Subcellular Localization of Bm4, the Protein Product of ORF4 of Bombyx Mori Nucleopolyhedrovirus

Abstract

Bm4, the protein product of open reading frame 4 (Bm4) of Bombyx mori nucleopolyhedrovirus (BmNPV), is a protein whose function is completely unknown. To unravel its biological function, its subcellular localization is a fi rst crucial step. In order to study the subcellular localization of Bm4 in the context of BmNPV infection, we used a recently developed BmNPV bacmid, which is infectious to Bm cell lines, to express Bm4-EGFP fusion protein. With EGFP fused to the 3’ terminus of Bm4 as the reporter gene and by using a confocal scanning microscope, we found that the green fl uorescent signal was localized primarily in the nucleus of Bm cells, however, a weaker signal was also observed in the membrane. Furthermore, bioinformatic analysis showed that there was no nuclear localization signal (NLS) or transmembrane helices (TMHs) in the amino acid sequence of Bm4, indicating that Bm4-EGFP fusion protein may be colocalized with host- or virus-encoded factors in the nucleus, and that Bm4 may have the ability to bind to certain protein located in the membrane. The western blot analysis using the antibody against EGFP detected only a specifi c band of about 67 kDa, which is consistent with the theoretical size of Bm4-EGFP fusion protein, confi rming that EGFP was correctly fused to Bm4. The localization of Bm4-EGFP fusion protein in the nucleus and membrane implies that Bm4 may have two different functions

Keywords

• Bac-to-Bac/BmNPV Baculovirus Expression System, EGFP, fusion protein, nuclear localization, membrane localization