Screening of candidate proteins interacting with IE-2 of Bombyx mori nucleopolyhedrovirus

IE-2 of Bombyx mori nucleopolyhedrovirus (BmNPV) has been shown to play important roles in baculovirus infection, which are involved in gene expression and viral replication. However, the mechanism remains unknown. In this paper, by TargetP software, four genes, i.e.-2, odv-e26, odv-e56 and BmNPV-gp101 (Ac-orf116) of BmNPV and Autographa californica multiple NPV (AcMNPV) were predicted to be located in mitochondria. By BLAST tool using BmNPV IE-2 protein sequence, 14 NPVs were found to have IE-2 homologues in GenBank, and most of them were predicted to be located in mitochondria, except for that of Antheraea pernyi NPV (AnpeNPV) and Anticarsia gemmatalis NPV (AngeNPV). To observe the subcellular localization of BmNPV IE-2, a recombinant virus overexpressed the IE-2 and eGFP fusion protein was constructed. In infected BmN cells, the fluorescence specifically enriched in the cellular mitochondria. This evidence was accordant with the prediction. Further, Pull-down assay was used to select protein candidates interacting with IE-2 in B. mori cells infected with BmNPV. Of several isolated protein components, sixteen candidates were identified by MALDI-TOF mass-spectrometry, eight baculoviral proteins (ALK-EXO, F protein, IAP-1, LEF-3, LEF-9, ODV-NC42, TLP, and VP39), and eight proteins from B. mori (Actin, ADP/ATP translocase, ATP synthase subunit beta, Beta-tubulin, DNA topoisomerase 2, Histone H4, Soluble guanylyl cyclae alpha-1 subunit, Transketolase). From the functional point of view, most of these proteins were generally divided into two groups, mitochondrial interaction proteins and viral DNA replication proteins. These results implied that the IE-2 had multiple functions involved in regulating viral gene expression, viral replication and also as a component of mitochondrial factors to regulate the cellular energy supply and apoptosis.