Analysis Of The Expression And The Interaction Proteins Of The Bombyx Mori Bidensovirus NS1

Bombyx mori bidensovirus(BmBDV)belongs to the new genus Bidensovirus in the new family Bidnaviridae,which is the unique single-stranded bipartite DNA virus encoding DNA polymerase.It had been belonged to Densovirinae in Parvoviridae.Similar to densovirus,BmBDV is a icosahedral no-enveloped virus which mainly infect the midgut columnar epithelial cells of the silkworm and cause the fatal densonucleosis.The BmBDV genome consists of two segments of the single stranded DNA molecule VD1(6.6 kb)and VD2(6.0 kb)in separate caspids.BmBDV NS1 is encoded by VD1-ORF2 with 316 amino acid residues.Its theoretical molecular weight is about 36 KDa and the isoelectric point is 8.70.The bioinformatics results indicate that the BmBDV NS1 is homologous to the parvovirus NS1.The previous study showed that Bm BDV NS1 protein is a multifunctional protein which pocesses the ATPase,helicase and specific DNA sequence binding activity.However,its expression pattern the interaction with the host or viral proteins have not been elucidated yet.In this study,12 peptide antigens which have12-amino acid were designed for the preparation of the BmBDV NS1 monoclonal antibodies.We finally obtained a monoclonal anti-NS1 antibody with high specificity and titer,which can specifically recognize the NS1 expressed in E.coli and the NS1 in the midgut of BmBDV-infected silkworm.The epitope of the antibodies is DIPPEEYRELRT.Analysis of expression indicated that ns1 gene was expressed at the late stage of viral infection.The transcript of ns1 gene is about 1.1 kb,which is detected from 48 hours post infection(hpi),and its abundance reached the peak at 72 hpi and then decreased at 96 hpi.The NS1 protein was detected from 72 hpi until 120 hpi,and the expression level of the NS1 is gradually increased.Besides the NS1 with expected size,there is a expression product with greater molecular weight which may be the phosphorylated NS1.Finally,we isolated and purified the interacting protein complex associated with NS1 in the midgut tissue of virus-infected silkworm using the anti-NS1 monoclonalantibody via co-immunoprecipitation.The results of LC-MS/MS suggested that 23 host factors were related to the interaction with NS1.Most of them are cytoskeletal proteins involved in maintaining the cell structure and morphology.Combining with that NS1 expressed at the late stage of virus infection,it is possible that NS1 plays a role in regulating the cell degeneration and lysis for release of progeny virus.Some of these interacting proteins are related to metabolism of substance and energy,substance transport,translation extension,protein degradation,immune and other biological process,indicating that NS1 could interact with relevant proteins regulating the corresponding biological process during the virus infection.