| Bombyx mori bidensovirus(BmBDV)was originally classified in the Parvoviridae subfamily Densovirinae.However the International Committee on Taxonomy of Viruses has reclassified this virus into the new genus Bidensovirinea of family Bidnaviridae,due to its unique genomic structure.BmBDV is currently the only member of the Bidnaviridae.Bombyx mori bidensovirus can cause densucaryosis in silkworms,which has caused great economic harm to the silkworm breeding industry.At present,the genome structure and transcription mechanism of the virus have been comprehensively reported,but the replication mechanism of the virus,the functions of various non-structural proteins,and the 3D structure of the viral capsid are still unclear.Studies have shown that non-structural proteins play an important role in the infection and replication cycle of viruses.At present,only the function of the relatively conservative non-structural protein NS1 have been reported in detail,while there are few studies on NS2 and NS3.The study of the function of structural proteins is crucial for elucidating the replication mechanism of viruses.Therefore,this subject takes the non-structural protein NS2 of BmBDV as the research object,and conducts a preliminary exploration of the function of this protein from three aspects: the cellular localization of the protein,the effect on the promoter activity of each gene,and the analysis of the interacting proteins.Firstly,we used bioinformatics software to analyze analyzed all reported invertebrate ssDNA virus nonstructural protein NS2,including BmBDV,and established a phylogenetic tree.Phylogenetic analysis shows that the NS2 of invertebrate ssDNA viruses is close to the current classification in evolutionary branches.The viruses in the same genus are clustered in one branch,suggesting that NS2 may have more conserved functions in the same genus.While BmBDV NS2 is evolutionarily close to Hamaparvovirnae subfamily.The NS2 protein of BmBDV was localized in the nucleus of Bm N cells when expressed in vitro,and software analysis indicated that there was a potential binary nuclear localization signal domain at the Cterminal of the protein.At the same time,we also verified the NS2 protein localization of other insect ss DNV viruses such as JcDV at the cellular level,and found that they were all localized in the nucleus,but there was no classical NLS domain in the amino acid sequence.Therefore,we also analyzed the NLS of the NS2 of invertebrate ssDNA viruses.The results showed that the NS2 proteins of each virus may contain nonclassical NLS domains,and the sequence and location are relatively conserved within the same genus.Therefore,we speculate that the nuclear localization signal domain of NS2 protein in invertebrate ssDNA viruses may have a unique arrangement,so as to ensure the functional conservation of NS2 protein.Based on the predicted results,we constructed a recombinant BmBDV NS2 fusion e GFP expression vector(p Fast-ie1-NS2-e GFP),and confirmed the functionality of the potential NLS domain through the truncation mutation table of the predicted region of the nuclear localization signal.The key amino acids of NS2 were found by point/multipoint mutation,and the results showed that the C-terminus of BmBDV NS2 protein has a dichotomous NLS domain,and the combination rule of this NLS is:(R)KR(K)X10-12 K,X represents any amino acid,and one of the basic amino acids in brackets should be left.The NS2 protein of invertebrate ssDNA virus is conserved in nuclear localization,suggesting that this protein may play an important role in viral replication and transcription.In order to explore whether the NS2 protein has a regulatory role in the transcription of other genes of the virus,we constructed the promoter recombinant plasmids and NS2 protein overexpression plasmids for all genes in BmBDV.The luciferase activity of Bm N cells transfected with the promoter plasmid alone or cotransfected with the NS2 expression plasmid was analyzed by the luciferase expression system,and the effect of NS2 overexpression on the promoter activity of each gene of the virus was analyzed.Results showed that NS2 could promote the promoter activity of NS3 and inhibit the promoter activity of VP,and having no regulatory effect on the promoter activities of other genes.It is speculated that NS2 may inhibit the expression of VP and increase the expression of NS3 at the late stage of infection,and together with NS3,promote virus assembly and virus release.In order to screen proteins that may interact with NS2 in host cells,we used a baculovirus expression system to link BmBDV NS2 with His tag,and constructed recombinant Bacmid(Bacmid-Ie1-e GFP-p5.5-NS2-His).After staining the cells and injecting the silkworm,the recombinant baculovirus and the midgut of the diseased silkworm were obtained respectively.Using Co-IP and LC-MS/MS techniques to analyze the interacting proteins of NS2,we obtained 1088 specific proteins,more than half of which had more than two unique peptide matches.Specific proteins mainly include immune-related apolipoproteins and heat shock proteins;translation elongation factors that function in viral replication;aminopeptidase that digests the midgut of silkworm;Phospholipase A2 for viral entry into cells.At the same time,we also found proteins with nuclear transport function among the interacting proteins,suggesting that the further research on the function of NS2 can start from the interaction with nuclear transport proteins.In conclusion,we conducted a preliminary study on the function of BmBDV NS2.Studies have shown that NS2 is located in the nucleus and has a unique nuclear localization signal,which may play a role in viral gene replication;NS2 may have the function of regulating the activities of NS3 and VP promoters;at the same time,we preliminarily analyzed the interaction proteins of NS2 in silkworm midgut.These preliminary results of the NS2 protein function of BmBDV provide data support for further in-depth research,and also lay a theoretical foundation for a comprehensive analysis of the virus’ s replication mechanism. |
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