| Objective Chinese honeybee larvae infected with Chinese sacbrood virus(CSBV)were analyzed using RNA-Seq to reveal the effects of CSBV infection on host physiological functions and immune mechanisms by comparing the differential m RNA expression levels of Chinese honeybee larvae before and after infection,and by combining GST This study was carried out to identify the host membrane proteins that interact with CSBV VP2 protein and validate their role in CSBV infection by combining GST pull-down and liquid chromatography-tandem mass spectrometry(LC-MS/MS)techniques.Methods Comparison of the transcriptomes of CSBV-infected Chinese honey bee larvae with healthy larvae showed that the transcriptomic expression levels of a total of 4832 genes were altered,of which 2643 genes were up-regulated and 2200 genes were down-regulated at the transcriptional level(difference multiplicity > 2);Among them,1,848 differentially expressed genes were enriched in Gene Ontology(GO),and the top 30 most significantly enhanced GO items mainly involved molecular functions,metabolic processes,and cellular components,among which the most significant changes in molecular procedures and metabolic processes,and significant changes in cellular components were mainly in extracellular region component(extracellular region and extracellular region part),membrane part and membrane protein complex.Region and extracellular region part),and membrane part and membrane protein complex;1121 differentially expressed genes were enriched in KEGG pathways,with the top 20 KEGG pathways significantly enriched,mainly involving metabolic processes,innate immunity,and apoptosis pathways.The main metabolism-related pathways include Carbon metabolism,Fatty metabolism,Amino Acid Metabolism,and Saccharometabolism;Immune pathways include the Mitogen-activated protein kinase(MAPK)signaling pathway,Lysosome signaling pathway,Hippo signaling pathway,and Toll and Imd signaling pathway.Analysis of the expression levels of the relevant host membrane proteins in the GO enrichment showed that initially,23 significantly different membrane proteins were obtained,with an up-regulation range of 1.18-10.5-fold and a down-regulation range of 1.13-5.02-fold.The GST pull-down combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS)technique was used to screen for host proteins that interact with CSBV VP2 proteins,and 17 proteins were initially identified as interacting with them.The potential host membrane protein ANX B9 interacting with CSBV VP2 was screened out based on the differential transcriptional expression of membrane protein and mass spectrometry analysis results.The interaction between host ANX B9 and CSBV VP2 was confirmed by yeast two-hybridization,Co-IP,and laser confocal analysis.The interaction sites of ANX B9 protein are about 178-183 aa,210-214 aa and VP2 protein are about 210-212 aa.Based on this,the copy number of CSBV in the experimental group BHK cells transfected with PTT5-ANX B9 and the control group cells was detected by the RT-qPCR method.The results showed that the copy number of the virus in the experimental group and the control group was 3.4±0.34*10~6 and 8.6±0.56*10~6, respectively,after 24 h inoculation with CSBV.48 h after inoculation with CSBV,the virus copy numbers of the experimental group and the control group were 1.5±0.21*10~7 and 3.6±0.77*10~7,respectively,with significant differences(P<0.01),indicating that host membrane protein ANX B9 plays an important role in the process of CSBV infection.Results Comparison of the transcriptome of CSBV-infected Chinese honey bee larvae with that of healthy larvae showed that the transcriptome expression levels of a total of 4832 genes were altered,of which 2643 genes were up-regulated and 2200 genes were down-regulated(difference multiplicity > 2 and P ≤ 0.05);Among them,1848 differentially expressed genes were enriched to Gene Ontology(GO),and the top 30 most significantly enriched GO items were mainly involved in molecular function,metabolic processes and cellular components,among which the most significant changes in molecular function and metabolic processes,and significant changes in cellular components were mainly concentrated in extracellular region component(extracellular region and extracellular region part),membrane part and membrane protein complex.region and extracellular region part),membrane part,and membrane protein complex;1121 differentially expressed genes were enriched in KEGG pathways,among which the top 20 KEGG pathways with significant enrichment differences were mainly involved in metabolic processes,innate immunity,and apoptotic cell death pathways.Among them,metabolism-related pathways mainly include Carbon metabolism,Fatty metabolism,Amino Acid metabolism,and Saccharometabolism;The immune pathways mainly include the Mitogen-activated protein kinase(MAPK)signaling pathway,Lysosome signaling pathway,Hippo signaling pathway,Toll and Imd signaling pathway.Analysis of the expression levels of the relevant host membrane proteins in the GO enrichment showed that initially,23 significantly different membrane proteins were obtained,with an up-regulation range of 1.18-10.5-fold and a down-regulation range of 1.13-5.02-fold.The host proteins interacting with CSBV VP2 protein were screened by GST pull-down combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS)technique,and 17 proteins were initially identified to interact with them.Based on the results of transcriptome differential expression membrane protein and mass spectrometry analysis,the host protein ANX B9 was verified by yeast two-hybrid,Co-IP and laser confocal,and it was confirmed that ANX B9 protein interacted with CSBV VP2 protein,and the bioinformatics software analysis showed that ANX B9 protein interacted at approximately 178-183 aa,210-214 aa and VP2 protein interacted at approximately 210-212 aa.The CSBV copy numbers of experimental BHK cells and control cells transfected with PTT5-ANX B9 were examined by RT-qPCR,and the results showed that the viral copy numbers of experimental and control cells were 3.4±0.34*10~6 and 8.6±0.56*10~6 after 24 h of CSBV inoculation,and the viral copy numbers of experimental and control cells were 1.5±0.21*10~7 and 3.6±0.77*10~7 after 48 h of CSBV inoculation,respectively.The differences were highly significant(P<0.01),indicating that the host membrane protein ANX B9 has an important function in the process of CSBV infection.Conclusions 1.Transcriptome analysis revealed that 4832 genes were altered in the transcript levels of CSBV-infected larvae compared to healthy larvae,with 2643 genes up-regulated and 2200 genes down-regulated(difference multiples > 2 and P ≤ 0.05).2.Analysis of the top 30 most significantly enriched GO terms showed that CSBV infection severely affected biological and metabolic processes in honey bee larvae and caused significant changes in extracellular and cell membrane protein genes;Analysis of the KEGG pathway showed that various metabolic processes were severely affected in the CSBV larvae 72 h after infection and that the host innate immune pathway was activated by the virus to exert antiviral effects;Analysis of the expression levels of host membrane proteins related to GO enrichment preliminarily identified 23 membrane proteins with significant differences.3.Combined transcriptome differentially expressed membrane proteins and mass spectrometry results screened for potential host membrane protein ANX B9 that interacts with CSBV VP2.The interaction between host ANX B9 protein and CSBV VP2 protein was confirmed by yeast two-hybrid,Co-IP,and laser confocal.4.PTT5-ANX B9 transfection of CSBV non-susceptible cells was able to alter the susceptibility of CSBV non-susceptible cells to CSBV and was able to promote the invasive effect of CSBV on non-susceptible cells. |
