A Preliminary Study On Bombyx Mori Nucleopolyhedrosis Virus Regulating The Host Through Ubiquitination And Causing Host Autophagy

Ubiquitination refers to the process that ubiquitin molecules classify intracellular proteins under the action of a series of special enzymes,select target protein molecules,and specifically modify the target protein.Ubiquitination is a post-translational modification of proteins that widely exists in eukaryotes and plays an important role in protein localization,metabolism,function,regulation and degradation.At the same time,it is also involved in the regulation of life processes including cell cycle,immune response,transcription,DNA repair,phagocytosis,autophagy and viral infection.Autophagy refers to the process of degrading abnormal proteins or organelles through lysosomes.It is an important way for eukaryotic cells to maintain homeostasis and is also tightly regulated by ubiquitination.Initial studies showed that autophagy was thought to be a cellular defense pathway that effectively degrades heterologous proteins produced by viral invasion.However,with continuous research,it is found that the virus can escape the degradation of the host’s autophagy pathway,and use the environment generated by the autophagy pathway to better complete its own replication and proliferation.BmNPV（Bombyx mori nucleopolyhedrovirus,BmNPV）is a large double-stranded DNA virus,which is one of the most important viral disease that causes the production reduction of the sericulture industry,and baculovirus is also an important foreign protein expression tool and potential biopesticides.Recent studies have shown that BmNPV is one of the few viruses that can encode its own ubiquitin gene,and ubiquitination plays an important role in the replication and proliferation of BmNPV,but the target and specific molecular mechanism of ubiquitination in the replication and proliferation of BmNPV are still unknown.This study will analyze the ubiquitination target proteins of the BmNPV ubiquitin gene,so as to improve the understanding of the interaction between BmNPV and the host.The main findings are as follows:（1）The ubiquitin gene in the BmBacmid bacmid of the wild-type BmNPV T3 strain was deleted using theλRed recombination system,and the ubiquitin-depleted bacmid BmT3DH10BΔubi-p MON7214 was constructed.Then through the Bac-to-Bac system,the viral ubiquitin gene carrying the his/myc tag was backfilled into BmT3DH10BΔubi-p MON7214 to obtain BmBacmid^{v Ubi-myc/his}.After two amplifications,a higher titer recombinant virus BmNPVv Ubi was obtained-myc/his.BmN cells were infected with this recombinant virus,and Western Blot detection showed that the constructed recombinant virus could express recombinant ubiquitin with his/myc tag,and it was found that it mainly existed in the form of mono-ubiquitination and di-ubiquitination in cells.（2）Bombyx mori was infected with the constructed BmNPV^{v Ubi-myc/his} recombinant virus,the infected whole silkworm samples were collected and lysed,and the target protein ubiquitinated by virus ubiquitin was obtained by purification under denaturing conditions using a nickel column.Through liquid chromatography-tandem mass spectrometry（LC-MS/MS）identification,it was found that a variety of silkworm or viral proteins were modified by viral ubiquitin.According to the GO/KEGG analysis,the host proteins regulated by viral ubiquitination are mainly related to the metabolic process,and the components and cell membrane components play the functions of binding and catalysis.Functionally,it is closely related to some sugar metabolism,cell movement,exogenous biodegradation metabolism,immune system,cytoskeleton regulation,mitophagy,apoptosis,etc.（3）BmN cells were infected with BmNPV^{v Ubi-myc/his} recombinant virus,viral ubiquitination interacting proteins were obtained by myc monoclonal antibody co-immunoprecipitation（Co-IP）,and viral polyhedra were identified by LC-MS/MS.Protein polyhedra,capsid protein VP80,and silkworm HSP/HSC proteins were abundantly present in samples after BmNPV infection.It is speculated that BmNPV infection may be related to the selective autophagy of the host.（4）BmNPV infection of BmN can cause the transformation of host LC3 protein from type I to type II and the degradation of p62 protein,indicating that BmNPV can induce autophagy in the host.Autophagic flux was inhibited after the addition of the autophagy-specific inhibitor Bafilomycin A1.Inhibition of autophagy did not affect BmNPV genome replication,but significantly reduced viral polyhedrin expression.It is suggested that the host autophagy pathway may be hijacked by BmNPV to facilitate the production of viral polyhedra.