Download Processing of Infectious Bursal Disease Virus (IBDV) pVP2

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
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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
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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
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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.
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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. EMs of the chromatographed isometric particles of fraction 30 and icosahedral
particles of fraction 33 and SDS-PAGE Characterization of IBDV-like Particles and subviral
Particles.
16
The purification of rVP2H particles using IMAC column. (A) Optical density profile
(OD280) showing separation of rVP2H protein using Ni2+ resin. The rVP2H protein was
purified as described in the text. The fractions represented by solid circles were analyzed in
Fig. 5B. (B) Western blot analysis of the purified rVP2H protein from IMAC column. Lane
M, molecular weight markers, Lanes 1-7 correspond to the fractions represented by solid
circles in Fig. 5A. The positions of rVP2H and molecular marker (35 kDa and 48 kDa) are
indicated. (C) Electron microscopy of the rVP2H particles eluted by pH 4 buffer.
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