Effects Of ESCRT And UPP-related Genes On Budded Virions Release Of Bombyx Mori Nucleopolyhedrovirus

Bombyx mori nucleopolyhedrovirus(Bm NPV)is a common double-stranded circular DNA envelope virus,it that can cause Bombys mori blood type of purulence disease which is the most harmful disease in sericulture production,and the outbreak of the disease seriously affects the efficiency and sustainability of sericulture production,so how to effectively prevent and control Bm NPV is an urgent production problem to be solved in the development process of the sericulture industry.In the process of infection,Bm NPV produce two kinds of virions during the infection cycle,in which occlusion-derived virion(ODV)mediates the spread of the virus between individuals,while budded virion(BV)mediates the systemic infection of the virus in Bombyx mori,which eventually leads to the death of Bombyx mori.The replication cycle of the virus includes multiple stages such as adsorption,penetration,decorating,biosynthesis,assembly,and release,and the blockade of any process can affect the infection and spread of Bm NPV to a certain extent,so elucidating the mechanism of BV release can provide a reference for prevention and control of the disease.Previous studies found that GP64 deficient in the N region of Bm NPV signal peptide is located in the cytoplasm,but it still generates infectious BV.Electron microscopy results showed that the multivesicular body(MVB)contains nucleocapsids with an envelope.After treatment with exosome inhibitor U18666 A,the release of progeny BV in infected cells decreased significantly,so it was preliminarily confirmed that Bm NPV has two kinds of envelope modes: directly egress from the plasma membrane and exosome transport after enveloping from MVB.This thesis further confirmed the existence of MVB envelope budding mode by studying the endosomal sorting complex required for transport(ESCRT)pathway and the ubiquitin-proteasome pathway(UPP),which are closely related to MVB and exosome formation and the role of these two pathways in the budding process is preliminarily explored.This thesis firstly examined vacuolar protein sorting 4(VPS4),vacuolar protein sorting 24(VPS24),VPS20 associated factor 1(VTA1),and sucrose nonfermentation protein 7(SNF7)in the ESCRT pathway,the results showed that the transcription levels of Vps4,Vps24,Vta1,and Snf7 increased significantly or were significantly downregulated at first,and then significantly upregulated with significant fluctuations,and then targeted by RNA interference to downregulate the expression levels of these genes in Bm N cells,which significantly reduced the yield of infectious BV.In addition,the study found that the gene expression phase of proteasome 26 S subunit non-ATPase 14(PSMD14),polyubiquitin,proteasome 20 S subunit alpha 6(PSMA6),and proteasome zeta subunit(PSMZ)in the UPP pathway were consistent with ESCRT,and downregulation of the expression of these genes also significantly reduced BV production.Further analysis showed that the interference of ESCRT pathway and UPP pathway did not affect the total expression of GP64,but significantly reduced the distribution of GP64 protein on the cell surface,and GP64 could not be correctly sorted and localized to the envelope site,resulting in a significant increase in the number of BV trapped in the cell,and the released viral amount and infectivity were also reduced.In this study,the effects of ESCRT and UPP pathway-related genes on Bm NPV BV release were revealed,and the results improved and developed the mechanism of envelop virus budding,which provided the theoretical reference for subsequent prevention and control research in the direction of budding and also contributed to an in-depth understanding of the transport mechanism of biomacromolecules.