Identification Of Sumoylated Proteins In The Silkworm And Research On The Bm Npv Early Protein Pe38 With Its Interactive Protein Of Host, Bombyx Mori

As we all know, Post-Translational Modifications have controlled a series of biological functions such as the bioactivity and function of proteins, subcellular localization, protein stability, protein-protein interactions and so on, to a great extent. It could play an important role on elaborate regulation when the body subjected to exogenous stimulus, and then contribute to generate rapid and effective stress responses. There are many kinds of post-translational modifications which all have really critical affection on normal biological function of cellular proteins such as ubiquitination, phosphorylation, acetylation and methylation modification etc.SUMO(small ubiquitin like modifier) is a type of newfound ubiquitin-related molecule, ubiquitination and SUMOylation are analogous, SUMO and ubiquitin are exactly similar in three-dimensional structureeven if they share only about 18 % amino acid sequence homology. Thus, the SUMO molecule participates in post-translational modifications as well as ubiquitin. Previous studies have shown SUMOylation is a part of important regulatory mechanisms that modify proteins in the nucleus and regulate multiple cellular processes such as cell cycle control syst, nucleo-cytoplasmic signal transduction, stress responses, subcellular localization of proteins, protein-protein interactions, and transcriptional activity of transcription factors.Silkworm is the only large-scale economic insect in captivity up to present,Bombyx mori nuclear polyhedrosis virus(BmNPV) is one kind of serious pathogens of silkworm which always cause lots of loss to sericulture, which mostly parasitizes in the nucleus of blood cells and various tissues. Asan immediately early viral gene, pe38 is quite critical to virus infection. PE38 is present during the early phase of infection as a nuclear 38-kDa protein, detailed analysis of protein PE38 subcellular localization has revealed that it is transported predominantly to the nucleus, where it appears in a punctate pattern.PE38 contains a putative nuclear localization signal near the N terminus, a C3HC4 or RING finger in the central portion, and a leucine zipper near the C terminus.Recent researches have demonstrated the level of virus DNA synthesis would significantly reduce in the absence of pe38. Thus, pe38 has a direct effect on the growth and replication of Baculovirus in cell culture and on virulence in insects.In this study, a recombinant baculovirus was constructed to express an enhanced greenfluorescent protein(eGFP)-SUMO fusion protein along with BmUBC9 according Bac-to-Bac baculovirus expression system. SUMOylation substrates fromBombyx mori cells infected with thisB. mori nuclear polyhedrosis virus were isolated by co-immunoprecipitation and identified by LC-ESI-MS/MS. The results display a total of 64 SUMOylated proteins were identified, of which 59 proteins were functionally categorized to gene ontology(GO) terms. The resultant proteins were classified into cellular component, molecular function, and biological process categories, according to the GO hierarchy. Under the “biological process” category, the proteins were involved in “cellular process”(19.39 %), “metabolic process”(16.48 %), “single organism process”(9.2 %), and “biological regulation”(8.05 %). Under the “cellular component” category, the proteins were classified as “cell”(21.56 %), “cell part”(21.56 %), “organelle”(16.5 %) and “macromolecular complex”(14.37 %). Under the “molecular function” category, the identified proteins were mainly involved in “binding”(48.15 %) and “catalytic activity”(35.8 %). Besides, for the sake of confirming the facticity of SUMOylation substrates, four SUMOylated candidates were verified by co-immunoprecipitation in Drosophila schneide 2 cells.On the other hand, our previous studies have revealed the involvement of specific host midgut proteins(such as serine protease, catalase, translation elongation factor 2 etc.) in the interactions between B. mori and BmNPV using the Y2 H system. In this paper, using similar Y2 H assays we verified the interaction between BmNPV PE38 and Bm SRPK protein. Our carefully chosen controls have ruled out the possibility of false positive results through self-activation and toxicity tests. Furthermore, qRT-PCR was used to analyze the differential expression of Bm eIF4 Eand Bm srpk in BmN cells infected with BmNPV. The results revealed that the relative expression levels of Bm eIF4 E and Bm srpk were significantly higher from 12 to 24 h postinfection(hpi). However, both the relative expression levels of these two genes were notably down-regulated after 48 h. Thus, we can infer that Bm eIF4 E and Bm SRPK could be critical for the early viral infection by interacting with the immediate early protein, PE38.