Study On The Mechanism Of Cell Autophagy And BmNPV Infection Of Bombyx Mori

Autophagy is an important biological process,which mediates a highly conserved and regulated self-degradation and circulation process.During autophagy,unnecessary organelles and dysfunctional proteins are sequestered into autophagosomes and finally delivered to lysosomes?animals?or vacuoles?plants and yeast?for degradation and recycling.Autophagy is involved in many physiological and developmental processes,including neurodegenerative diseases,cancer,and hunger,bacterial or viral infection.The research of connections of virus infection and autophagy has important biological significance in understanding the interaction mechanism between host and virus.Bombyx mori?B.mori?,as an important economic insect,has long escaped from natural selection owing its long-term domestication and breeding.So silkworm is a good material for studying the interaction between virus and host.Bombyx mori nucleopolyhedrovirus?BmNPV?belongs to baculovirus genus,which is a class of double stranded circular DNA virus with envelope.In the process of infection,a variety of mechanisms are regulated by virus to facilitate its reproduction and replication,as well as accompanying the occurrence of various stress responses of the host.In recent years,some host resistance mechanisms have been resolved along with the intensive study of the mechanism of BmNPV infection,but the interaction network of virus and host is still unclear.Using the model silkworm and BmNPV,this study discovered the effect of BmNPV infection on autophagy in silkworm cells;then,overexpression and CRISPR/Cas9 gene knockout technology were used to confirm the autophagy related gene of virus infection and replication at both the cellular level and the individual level;finally,through immunoprecipitation and mass spectrometry,we studied and explained the molecular mechanism of autophagy related genes affecting viral infection.Through this study,we can provide experimental and theoretical basis for revealing the interaction mechanism of autophagy and BmNPV infection in silkworm.The main results and conclusions obtained in this paper are as follows:1.The effect of BmNPV infection on autophagy in silkworm cellsThrough a variety of classical autophagy detection methods,this study demonstrated that BmNPV infection could induce autophagy in the BmN-SWU1 cells,which was derived from B.mori ovarian tissue.Transmission electron microscopy?TEM?observation revealed that there were a large number of double-membrane vesicles in BmNPV-infected cells,which was similar to autophagosome induced by starvation as the positive control.But there was almost no vesicle in normal cultured cells as the negative control.The formation of ATG8-GFP?autophagosome-specific membrane marker?green fluorescence dots were detected by immunofluorescence.The results showed that a considerable number of ATG8-GFP punctate accumulated in BmNPV infected cells in the early stage of infection?6h?,but decreased in the later stage of infection;statistical results showed that the percentage of cells containing green fluorescence punctates and the number of punctates per cell both increased significantly in the early stage of infection.Western blotting analysis showed that the transformation from ATG8 to ATG8-PE?membrane binding capacity of ATG8 protein?significantly enhanced in the early stage of viral infection.The ratio of ATG8-PE to Tubulin also showed the similar results.The presence of autophagosome does not indicate that it will eventually fuse with lysosomes to form a complete autophagic flux.Therefore,we constructed the ATG8-GFP-RFP autophagy tandem sensor to monitor the autophagic flux.The confocal microscope results of the infected cell showed that there are lots of red fluorescence dots without green dots in the same location?this is autolysosome?.While in the control the red fluorescence is completely coincident with the green.p62 is one of the most clearly studied selective autophagy receptor and its degradation is also a typical sign of autophagic flux.Through the detection and analysis of p62 protein,we found a significant degradation of p62 in the early stage of virus infection.The above results showed that BmNPV infection did induce the complete autophagy in the BmN-SWU1cells.2.The effect of autophagy-related genes on BmNPV infection in cellsAutophagy-related genes?Atgs?are a class of conserved genes that play a role in autophagy pathways.Through the prediction of the conservative domain of the protein encoded by 15 BmAtgs?Atg1-9,Atg11-14,Atg16 and Atg18?,we found all of them showed conserved domains and functions of their mammalian homologs.Quantitative detection showed that the expression of all autophagy-related genes fluctuated significantly in the process of viral infection.Most of them exhibited a tendency to increase at the early stage and then decrease,which was consistent with the trend of autophagy induced by virus infection.After overexpressing or knocking out of a single Atg gene before BV infection,cells were collected at different hours post infection.Real-time PCR detection showed that overexpression of a single Atg gene up-regulated the expression level of the ie-1 gene?a BmNPV immediate early gene?,while a single Atg gene knockout significantly reduced ie-1 gene expression;overexpression and knockout samples of Atgs gene were further partly selected to detect the expression level of vp39?a BmNPV late gene?and p10?a BmNPV very late gene?,and we found their expression showed a similar trend.The above results show that the change of a single autophagy related gene can affect the expression of the viral key genes.Then expression of downstream Atg8 and Atg12 genes was detected by qRT-PCR post overexpression of a single Atg gene.The result showed that overexpression of Atg gene could increase the expression level of downstream Atg8 and Atg12 genes.By selecting autophagy related genes that function at different stages of the autophagy pathway,combining with the significant impact of early results on virus infection and the effect on autophagy itself,we finally chose Atg7,Atg9 and Atg13 as the key genes for subsequent research.3.The effect of autophagy-related genes on the BmNPV infection in individualsIn order to further identify the effects of autophagy-related genes on virus infection,transgenic silkworm was obtained through transgenic technology and its efficiency of virus infection was analyzed subsequently.Firstly,we constructed the transgenic vectors.Through transgenic injection and screening,three transgenic overexpressing strains were successfully obtained,named BmAtg7-OE,BmAtg9-OE and BmAtg13-OE.qRT-PCR showed that transgenic lines could significantly overexpress the target genes.In addition,by inhering with the previously preserved Cas9-EGFP strain in our laboratory,a double light knockout strain was screened and obtained,named BmAtg13-KO.The genomic was extracted and sequenced in T-vector,indicating that the knock-out efficiency was up to 70%.Then the day 1 4^th-instar larvaes were fed with 10⁶ viruses.Mortality rate showed that transgenic overexpressed strains showed a significantly higher mortality contrasted with Dazao,while BmAtg13-KO knockout lines showed some resistance in a certain degree.The analysis of the viral genome copies and the expression of the key virus genes showed the same result as the mortality rate.The above results fully indicate that autophagy related genes significantly affect the process of virus infection,which is consistent with the previous results in cells.4.Researches on the mechanism of BmAtg13 affecting the efficiency of BmNPV infectionIn order to explore the molecular mechanism of autophagy related genes affecting virus infection,combined with previous experimental results,we selected Atg13 gene?an important member of ULK1 complex,an autophagic initiation complex?from many Atgs genes as the target gene for further researches.Firstly,we confirmed the conservatism of Atg13 gene by amino acid sequence alignment,conservative domain molecular model prediction and the phylogenetics analysis.Then through co-immunoprecipitation and mass spectrometry,we obtained many ATG13 interaction candidate proteins including host protein polyubiquitin,ATG7,Hsp70,lots of ribosome proteins and virus protein Orf5,Orf10 and Bro-B.The interaction between ATG13 and Bro-B,ATG13 and ATG7 was further confirmed by immunofluorescence co-localization and co-immunoprecipitation.Bro-B is a delayed early protein of baculovirus,and it owns nucleic acid binding activity and affect the synthesis of host proteins to benefit BmNPV infection.The interaction between ATG13 and Bro-B may involve in the mutual regulation of the host and the virus.ATG7 is a key modifier enzyme for the two ubiquitin conjugated systems,which is crucial to the formation and extension of autophagic membrane.The interaction of ATG13 and ATG7 may be related to the function of ATG13 as a member of the initiating complex of autophagy,which induces and initiates autophagy.We analyzed the interaction domain between Bro-B and ATG13 by truncation analysis.We found that the N-terminal HORMA domain of ATG13 can co-located with N-terminal of Bro-B,while the C-terminal disordered structure of ATG13 can be co-located with both N and C-terminal of Bro-B.Finally,we found that BmAtg13 was in the upstream of Bro-B by detecting the mutual regulation relationship between them.Overexpression of BmAtg13 inhibited the expression of Bro-B,and knockout of BmAtg13 promoted the expression of Bro-B.The above researches confirmed the interaction between BmAtg13 and virus infection,which provided a theoretical reference and direction for further research on the mechanism of autophagy and virus interaction.