Yan WU, Bing LI, Wu-Jie SU, Wei-De SHEN, Guang GAO, Wen-Bing WANG

Subcellular localization of Bm4, the protein product of ORF4 of Bombyx mori nucleopolyhedrovirus

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.

PDF

___

[1] Wang D, Zhang CX. 2006. HearSNPV orf83 encodes a late, nonstructural protein with an active chitin-binding domain. Virus research. 117: 237-243.
[2] Katsuma S, Deng XT, Zhou LE, Iwanaga M, Noguchi Y, Kobayashi M, Maeda S. 2000. Identifi cation of Novel Residues Involved in Nuclear Localization of a Baculovirus Polyhedrin Protein. VIRUS GENES. 21(3): 233-240.
[3] Wang F, Zhang CX, Kumar VS, Wu XF. 2005. Infl uences of chitinase gene deletion from BmNPV on the cell lysis and host liquefaction. Archives of virology. 150: 981-990.
[4] Gomi S, Majima K, Maeda S. 1999. Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus. Journal of general virology. 80: 1323-1337.
[5] Iwanaga M, Takaya K, Katsuma S, Ote M, Tanaka S, Kamita SG, Kang WK, Shimada T, Kobayashi M. 2004. Expression profi ling of baculovirus genes in permissive and nonpermissive cell lines. Biochemical and biophysical research. 323: 599–614.
[6] Motohashi T, Shimojima T, Fukagawa T, Maenaka K, Park EY. 2005. Effi cient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system. Biochemical and biophysical research. 326: 564–569.
[7] Miao YG, Zhang YS, Nakagaki K, Zhao TF, Zhao AC, Meng Y, Nakagaki M, Park EY, Maenaka K. 2006. Expression of spider fl agelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system. Applied microbiology and biotechnology. 71: 192–199.
[8] Yue WF, Miao YG, Li XH, Wu XF, Zhao AC and Nakagaki M. 2006. Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac/BmNPV Baculovirus expression system. Applied microbiology and biotechnology. 73: 181–186.
[9] Dardalhon V, Noraz N, Boyer M, Bakker AQ, Pollok K, Rebouissou C, Spits E, Taylor N. 1999. Green Fluorescent Protein as a Selectable Marker of Fibronectin- Facilitated Retroviral Gene Transfer in Primary Human T Lymphocytes. Human Gene Therapy. 10(1): 5-14.
[10] Belyavskyi M, Braunagel SC, Summers MD. 1998. The structural protein ODV-EC27 of Autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin. Proc. Natl. Acad. Sci. USA. 95: 11205-11210.
[11] Takramah D, Seiffert BM, Schaller S, Vigneron M, Häcker G. 2003. Baculovirus P35 interacts with a subunit of human RNA polymerase II and can enhance promoter activity in human cells. Journal of general virology. 84:3011-3019.
[12] Vanarsdall AL, Okano K, Rohrmann GF. 2006. Characterization of the Role of Very Late Expression Factor 1 in Baculovirus Capsid Structure and DNA Processing. Journal of virology. 80(4): 1724-733.1