In order to explore the mechanism of orf126 of Bombyx mori nucleopolyhedro virus, the subcellular localization of ORF126 was conducted. The egfp gene was fused with the C-terminal of orf126 genes, BmN cells were transfected with different plasmid DNA and the superinfection were performed at 12 h post transfection. The fluorescence was examined by confocal laser scanning microscopy at different time point after transfection. The results show that EGFP protein was uniformly present throughout the cytoplasm and nucleus either in expression alone or superinfection, however, the fluorescence of EGFP linked to ORF126s were present barely in the cytoplasm.Key words: BmNPV, orf126, transient expression, subcellular localization

Bifang, H

Meng, W

XiaoFang, G

Xingjia, S

English

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African Journal of Biotechnology Vol. 10(44), pp. 8877-8880, 15 August, 2011     Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.1213 ISSN 1684–5315 © 2011 Academic Journals     Full Length Research Paper  Subcelluar localization of orf126 of Bombyx mori nucleopolyhedrovirus  HAO Bifang1*, GUO XiaoFang2, WANG Meng1 and SHEN Xingjia1  1Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang Jiangsu 212018, China. 2School of Computer Science and Engineering, Jiangsu University of Science and Technology, Zhenjiang Jiangsu 212003, China.  Accepted 1 July, 2011  In order to explore the mechanism of orf126 of Bombyx mori nucleopolyhedro virus, the subcellular localization of ORF126 was conducted. The egfp gene was fused with the C-terminal of orf126 genes, BmN cells were transfected with different plasmid DNA and the superinfection were performed at 12 h post transfection. The fluorescence was examined by confocal laser scanning microscopy at different time point after transfection. The results show that EGFP protein was uniformly present throughout the cytoplasm and nucleus either in expression alone or superinfection, however, the fluorescence of EGFP linked to ORF126s were present barely in the cytoplasm.  Key words: BmNPV, orf126, transient expression, subcellular localization.   INTRODUCTION  Baculoviruses are double-stranded DNA viruses that are pathogenic for invertebrates, particularly insects of the order Lepidoptera. Bombyx mori nucleopolyhedrovirus (BmNPV), a member of baculovirus, is an important pathogen of silkworm, which results in about 5 to 15% lost in the sericulturer in China. A number of genes’ function have been depicted since its genomics sequence was reported (Gomi et al., 1999); these results rebound to keep abreast the virus life cycle and to prevent the occurrence of the disease. Our previous research revealed that BmNPV orf126 was not a necessary gene for virus replication (Hao et al., 2009) and two subtypes (SX126 and GD126) were found in seven wild isolates collected from different provinces of China, which exhibited different function in the virus replication cycle; GD126 can enhance the polyhedrin expression signifi-cantly, but its mechanism still remain unclear, thus further investigation is required for exploring the mechanism of orf126. Baculovirus gene expression is regulated mainly at the transcriptional level, and involves sequential expression of early, late and very late genes. The function of early genes is the initiation of infection, and they are required to accelerate replication events  and  to  prepare    *Corresponding author. E-mail: bifanghao@gmail.com the host cell for virus multiplication (Friesen, 1997). Orf126 is an early gene, whose product was detected from 6 h post infection (Hao et al., 2009), thus Orf126 was supposed to be involved in the replication of virus or interacted with host cell. Comprehensive knowledge of the location of proteins within the cellular micro-environments is critical for understanding their functions, and the location will provide the necessary information for the interaction of protein and the evolution. However, the location of ORF126 exerting its function which is required for the functional assay of ORF126 in the host cell is still unclear.  Together with various experimental approaches to determine the subcellular localization of proteins, many powerful computational tools have been developed for predicting the subcellular localization of proteins in various organisms based on the sequence information alone (Chou and Shen, 2008; Chou and Elrod 1999; Chou et al., 2011; Chou, 2001; Jiang et al., 2008; Li et al., 2008; Zeng et al., 2009) as well as a long list of references cited in a comprehensive review (Chou and Shen, 2007). Among the aforementioned predictors, the predictor iLoc-Euk (Chou et al., 2011), some predictors in the Cell-PLoc package (Chou and Shen, 2008) and all the predictors in the Cell-PLoc 2.0 package (Chou and Shen, 2010) can be used for not only the single-location or ‘‘singleplex’’ proteins but  also  the  multiple-location  or  8878        Afr. J. Biotechnol.      Figure 1. Procedure of the construction of transfection vector.    ‘‘multiplex’’ proteins that may simultaneously reside at, or move between two or more different subcellular locations. Proteins with multiple location sites or dynamic feature of this kind are particularly interesting because they may have some unique biological functions (Glory and Murphy., 2007 ). Particularly, as pointed out by Millar et al. (2009), recent evidences have indicated that an increasing number of proteins have multiple locations in the cell. EGFP was an efficient molecular probe; it was applied widely in the localization analysis through its fusion with other proteins (Cheng et al., 2009; Huang et al., 2008; Petry et al., 2006). In this study, the localization of ORF126 was explored by the EGFP probe, the location was examined through the scanning of EGFP in the BmN cell by laser confocal microscope, and the results provided the subcelluar location of ORF126 where it exerted its function.    MATERIALS AND METHODS  Insect cell line and virus   BmN cells were maintained at 28°C in TC-100 insect medium (PAA Company) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL). The wild-type (wt) BmNPV strains used in the experiments were originally isolated from the cadavers of B. mori larvae collected in Guangdong, Shaanxi provinces of China and were designated BmNPV-GD and BmNPV-SX, respectively, and vBmBacJS13-ph was constructed by Huang et al. (2007).   Construction of the transient expression vector   The plasmids containing the different subtypes of orf126-egfp fusion gene were constructed to study the localization of ORF126 in BmN cells (Figure 1). A fragment containing the egfp gene was digested from pEGFP-N1 (Clontech) with EcoRI and NotI, and subcloned into pIZ/V5-His (Invitrogen) to generate pIZ/V5-egfp. The SX126 or GD126 sequence without the stop codon (TAA) was amplified by PCR from viral DNAs which were extracted from purified OBs of isolates BmNPV-GD and BmNPV-SX as described by Sun and Zhang (1994) using the primer pair 126SF (5’-GCGAAGCTTA TGTTCAAACCCAACAT GATTAGA-3’; HindIII site underlined) and 126SR (5’-GCGGGATCCGCATAAATTTTATTTTTATTTTTGTTTT GGTT-3’; BamHI site underlined). The PCR products were purified and cloned into the pIZ/V5-egfp to generate pIZ/V5-SX126-EGFP or pIZ/V5-GD126-EGFP.   Localization analysis of ORF126   Plasmid pIZ/V5-SX126-EGFP or pIZ/V5-GD126-EGFP was tran-sfected  into  BmN  cells  with  lipofectin  (Invitrogen)  following  the manufacturer’s   protocol.   Plasmid   pIZ/V5-egfp   was  used  as  a        Figure 2. Localization of the SX126 and GD126 EGFP-fusion proteins in BmN cells with or without virus infection. BmN cells were transfected with the control plasmid pIZ/V5-egfp (a and b), the plasmid pIZ/V5-SX126-EGFP (c and d) or with pIZ/V5-GD126-EGFP (e and f). BmN cells (b, d and f) were infected with BmBacJS13-ph at 12 h p.t., the nuclear DNAs of the cells were stained with Hoechst and were examined by confocal laser scanning microscopy for fluorescence at 60 h p.t. All magnification and exposure times were identical and bars = 10 µm.    negative control. Superinfection was conducted with vBmBacJS13-ph (MOI of 10 TCID50 units per cell) at 12 h post-transfection (p.t.). The sample was observed at a time interval of 12 h. The cell samples for confocal examination were fixed by paraformaldehyde of 4% for 15 min, rinsed with PBS, stained with Hoechst (Beyotime) dye for 5 min, washed with PBS for two times and observed under a confocal laser-scanning microscope (Leica).   RESULTS AND DISCUSSION  The transient expression vector was identified by double digestion of restriction endonuclease analysis (data not shown),  then   the   correct   plasmids   were   applied  to  Hao et al.         8879    transfect BmN cell, and superinfection was conducted at 12 h p.t. The fluorescence was examined by confocal laser-scanning microscopy at 60 h p.t. The results show that both the intracellular localization of SX126 and GD126 ORF126 were localized in the cytoplasm of transfected cells through transient expression, and fluorescence congregation was also observed in the cytoplasm. As shown in Figure 2, the fusion proteins of SX126 or GD126 were located exclusively in the cytoplasm of transfected cells (Figure 2c and e). When BmN cells were superinfected with vBmBacJS13-ph, fluorescence remained in the cytoplasm of infected cells (Figure 2d and f), hereby, their location was not affected by other factors of the virus. However, in the negative controls, EGFP showed homogeneous fluorescence in the cytoplasm and nucleus when expressed alone (Figure 2a) or when superinfected with vBmBacJS13-ph (Figure 2b). Therefore, ORF126 is likely to be localized in the cytoplasm of the host cells. The bioinformatics analysis showed that ORF126 has a endoplasmic reticulum location signal (KNKNKIY) in the C-terminal, which was not in the Ac150, however, the truncated ORF126 localization was not changed (data not shown), so ORF126 might contain other cytoplasm location sequence, which will be distinguished by further investigation. Though there are small different amino acid difference between SX126 and GD126, their phenotype is completely different, but the similar subcellular location indicated that they were not involved in the virus replication cycle since they did not enter the nuclear. Combined with the cytopathic effect of the cell infected with GD126-contained virus (cell lysed time or the ST50 of the infected larvae), ORF126 may interact with the host cell factor in the cytoplasm, but further investigation is needed.   REFERENCES  Cheng F, Mclaughlin PJ, Verderame MF, Zagon IS (2009). Dependence on nuclear localization signals of the opioid growth factor receptor in the regulation of cell prolification. Exp. Biol. Med. 234(5): 532-541. Chou KC, Wu ZC, Xiao X (2011). iLoc-Euk: A Multi-Label Classifier for Predicting the Subcellular Localization Singleplex Multiplex Eukaryotic Proteins. PLoS One. 6: e18258. Chou KC (2001). Prediction of protein cellular attributes using pseudo amino acid composition. Proteins: Structure, Function,  Gen. (43):  246-255. Chou KC,  Shen HB (2010). Cell-PLoc 2.0: An improved package of web-servers for predicting subcellular localization of proteins in various organisms. Natural Sci (2): 1090-1103. Chou KC, Elrod DW (1999). 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Subcelluar localization of orf126 of Bombyx mori nucleopolyhedrovirus

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Subcelluar localization of orf126 of Bombyx mori nucleopolyhedrovirus

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