similarly to h NPC1 protein,the putative BmNPC1 protein was predicated to contain 3 domains,the N-terminal domain?NTD?,the Sterol-sensing domain?SSD?,and the patched domain PTC).Multi-sequences alignment reflected significant identity among vertebrates and invertebrates,suggested npc1 were conserved.Three-dimensional modeling showed BmNPC1 was similar to h NPC1.Phylogenetically,BmNPC1 can be grouped in a single clade with Apis cerana,Drosophila melanogaster,and is relatively distant from NPC1 s from Saccharomyces cerevisiae and Toxoplasma gondii.2.The identification and localization of BmNPC1 in B.mori.To further study the biological function of BmNPC1 and its role in baculovirus infection in insect cells,the full length?4 kb?coding sequence?CDS?of BmNPC1 was cloned from the Bombyx mori c DNA library.Microarray data showed Bmnpc1 was transcribed in all tissues of silkworm,and highly expression in midgut,malpighian tubule and testis,low expression in fatbody and sericterium.Furthermore,a rabbit polyclonal antibody against h NPC1?Pc Ab-h NPC1?was used to detected protein expression and localization of BmNPC1 in BmE cells,with a single band of ¹40 k Da detected in BmE whole cell lysate by Western Blot.Proteins localized in the plasma membrane,vesicle lumen and membranes in BmE cells as revealed by IFA.Taken together,we demonstrate that the putative BmNPC1 gene?XP₀12544312.1?was transcribed and translated in BmE cells,with the protein abundantly expressed in the plasma membrane and intracellular vesicles.The sequences of BmNPC1 extracellular loop A?31 to 264 amino acids?or I?881 to1152 amino acids?were amplified with primer pairs BmNPC1-A-PE and BmNPC1-I-PE and were then cloned into p ET32a?+?plasmid for protein expression in E.coli.The recombinant BmNPC1-A,BmNPC1-I proteins with His tag were purified with a His Trap HP 5 ml column according to the manufacturer's instructions.The purified recombinant protein BmNPC1-A and BmNPC1-I were later used as an immunogen to immunize 7-week-old BALB/c mice with Freund's complete adjuvant.The TDPVELWASPTSRS polypeptide from BmNPC1?409 to 422 amino acids?coupled with the KLH tag were synthesized by Gen Script and used to immunize New Zealand rabbits with Titermax Gold adjuvant.The anti-BmNPC1-A and anti-BmNPC1-I mouse serum,anti-BmNPC1 rabbit serum,and control sera from both na?ve mice and rabbits immunized with KLH tag only were collected and stored at-20?C until use.3.Baculovirus BmNPV infection depended on BmNPC1.To investigate whether BmNPC1 is involved in BmNPV infection in insect cells,we first examined the effect of two small molecule NPC1 antagonists on BmNPV-GFP infection in BmE cells.BmE cells were pretreated with imipramine or U18666 A before the addition of BmNPV viral inoculum,and resulted in a dose-dependent reduction in viral infectivity as demonstrated by the percentage of GFP positive cells.For BmE cells pretreated with 100 ?M of imipramine or 10 ?M of U18666 A,the viral load was reduced to 27% and 10% respectively compared to the control.It was suggested thatblockage of BmNPC1 function by small molecules can efficiently reduce BmNPV infection in BmE cells.To further study the role of BmNPC1 in BmNPV infection,RNAi was performed,and viral infectivity as indicated by GFP expression was decreased significantly in BmNPC1 RNAi treated cells.Viral loads in BmNPC1 RNAi treated cells were less than 10% of that in control RNAi-treated cells.The viral titer?5.6log10TCID50/ml?at 96 h p.i.as quantified by TCID50 in BmNPC1 RNAi treated cells was significantly lower compared to that in control cells?10.8 log10TCID50/ml?.Meanwhile,we generated a BmNPC1 null stable cell line by using CRISPR-Cas9 technology,partial BmNPC1 gene was replaced by ie1-Ds Red and A3-Neo expression cassettes.We tested the susceptibility of the NPC1-mutant cells to BmNPV-GFP virus infection,and found that the percentage of GFP positive cells among BmNPC1-null cells as indicated by the expression of red fluorescence was less than 2% of that of the control cells,the virus load was reduced to 20% of that in control cells.Altogether,these data confirmed that BmNPC1 is required for BmNPV infection,and the cells lacking functional BmNPC1 do not support BmNPV infection.4.The mechanism of BmNPC1 involved in BmNPV infection.It was showed that functional BmNPC1 might be required for BmNPV infection in insect cells.To investigate whether BmNPC1 contributes to BmNPV infection by interacting with glycoprotein GP64 during viral entry,Co-immunoprecipitation was performed.The results suggest that domain C of BmNPC1 is sufficient for GP64 binding,while in comparison,the binding affinity of BmNPC1-A or BmNPC1-I to GP64 is relatively low.Interesting,BmNPC1-C binding affinity to BmNPV GP64 was enhanced at a lower p H environment.To further confirm these interactions,we employed a yeast two-hybrid system?Y2H?,and the results revealed that BmNPC1-C interacted with full-length GP64,whereas BmNPC1-A or BmNPC1-I bound to GP64 with a weak affinity.Collectively,these results demonstrate a specific interaction between BmNPC1 domain C and BmNPV GP64.IFA co-localization techniques using anti-h NPC1 and anti-GP64 staining on host cells demonstrated shows the overlay of BmNPC1 with BV,indicated that baculovirus GP64 could interact with BmNPC1 at the surface of BmE cell.To further confirm which domain of BmNPC1 was most critical for BmNPV infection,we assessed the susceptibility of BmNPV infection in the presence of BmNPC1 domain specific antibodies.The results showed BmNPV infection was significantly reduced in BmEcells treated with antibodies specific for BmNPC1-C,but not BmNPC1-A or BmNPC1-I as measured by GFP positive cells and viral gene expression by q PCR.These data suggest that the interaction between BmNPC1 domain C and BmNPV GP64 is essential for BmNPV infection in BmE cells.In summary,our study demonstrated that BmNPV entry requires the cholesterol transporter BmNPC1 as the receptor for baculovirus infection.In this model,baculovirus binds to the cell surface with the possibility of using BmNPC1 as one of the attachment factors,and is then internalized and trafficked to late endosomes.In the caustic p H environment of endosomes,GP64 undergoes conformational changes to expose certain domain?s?for better access by BmNPC1.The specific binding between GP64 and BmNPC1 subsequently facilitates viral fusion events to allow nucleocapsids to be released into the cytoplasm.Our work fills an important gap in baculovirus research,and identifies a new antiviral target against baculovirus infection.Elucidation of baculovirus entry mechanisms will further facilitate the application of baculovirus systems in eukaryotic gene delivery.As the cholesterol transporter NPC1 is shared by several viral families,this work provides a new avenue of inquiry that NPC1 may represent a common entry factor for many enveloped viruses infected cells.