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Processing of Infectious Bursal Disease Virus (IBDV) pVP2 Promotes SelfAssembly of IBDV-like Particles in Hi-5 Cells Jin-Yi Ho, Meng-Shiou Lee1, Shyue-Ru Doong4, Min-Ying Wang1 and Su-Yuan Lai3* 1 Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan 40227 ROC 3 4 Department of Food Science, Chungtai Institute of Health Sciences and Technology, Taichung, Taiwan, 40605 ROC Department of Chemical Engineering, National Central University, Taichung, Taiwan, ROC *To whom correspondence should be addressed Mailing Address: Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 40227 Taiwan Telephone: 886-4-285-6697 Fax: 886-4-285-6697, 886-4-285-3527 E-mail address: [email protected] Running title: Production of IBDV-like Particle. Abstract 1 The capsid of infectious bursal disease virus (IBDV) with a size of 60-65 nm is formed by the processing of a large polyprotein (pVP2-VP4-VP3) and subsequent assemblage of pVP2/VP2 and VP3. When polyprotein was expressed in Sf9 insect cells, the processing of polyprotein was restrained, leading to a limited production of capsid, i.e., IBDV-like particles. In the present study, another insect cell line, High-FiveTM (Hi-5) cells, was adopted for the expression of IBDV polyprotein. In this system, polyprotein was processed to VP3 protein and the matured form of VP2 protein. Consequently, significant amounts of IBDV-like particles and smaller subviral particles were produced, suggesting that the maturation of VP2 played a key role in capsid assemblage. The size and morphology of subviral particles were similar to T=1 icosahedral particles formed by VP2 protein, suggesting that when there was not enough VP3 protein for the formation of IBDV-like particles, the excessive VP2 protein formed subviral particles. Furthermore, in the present study, an efficient purification process was also established to obtain highly pure IBDV-like particles. These particles can facilitate research for the understanding of IBDV structural biology by determining its high-order structure, as well as the development of vaccines or diagnostic kits. Key words: infectious bursal disease virus; capsid assembly; protease degradation; baculovirus expression system. INTRODUCTION 2 Infectious bursal disease virus (IBDV), a small, non-enveloped virus with a diameter of about 60 nm, can cause a highly contagious disease in young chickens (recently reviewed by (Müller et al., 2003; Nagarajanand Kibenge, 1997). This virus belongs to family Birnaviridae and is .characterized by a bisegmented dsRNA genome, in which the two segments are linked by VP1 protein, an RNA-dependent RNA polymerase (Nagarajanand Kibenge, 1997; von Einem et al., 2004). An open reading frame in the larger segment encodes a 115 kDa polyprotein, generally having 1012 amino acids (aa). This polyprotein can be digested to pVP2 (1-512 aa), VP4 (513-791 aa), and VP3 (792-1012 aa) proteins by an autoproteolysis of VP4 ((Azad et al., 1987; Lejal et al., 2000; Sanchezand Rodriguez, 1999), prior to the beginning of viral assembly. Encapsidation is presumably followed by the interaction of pVP2 and VP3, while VP3 can also interact with VP1 and the viral dsRNA (Maraver et al., 2003; Maraver et al., 2003; Ona et al., 2004; Tacken et al., 2000). Then, precursor pVP2 undergoes a second proteolytic step leading to the formation of a smaller matured product, VP2 (1-441 aa) protein (Chevalier et al., 2002; Da Costa et al., 2002; Kibenge et al., 1988; Nagarajanand Kibenge, 1997). VP2 and VP3 proteins, the major components of the mature virus, respectively, form the outer and inner capsid of the virus, respectively (Bottcher et al., 1997; Caston et al., 2001). The structure of the virus is based on a T = 13 lattice and the capsid subunits are predominantly trimer clustered. Recently, a better understanding of the structural and molecular biology of IBDV and its assemble pathway has been obtained through studies of recombinant IBDV polyprotein expressed using either vaccina or baculovirus expression systems (Chevalier et al., 2002; Muller et al., 2003). In the baculovirus expression system, an inefficient assembly of IBDVlike icosahedral capsids was reported (Dybingand Jackwood, 1997; Kibenge et al., 1999; Martinez-Torrecuadrada et al., 2000). The reason was attributed to that (i) pVP2 was not 3 efficiently processed to VP2 and (ii) VP3 was proteolytically cleavaged and resulted in a VP3 mutant lacking its C-terminal 13 amino acid residues (Maraver et al., 2003; Maraver et al., 2003). Very recently, the last C-terminal residue of VP3, i.e., glutamic acid 257, has been proposed to control viral capsid assembly in Sf9 cells (Chevalier et al., 2004). Therefore, researchers have either used methods such as fusion of green fluorescent protein (GFP) to the C-terminal domain of polyprotein pVP2-VP4-VP3 to promote the processing of pVP2 (Chevalier et al., 2004; Chevalier et al., 2002) or the coexpression of VP1 with polyprotein to prevent the degradation of VP3 and enhance the assembly of IBDV in Sf9 cells (Lombardo et al., 1999). We have demonstrated that an insect cell line, High-FiveTM (Hi-5) cells, has the capability to process pVP2 in the absence of VP4 (Lee et al., 2004). In the present work, we studied IBDV polyprotein processing in Hi-5 cells and demonstrated that this polyprotein was efficiently processed to yield VP2 and VP3 proteins in the infected Hi-5 cells. Consequently, significant amount of IBDV-like particles was produced, proving that Hi-5 cells are a better host for the expression of IBDV polyprotein than Sf9. Additionally, other heterogonous morphologies, such as type I and II tubules and some smaller icosahedral particles (refered as subviral particles later) with a diameter of, approximately, 20 nm, have been observed. Among these structures, IBDV-like and subviral particles were found extracellularly and in a large of quantity, a phenomenon not reported previously. Finally, the highly pure IBDV-like and subviral particles were separated to address their composition and the origin and biological significance of subviral particles are also discussed. MATERIALS AND METHODS 4 Viruses and Cells. The recombinant proteins expressed by the three recombinant baculoviruses used in this study are given in Figure 1. Two of these baculoviruses, vIBDVJ6 expressing IBDV polyprotein pVP2-VP4-VP3 and vP3009VPX expressing pVP2 protein, were described previously (Ho et al., 1999; Lee et al., 2004). The construction and generation of the third baculorvirus, vP3009mVP2, expressing a matured form of VP2 protein (mVP2, residues 1-441), will be published elsewhere. Two insect cell lines, including Sf9 cells and High-fiveTM (Hi-5) cells (Invitrogen, ) were routinely cultured and passaged as described previously (Wang et al., 2000). All recombinant viruses were propagated in Spodoptera frugiperda (Sf9) cells to generate viral stocks for protein expression. End-point dilution method was chosen to determine the titer of a recombinant virus in the viral stock. Total insect cell counts and viability were determined as described by Wang and Doong (Wangand Doong, 2000). Expression of recombinant proteins. Sf9 or Hi-5 cells were cultured in shaking flasks at 28 o C (O’Reilly et al., 1992) and were infected with a recombinant baculovirus at a multiplicity of infection (MOI) of 10 for the protein expression. The infected cells were daily collected postinfection and the cell broths were subjected to centrifugation. The resulting pellet and supernatant were separately examined for the expression pattern of either polyprotein or pVP2 using Western-blotting analysis. Production of IBDV-lik particles. For the production of recombinant IBDV-like particles, Hi-5 cells were seeded at a density of 5x105 viable cells/ml in 500 ml growth media, ESF 921, (expression system, CA) in a 500 ml spinner flask (Bellco Glass, Inc., Vineland, NJ), and incubated at 27 0C on a magnetic stirrer at a speed of 150 rpm. A porous HPLC solvent sparger (Hitachi Ltd., Tokyo, Japan) connected to no. 16 silicon tubing (MasterFLex(R), Cole- 5 Parmer, Niles, IL) was used to provide air bubbles to enhance the oxygen transfer rate. Air was introduced with a pump and the volumetric air flow was set at, approximately, 67 ml/min. Under this circumstance, oxygen supply is adequate for the high level of protein production (Wangand Doong, 2000). The virus was directly added to spinner flask cultures at an MOI of 0.1, 1, or 10 when cell density reached 1x106 viable cells/ml and the infected insect cells were harvested when the viability dropped below 60%. Purification of IBDV-like Particles and Subviral Particles Using Ultracentrifugation. The harvested Sf9 or Hi-5 cell broths were centrifuged at 5000x g for 20 minutes. The resulting cell pellets were resuspended in 5 ml of TNE buffer (10mM Tris, 100mM NaCl, 1mM EDTA, pH7.6) and, then, sonicated on ice three times for 10 seconds with a 30% pulsed duty cycle (Vibra cell, Sonics & Materials, Inc., Newtown, CT). Insoluble cell debris was removed by centrifugation at 11160xg for 35 minutes and the supernatant was loaded on a 25% (w/v) sucrose solution and subjected to centrifuging at 35,000 rpm and 4 0C for 3 hours in an SW41 Ti rotor (Beckman Instruments, Inc., Fullerton, CA). After sucrose cushion, the pellet was resuspended in 1 ml of TNE buffer, and then subjected to 20~40% cesium chloride (CsCl) gradient centrifugation for 24 hrs at 35,000 rpm and 4 0C. The solution in the centrifuge tube was collected at 0.5 ml/fraction and each fraction, verified by buoyant density measurement, was analyzed using either Western blot or EM for morphology observation. To purify extracellular IBDV-like and subviral particles, the culture supernatant was first microfiltered through a 0.45 µm cellulose acetate membrane (Millipore, Rochester, NY, SteritopTM). Then, both a hollow fiber apparatus (A/G Technology Corporation, Needham, MA) and 10 kDa cut-off membrane (Millipore, Rochester, NY) were used to concentrate the filtrate. To minimize extra proteolytic degradation, protease inhibitor (Rocher) was added to 6 the filtrate. After concentration, the particles were purified by ultracentrifugation as described above. Following ultracentrifugation, gel-filtration chromatography was further performed to separate IBDV-like particles from subviral particles. Separation of IBDV-like Particles with Size Exclusion Chromatography. After ultracentrifugation, the fractions having a mixed population of particles were pooled and concentrated until concentrations between 0.4 and 3.5 mg/ml as determined by a BCA kit. Then aliquots of samples (20 to 100-µl) were injected to an analytical high-pressure liquid chromatographic (HPLC) column (14-ml volume; TSK-gel G5000PWXL: inner diameter 7.8 cm, length 30 cm; TosoHaas) connected to a FPLC system (Beckman, Piscataway, NJ, USA). The column was packed with methacrylate resins (Tosoh Corporation, Tokyo, JAPAN), which are routinely applied to separate globular proteins with molecular weights ranging from 4 to 1x104 kDa. A phosphate-buffered saline (3 mM Na2HPO4, 1 mM KH2PO4, 150 mM NaCl, [pH 7.5]), at a flow rate of 0.1 ml/min, was employed as a running buffer. Following chromatography, fractions were collected and the reactivity of each fraction was measured using enzyme-linked immunosorbent assay (ELISA) (Cheng et al., 2001). Fractions with a strong ELISA response were analyzed by SDS-PAGE or Western blot, and concentrated for electron microscopic (EM) observation (Ho et al., 1999). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described previously (Cheng et al., 2001) with slight modification. Briefly, samples were mixed with reducing sample buffer and boiled, centrifuged, and loaded to a 12.5% slab gel. Following electrophoresis, the gel was stained by sliver nitrate, or used for Western blotting analysis. For the latter, the gel was electroblotted onto a PVDF membrane, 7 which was then reacted with either anti-VP2 or anti-VP3 polyclonal antibody, prepared by immunizing rabbits using the expressed IBDV VP2 fragment (amino acid residues 1 to 389) in E. coli or VP3 fragment (amino acid residues 729 to 1012), respectively. Following washing, the membrane was incubated with secondary antibody (alkaline phosphatase conjugated affinipure goat anti-rabbit IgG (JacksonImmunoResearch, West Grove, PA) and the colour developed as described by Cheng et al. (Cheng et al., 2001). Transmission Electron Microscopy Negative-stain electron microscopy was used to investigate the presence, size, and morphology of particles formed and direct observation through negative staining was performed as previously described (Wang et al., 2000). Briefly, 300-mesh grids covered with Formvar/carbon membranes were incubated with particles in phosphate buffer, pH 8. Following decantation, the grids were floated on an aqueous solution of 2% uranyl acetate to achieve contrast. After decantation and drying, the grids were observed through a transmission electron microscope (JEOL JEM 1200 EX-2) with a voltage acceleration of 120 kV. RESULTS AND DISCUSSION Efficient processing of IBDV ORFA1 polyprotein in Hi-5 cells. The expression patterns, analyzed by Western blot, of IBDV polyprotein in two different insect cells, Sf9 and Hi-5, were shown in Fig. 2A. As expected, pVP2 protein was expressed and accumulated in both Sf9 and Hi-5 cells at 48 h post-infection. Notably, the expressed pVP2 protein was gradually processed to VP2 protein in Hi-5 cells. However, the processing of pVP2 was hardly detected in Sf9 cells, suggesting the processing of polyprotein to VP2 was incomplete. This result is 8 consistent with other reports (Chevalier et al., 2002; Ho et al., 1999; Kibenge et al., 1999; Martinez-Torrecuadrada et al., 2000). At 72 h post-infection, most of pVP2 in Hi-5 cells were converted into VP2, whereas most of pVP2 in Sf9 cells remain intact. Part of the product in both cell lines was released into culture media due to cell lysis. Another processed product of polyprotein, VP3, appeared at the same time as that of pVP2(VP2). As expected, VP3 was in the form of doublet in both cell lines. The degradation of VP3 has been attributed to a significant factor for the inefficient particle assembly (Maraver et al., 2003). To demonstrate that Hi-5 cells have the capability to process pVP2 in the absence of VP4, the protease encoded by IBDV and supposedly responsible for the processing of pVP2, either Hi-5 or Sf9 cells were infected by another recombinant baculovirus, vP3009VPX, which contains pVP2 gene. The results (Fig. 2B) indicated that pVP2 expressed alone could be converted to VP2 in Hi-5 cells but not in Sf9, suggesting that cellular proteases in Hi-5 cells play a role in the maturation of pVP2 to VP2. Based on this result, we hypothesize that proteases produced by Hi-5 cells as well as VP4 were involved in the processing of IBDV polyprotein. Formation of IBDV-like Particles and Subviral Particles in the infected Hi-5 Cells. We have demonstrated that the processing pattern of polyprotein in Hi-5 cells was significantly different from that in Sf9 cells. To examine the particles generated in the two infected cells, the collected cell lysates and culture medium were concentrated and ultracentrifuged. EM observation showed that fractions with the expressed proteins as demonstrated by Western blot (data not shown) and buoyant densities between 1.29 to 1.30 g/cm3 contained a mixed population of particles (Figs. 3). Most particles generated in Sf9 cell lysates were rigid IBDV type I tubules with 40~45 nm in diameter mixed with very few of type II tubules with 23 nm in diameter and spherical IBDV-like particles (Figs. 3A-B). As 9 expected, type I tubules were released into the culture medium of the infected Sf9 cells (Fig. 3C). On the other hand, particles generated in the Hi-5 cell pellet (Figs. 3D-E) exhibited more heterogonous morphologies, including type I and II tubules, spherical IBDV-like particles and some smaller icosahedral particles, refered as subviral particles by Faragher (1971). Remarkably, a great number of IBDV-like and subviral particles were found in the culture medium of Hi-5 cells but not in that of Sf9 cells (Fig. 3F). To further confirm the appearance of IBDV-like and subviral particles, three more batches of Hi-5 cells were infected with different MOIs (0.1, 1, and 10) of vIBDVJ6 recombinant baculovirus. All three batches confirmed that the two icosahedral particles were simultaneously produced in the culture media and their yields were MOI-dependent (Figs. 4). The size (ca. 25 nm in diameter) and morphology of the subviral particles were similar to those of VP2-formed particles (Wang et al., 2000), suggesting that subviral particles were assembled by VP2 protein alone. To characterize the composition of subviral particles, these two icosahedral particles were further separated using gel-filtration chromatography. Separation and characterization of IBDV-like Particles and subviral Particles. Two major types of particles, e.g. IBDV-like and subviral particles, were formed extracellularly when the polyprotein was expressed by the Hi-5 cells. Further separation of these particles using sedimentation method was unsuccessful. Thus, gel-filtration chromatography was adopted. A TSK gel-filtration column was selected for this purpose due to the different size between subviral and IBDV-like icosahedral particles, i.e. 25 nm vs 65 nm. This column was first calibrated with BSA protein and rVP2H particles ca. 23 nm in diameter obtained as previously described (Cheng et al., 2001). Following chromatography, representative fractions collected in the first two peaks were observed under EM (Figs. 6). The electron micrographic observation demonstrated that IBDV-like particles were eluted first (fraction 10 27, Fig. 6A), followed by the subviral icosahedral particles (fractions 36, Figs. 6B). Western blotting analysis confirmed that the IBDV-like particles (Fig. 5, fraction 26-30) contained pVP2, VP2 and VP3 proteins; however, VP2 protein with only trivial amount of pVP2 protein were found in the small subviral particles (Fig. 5, fraction 34-38). This result confirms that IBDV-like particles could be separated from subviral particles by gel-filtration chromatography and, practically no VP3 was found in the subviral particles. Our result implies that when there was not enough VP3 to interact with pVP2/VP2 to form IBDV-like particles, excessive pVP2/VP2 proteins formed subviral particles. Notably, VP2 (the processed product of pVP2) generated in either the assembled IBDV-like particles or subviral particles co-migrated in SDS-PAGE with the expressed mVP2 that has 441 amino acid residues (Fig. 7, right panel), suggesting that the processing of polyprotein in Hi-5 cells was complete (Da Costa et al., 2002). Our results address that a complete processing of polyprotein is critical for viral assembly and the formation of IBDV-like particles. In Hi-5 cells, unidentified cellular proteases efficiently processed pVP2 in the absence of VP4 (Fig. 2B). We hypothesize that the same proteases act on the second stage processing of polyprotein, i.e. processing pVP2 to VP2 and leading to the formation of IBDV-like Particles. In contrast, these proteases are absent or inactive in Sf-9 cells, leading to an inefficient processing of pVP2 and a generation of irregular particles, namely, type I and II tubules. Coexpression of pVP2 and VP3 in Hi-5 cells is sufficient for the formation of IBDV-like particles (Ona et al., 2004), further supporting our hypothesis that cellular proteases, instead of VP4, were involved in the processing of pVP2. Although some proteases of Hi-5 cells promote IBDV assembly, proteolytic degradation of VP3 protein limits the production of IBDV-like particles. As shown in Fig. 6B, the degraded VP3 was not found in the assembled IBDV-like particles, indicating that 11 pVP2 did not assemble with the degraded VP3. Under this circumstance, the amount of VP3 was not enough to assemble with pVP2, and the excessive pVP2 protein was digested to VP2 that forms subviral particles. Subviral particles were first described by Faragher (1971) on negatively stained preparations of IBDV-infected bursal homogenate. In that study, two populations of viruses of mean diameter 56 nm and 20 nm were found, the former being IBDV capsids and the latter being subviral particles. Subsequently, Almeida and Morris (1973) confirmed the existence of these two particles. This is the first work to report simultaneous production of IBDV and subviral particles using a heterogeneous expression system. Our result clearly demonstrates that subviral particles were by-products of IBDV assembly and formed by pVP2/VP2. This answers the origin of subviral particles questioned by Almeida and Morris (1973). VP2 is the primary host-protective immunogen of IBDV and contains the antigenic regions responsible for elicitation of neutralizing antibodies (Becht et al., 1988; Fahey et al., 1989; Heine et al., 1991). Furthermore, it displays as trimeric clusters of outer protruding structures on IBDV capsids (Böttcher et al., 1997). Thus, it is reasonable that Almeida and Morris (1973) suggest there was an antigenic relationship between them. In conclusion, since the processing of pVP2 to VP2 can be performed, presumably through the action of some unidentified cellular proteases in Hi-5 cells, they are a better cell line than Sf9 cells in terms of expression and formation of IBDV-like particles. This study also confirms that the maturation of pVP2 is a limiting step for the assembly of IBDV polyprotein in insect cells. ACKNOWLEDEMENT This research was supported by grants from the National Science Council (Grant No. NSC 87-2313-B-005-081). Critical review of the manuscript by Dr. Su-Yuan Lai is gratefully acknowledged. 12 Figure legends Figure 1. Three recombinant baculoviruses, including vIBDVJ6, vP3009VPX, vP3009mVP2, were used in this study. Figure 1 described the recombinant proteins expressed by these three recombinant viruses. The recombinant baculoviruses, either vIBDVJ6 [Ho et al., 1999], expressing IBDV polyprotein, pVP2-VP4-VP3, or vP3009VPX [Lee et al., 2004], expressing VPX protein, under the control of the polyhedrin promoter was described previously. The construction and generation of vP3009mVP2, expressing a matured form of VP2 protein (mVP2, residues 1-441) will be published elsewhere. Location of primer target sequence on 13 the VP2 gene of pP3009-2, the plasmid containing the coding region for the complete polypeptide of IBDV P3009. The expected size of the amplified product is about 1.4 kb. Figure 2. (A) Western blotting analysis of the expression patterns of IBDV polyprotein in Sf9 and Hi-5 cells. The cells were infected with a recombinant baculovirus, vIBDVJ6, which contains the gene of IBDV ORF A1 that encodes polyprotein (VPX-VP4-VP3). To examine the expression patterns of IBDV polyprotein in Hi5 or Sf9 cells, cell pellets collected at 24, 48, 72 hrs postinfection were separated from the culture medium and both pellet and medium were analyzed using either anti-VP2/VPX or anti-VP3 polyclonal antibodies. Lane M, molecular weight markers (104, 82, 48, 35, 28, 19 kDa); Lane 1, IBDV P3009infected CEF lysates; Lanes 2-3, putative recombinant baculovirus vP3009VP2- and vP3009VP2H-infected Sf9 cells lysates, respectively. The position of rVP2H in lane 3 is slightly higher than that of rVP2 in lane 2; Lane 4, uninfected Sf9 cells lysates; Lane 5, AcMNPV baculovirus-infected Sf9 cells. (B) Western blotting analysis of VPXH protein processing in recombinant baculovirusinfected Sf9 and Hi-5 cells. Lane M, molecular weight markers (112, 81, 50, 36, 29, 21 kDa); Lanes 1 and 6, recombinant baculovirus vP3009VPXH-infected Sf9 and Hi-5 cells lysates, respectively; Lane 2, IBDV P3009-infected CEF lysates; Lanes 3 and 7, uninfected Sf9 and Hi-5 cells lysates, respectively; Lanes 4 and 8, AcMNPV baculovirus-infected Sf9 and Hi-5 cells lysates, respectively; Lane 5, purified rVP2H. (B) Western blotting analysis of the Cterminal degradation of rVPXH protein. Lane 1, purified rVP2H; lanes 2 and 3, recombinant baculovirus vP3009VPXH-infected Sf9 and Hi-5 cells lysates, respectively. Figure 3. EMs of the particles generated in the Sf9 and Hi-5 cells infected with vIBDVJ6 recombinant baculovirus as described in Fig. 2. To examine the particles generated in the 14 two infected cells, either the collected Sf9 cell lysates and culture medium or Hi-5 cell lysates and culture medium were concentrated and ultracentrifuged. EM observation showed that the fractions having the expressed proteins as demonstrated by Western blot (Fig. 4 and 5) and having buoyant densities between 1.29 to 1.30 g/cm3 contained a mixed population of particles. (B) EMs of the chromatographed isometric particles of fraction 30 and icosahedral particles of fraction 33 (C) and fraction 39 (D). Bar scale is 50 nm. (E) Western blotting analysis of the composition of isometric and icosahedral particles. The fraction numbers eluted from column are indicated. Lane C, Hi-5 cell expressed rVP2H. Figure 4. EMs of the particles generated in the Hi-5 cells infected with vIBDVJ6 at different MOIs. To examine the particles generated in the two infected cells, the collected Hi-5 cell culture media were concentrated and ultracentrifuged. EM observation showed that the fractions having the expressed proteins as demonstrated by Western blot (Fig. 4 and 5) and having buoyant densities between 1.29 to 1.30 g/cm3 contained a mixed population of particles. (B) EMs of the chromatographed isometric particles of fraction 30 and icosahedral particles of fraction 33 (C) and fraction 39 (D). Bar scale is 50 nm. (E) Western blotting analysis of the composition of isometric and icosahedral particles. The fraction numbers eluted from column are indicated. Lane C, Hi-5 cell expressed rVP2H. Figure 5. Separation of IBDV-like Particles and subviral Particles by gel-filtration chromatography. An TSK gel-filtration column was selected for this purpose due to the different size between subviral and IBDV-like icosahedral particles. This column was first calibrated with BSA protein and rVP2H particles ca. 23 nm in diameter (A). Following chromatography, fractions collected in the first two peaks were detected by ELISA with polyclonal rabbit anti-VP2 and anti-VP3 antibodies. The electron micrographic observation 15 and Western blotting analysis demonstrated that IBDV-like particles were eluted first and contained pVP2, VP2 (B) and VP3 proteins (C), followed by the subviral icosahedral particles which were formed by VP2-like proteins with only trivial amount of pVP2 protein (B) but not VP3 (C). (A) Chromatographic profile of IMAC-purified particles detected by ELISA with polyclonal rabbit anti-VP2 antibody. Peaks 1 and 2 indicate the elution profiles of isometric and icosahedral particles, respectively. (B) EMs of the chromatographed isometric particles of fraction 30 and icosahedral particles of fraction 33 (C) and fraction 39 (D). Bar scale is 50 nm. (E) Western blotting analysis of the composition of isometric and icosahedral particles. The fraction numbers eluted from column are indicated. Lane C, Hi-5 cell expressed rVP2H. Western blotting analysis of the purified rVP2 particles. The purification of rVP2 particles was performed by using sucrose density gradient ultracentrifugation, as described in Figs. 3A-D; the fractions collected were indicated as in Fig. 3D. Lane M, molecular weight markers; Lane 1, AcMNPV baculovirus-infected Sf9 cells; Lane 2, the supernatant loaded for the purification of rVP2 particles; Lanes 3-7, fractions 1 to 5, respectively, generated by sucrose density gradient ultracentrifugation as indicated in Fig. 3D. Notably, the degraded rVP2 protein (~ 25 kDa) was seen in lanes 3 and 4, which was barely present in cell lysate before purification (lane 2). The positions of rVP2H and molecular marker (34 kDa and 47 kDa) are indicated. Figure 6. 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