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Journal of Virology, January 2001, p. 61-72, Vol. 75, No. 1
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.1.61-72.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Roles of Disulfide Linkage and Calcium Ion-Mediated
Interactions in Assembly and Disassembly of Virus-Like Particles
Composed of Simian Virus 40 VP1 Capsid Protein
Ken-Ichiro
Ishizu,1
Hajime
Watanabe,2
Song-Iee
Han,1
Shin-Nosuke
Kanesashi,1
Mainul
Hoque,1
Hiroaki
Yajima,3
Kohsuke
Kataoka,4 and
Hiroshi
Handa4,*
Frontier Collaborative Research
Center4 and Faculty of Bioscience and
Biotechnology,1 Tokyo Institute of
Technology, Midori-ku, Yokohama 226-8501, Center for Integrative
Bioscience, Okazaki National Research Institutes, Myodaiji, Okazaki
444-8585,2 and Kirin Brewery Co., Ltd.,
Kanazawa-ku, Yokohama 236,3 Japan
Received 17 July 2000/Accepted 25 September 2000
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ABSTRACT |
The simian virus 40 capsid is composed of 72 pentamers of VP1
protein. Although the capsid is known to dissociate to pentamers in
vitro following simultaneous treatment with reducing and chelating agents, the functional roles of disulfide linkage and calcium ion-mediated interactions are not clear. To elucidate the roles of
these interactions, we introduced amino acid substitutions in VP1 at
cysteine residues and at residues involved in calcium binding. We
expressed the mutant proteins in a baculovirus system and analyzed both
their assembly into virus-like particles (VLPs) in insect cells and the
disassembly of those VLPs in vitro. We found that disulfide linkages at
both Cys-9 and Cys-104 conferred resistance to proteinase K digestion
on VLPs, although neither linkage was essential for the formation of
VLPs in insect cells. In particular, reduction of the disulfide linkage
at Cys-9 was found to be critical for VLP dissociation to VP1 pentamers
in the absence of calcium ions, indicating that disulfide linkage at
Cys-9 prevents VLP dissociation, probably by increasing the stability
of calcium ion binding. We found that amino acid substitutions at
carboxy-terminal calcium ion binding sites (Glu-329, Glu-330, and
Asp-345) resulted in the frequent formation of unusual tubular particles as well as VLPs in insect cells, indicating that these residues affect the accuracy of capsid assembly. In addition, unexpectedly, amino acid substitutions at any of the calcium ion binding sites tested, especially at Glu-157, resulted in increased stability of VLPs in the absence of calcium ions in vitro. These results suggest that appropriate affinities of calcium ion binding are
responsible for both assembly and disassembly of the capsid.
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INTRODUCTION |
Simian virus 40 (SV40), a member of
the Papovaviridae, is a small, nonenveloped tumorigenic
virus. Its genome consists of double-stranded circular DNA of 5,243 bp,
which encodes three structural proteins (VP1, VP2, and VP3) and two
nonstructural proteins (large T antigen and small T antigen). The
structural proteins are expressed in late infection, and viral capsids
are formed in the nucleus of infected susceptible host cells
(32). The SV40 capsid is about 45 to 50 nm in diameter,
and its major component is VP1. The capsid is formed by the arrangement
of 72 VP1 pentamers in a T=7d icosahedral lattice. The
three-dimensional structure of the SV40 virion has been elucidated by
X-ray crystallography, which showed that the capsid is formed from
three nonequivalent types of interactions between pentameric capsomeres
(
-', 
-', and 
-) (20, 30).
SV40 capsids, as well as those of the closely related murine
polyomavirus, dissociate to VP1 pentamers following treatment with
dithiothreitol (DTT) and EGTA in vitro (2-4, 7),
indicating that disulfide linkage and calcium ion-mediated interactions
between pentamers are important for capsid formation in these viruses. Consistent with these observations, crystallographic analysis of SV40
virions has identified disulfide linkage between Cys-104 and Cys-104 in
-' interpentameric interactions and has shown that the two
calcium ions which are bound per VP1 molecule are also involved in
interpentameric interaction (20, 30). In spite of these
observations, however, the functional roles of these interactions in
association and dissociation of the capsid are largely unknown.
We have previously reported that recombinant SV40 VP1 protein expressed
in a baculovirus expression system assembled into virus-like particles
(VLPs) in insect cells in the absence of VP2, VP3, T antigens, and
genomic DNA. The VLPs were morphologically indistinguishable from
wild-type SV40 and could be dissociated into VP1 pentamers by treatment
with DTT and EGTA in vitro (19). Thus, the structural
properties of the recombinant VLPs were very similar to those of
wild-type SV40 particles, making this system a useful tool with which
to study the assembly and disassembly of the SV40 capsid.
To elucidate the functional roles of disulfide linkage and calcium
ion-mediated interactions, we produced a series of VP1 proteins mutated
at cysteine residues and putative calcium ion binding sites, and
analyzed the assembly in insect cells and disassembly in vitro of the
VLPs formed from these mutant proteins.
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MATERIALS AND METHODS |
Construction of recombinant baculovirus.
The construction
and structure of baculovirus expressing wild-type VP1 are described
elsewhere (19). Point mutations were introduced by
PCR-based site-directed mutagenesis (8) using pUCVP1
(19) as the initial PCR template. The primers used were as
follows (mutation sites are underlined): WT5'T (terminal) S (sense)
(5'-AAG GGT CGA CAT GAA GAT GGC CCC AAC AAA AAG-3'), C9A S (5'-AAG GGT
CGA CAT GAA GAT GGC CCC AAC AAA AAG AAA AGG AAG TGC TCC AGG
GGC-3'), C104S S (5'-GGA CTT AAC CTC TGG AAA TAT TTT G-3'),
C104S AS (anti-sense) (5'-CAA AAT ATT TCC AGA GGT TAA GTC C-3'), C207S S (5'-CCA GTG GAG TCC TGG GTT CCT G-3'), C207S
AS (5'-CAG GAA CCC AGG ACT CCA CTG G-3'), C254S S (5'-GGG
CCC TTG TCC AAA GCT GAC-3'), C254S AS (5'-GTC AGC TTT
GGA CAA GGG CCC-3'), E46,48Q dm (double mutation) S (5'-CTT
CAC TCA GGT GCA GTG CTT TTT AAA TCC-3'),
E46,48Q dm AS (5'-AAA AAG CAC TGC ACC TGA GTG AAG CTG TC-3'), E157Q S (5'-GTT GGT GGG CAA CCT TTG GAG
C-3'), E157Q AS (5'-GCT CCA AAG GTT GCC CAC CAA C-3'),
E160Q S (5'-GGA ACC TTT GCA GCT GCA GGG-3'), E160Q AS
(5'-CCC TGC AGC TGC AAA GGT TCC-3'), E157,160Q dm S (5'-GTT
GGT GGG CAA CCT TTG CAG CTG CAG GG-3'),
E157,160Q dm AS (5'-CCC TGC AGC TGC AAA GGT TGC
CCA CCA AC-3'), S213A,K214A,E216Q tm (triple mutation) S (5'-CCT GAT CCA GCT GCA AAT CAA AAC ACT AGA TAT
TTT GGA ACC TAC-3'), S213A,K214A,E216Q tm AS (5'-GTG TTT
TGA TTT GCA GCT GGA TCA GGA ACC CAG
CAC TCC-3'), E329,330Q dm S (5'-CCT CTC AAG TAC
AGC AGG TTA GGG-3'), E329,330Q dm AS (5'-CCC TAA CCT
GCT GTA CTT GAG AGG-3'), D345N S (5'-GGG GAT
CCA AAC ATG ATA AGA TAC-3'), D345N AS (5'-GTA TCT TAT CAT GTT TGG ATC CCC-3'), WT3'T AS (5'-CCG GCT CGA GTC ACT GCA
TTC TAG TTG TGG TTT G-3'). The WT5'T S and WT3'T AS primers were used in the second round of PCR. A SalI restriction site was
introduced at the 5' end of the mutated DNA fragments to facilitate
further subcloning. The amplified products were digested with
SalI and inserted into the SalI and blunt-ended
HindIII sites of pBluescriptII to make pBSmt1 to pBSmt6
and pBSmtA to pBSmtH. The nucleotide sequences of the entire coding
regions of all mutants were confirmed by dideoxynucleotide sequencing analysis.
For baculovirus expression, pBSmt1 to pBSmt6 and pBSmtA to pBSmtH were
digested with SalI and EcoRV and the inserts were
recloned into the SalI and blunt-ended
HindIII sites of pFastBac1 (Gibco BRL). Recombinant
baculoviruses were produced using the Bac-To-Bac system (Gibco BRL).
Expression of VP1 proteins in insect cells.
Spodoptera
frugiperda (Sf9) cells were maintained in spinner culture flasks
at 27°C in Sf-900II medium (Gibco BRL) supplemented with
streptomycin, penicillin, and 0.3% heat-inactivated fetal bovine serum.
For protein expression, Sf9 cells were seeded onto tissue culture
dishes at 2 × 10
5 cells/cm
2 and allowed
to attach for at least 1 h at room temperature. Recombinant
baculoviruses encoding mutant VP1 genes were used to infect the
attached Sf9 cells (5 × 10
7 cells at the time of
infection) at a multiplicity of infection
of 5 to 10. After 1 h,
TC-100 medium (Gibco BRL) supplemented
with streptomycin, penicillin,
and 10% heat-inactivated fetal
bovine serum was added and the cells
were incubated at 27°C. The
infected cells were harvested at 72 h postinfection (p.i.). After
being washed twice with
phosphate-buffered saline, the cells were
sonicated in 1 ml of
sonication buffer (20 mM Tris-HCl [pH 7.9],
1% sodium deoxycholate,
2 mM phenylmethylsulfonyl fluoride (PMSF),
1 µg of chymostatin per
ml, 1 µg of aprotinin per ml, 1 µg of
leupeptin per ml, 1 µg of
antipain per ml, 1 µg of pepstatin per
ml) and centrifuged at
15,000 ×
g for 10 min at 4°C, and the supernatants
were collected. For cysteine mutants mt1, mt5, and mt6, nuclear
extracts of the infected Sf9 cells were prepared as described
elsewhere
(
29) with some modifications. A 1-ml volume of buffer
A
(10 mM HEPES-NaOH [pH 7.9], 10 mM KCl, 2 mM CaCl
2, 0.5 mM
PMSF,
1 µg of chymostatin per ml, 1 µg of aprotinin per ml, 1 µg
of
leupeptin per ml, 1 µg of antipain per ml, 1 µg of pepstatin per
ml), 62.5 µl of 10% Nonidet P-40 (NP-40), and 250 µl of buffer
C
(20 mM HEPES-NaOH [pH 7.9]), 0.4 M NaCl, 2 mM CaCl
2, 1 mM
PMSF,
1 µg of chymostatin per ml, 1 µg of aprotinin per ml, 1 µg
of
leupeptin per ml, 1 µg of antipain per ml, 1 µg of pepstatin per
ml) were used per 200 µl of packed
cells.
Purification of VP1 proteins.
Whole-cell lysates or nuclear
extracts (mt1, mt5, and mt6) of infected Sf9 cells (400 µl) were
gently loaded onto 4 ml of preformed 20 to 50% (wt/vol) cesium
chloride density gradients in 20 mM Tris-HCl (pH 7.9) and centrifuged
at 35,000 rpm for 3 h at 4°C in an SW55Ti rotor (Beckman). After
centrifugation, fractions were collected from the tops of the tubes and
a small aliquot of each fraction was separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were
visualized by staining with Coomassie brilliant blue (CBB). Fractions
containing VLPs were filled up with 37% (wt/vol) cesium chloride
solution in 20 mM Tris-HCl (pH 7.9) and centrifuged at 50,000 rpm for
20 h at 4°C in an SW55Ti rotor. After centrifugation, fractions
were collected as described above. For mt1, mt5, and mt6, 2 mM
CaCl2 was added to all cesium chloride solutions. Finally,
fractions containing VLPs were adjusted to a final concentration of
0.1% NP-40 and dialyzed against 20 mM Tris-HCl (pH 7.9)-0.1% NP-40
at 4°C for at least 15 h with several buffer changes. After
dialysis, samples were collected and centrifuged at 15,000 × g for 10 min at 4°C, and the supernatants were stored at
80°C.
Electron microscopy.
Baculovirus-infected Sf9 cells were
harvested, washed with 100 mM cacodylate (pH 7.2)-2.5 mM
CaCl2, and fixed with glutaraldehyde at 8 days p.i. The
cells were then fixed with osmium tetroxide, stained with uranyl
acetate, and processed for thin sectioning. For negative staining of
purified VLPs or pentamer, 3 µl of each sample was absorbed onto
glow-discharged carbon-coated copper grids. The grids were washed with
water, stained with 2% uranyl acetate, and air dried. The specimens
were examined with an H-7500 electron microscope (Hitachi) at 80 kV.
Sucrose gradient centrifugation.
A 20-µl volume of each
cell lysate was diluted tenfold with 20 mM Tris-HCl (pH 7.9) and loaded
onto a 0.6-ml preformed 10 to 30% sucrose gradient in 20 mM Tris-HCl
(pH 7.9) in a 5- by 41-mm open-top tube (Beckman). Using the
appropriate adapters, the tubes were centrifuged at 45,000 rpm for
1 h at 4°C in an SW55Ti rotor. After centrifugation, 55-µl
fractions were collected from the tops of the tubes and the bottom of
each tube was washed with 55 µl of 2× SDS sample-loading buffer. A
7-µl volume of each fraction was separated by SDS-PAGE and
immunoblotted with anti-SV40 VP1 polyclonal antibody (courtesy of M. Ikeda and I. Tamai, MBL, Nagoya, Japan). Immunoreactive bands were
detected using the enhanced chemiluminescence system (Amersham).
For preparation of VP1 pentamer, purified VLP preparations were
adjusted to final concentrations of 25 mM EGTA and 30 mM DTT,
incubated
for 1 h at 37°C, and separated on a Superdex 200 gel
filtration
chromatography column (Pharmacia) in 20 mM Tris-HCl
(pH 7.9)-150 mM
NaCl-5 mM EGTA-5 mM DTT at 4°C. The peak fractions
corresponding in
size to approximately 200 kDa were collected
and stored at
80°C.
SDS-PAGE under nonreducing conditions.
Purified VLPs (10-ng
portions) were incubated in 20 mM CHES
(N-cyclohexyl-2-aminoethanesulfonic acid) (pH 9.0)-1%
SDS-2 mM N-ethylmaleimide at 37°C for 10 min to block
free thiols. Samples were boiled for 5 min after addition of SDS
sample-loading buffer containing no -mercaptoethanol and then boiled
again in the presence or absence of 100 mM DTT. The proteins were
separated by SDS-PAGE (8% polyacrylamide) and immunoblotted with
anti-VP1 antibody.
Degradation by proteinase K.
Proteinase K (0.1, 0.3, or 1 ng) was added to 10 ng of purified VLPs, and the reaction mixtures were
incubated at 37°C for 15 min. The reactions were stopped by addition
of SDS sample-loading buffer and immediate boiling. The samples were
separated by SDS-PAGE (12% polyacrylamide) followed by immunoblotting
with anti-VP1 antibody.
Native agarose gel electrophoresis.
Purified VLPs (20-ng
portions) were incubated for 1 h in 20 µl of 20 mM Tris-HCl (pH
7.9) with or without 30 mM DTT in combination with 2 mM
CaCl2 or 25 mM BAPTA (a highly selective calcium
ion-chelating agent) (33). Samples containing BAPTA or DTT
were incubated at 37°C, and all other samples were incubated at
4°C. After incubation, 5 µl of sample-loading buffer (250 mM
Tris-acetate [pH 8.1], 25% [vol/vol] glycerol, 0.125% bromophenol
blue) was added to each sample and 7 µl of sample was loaded onto
0.8% agarose gels in 50 mM Tris-acetate (pH 8.1) with or without 2 mM
CaCl2. Electrophoresis was carried out at 4°C in the same
buffer as in the gel until the dye reached the bottom of the gel
(approximately 2 h). After electrophoresis, the proteins were
transferred to polyvinylidene difluoride membranes in 50 mM NaOH for 3 to 8 h at room temperature by a standard capillary transfer method
and then immunoblotted with anti-VP1 antibody.
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RESULTS |
SV40 VP1 VLP formation in the nucleus of insect cells and
dissociation to pentamers in vitro.
We previously constructed a
recombinant baculovirus expressing VP1 and showed that VP1 was
efficiently accumulated and assembled into VLPs in insect cells
(19). We further assessed this system for use as a model
of assembly and disassembly of the SV40 capsid. Figure
1 shows an electron microscopic view of
baculovirus-infected Sf9 cells. Spherical particles approximately 45 nm
in diameter accumulated abundantly in the nucleus, which is the
location of wild-type SV40 particle formation in susceptible host
cells. No such particles were formed in the cytoplasm or in
mock-infected Sf9 cells (data not shown).
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FIG. 1.
(A) Electron micrograph of an ultrathin section of an
Sf9 cell 8 days after infection with baculovirus expressing VP1. (B)
Higher magnification of the region indicated in panel A. Scale bars,
530 nm (A) and 220 nm (B).
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The VLPs were easily and efficiently purified by cesium chloride
density gradient ultracentrifugation (Fig.
2A). We obtained
6 to 8 mg of VP1 protein
at more than 90% homogeneity from 5 ×
10
8 insect
cells. Electron microscopic observation confirmed that
VP1 formed VLPs
which were morphologically indistinguishable from
wild-type SV40
particles (Fig.
2B).
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FIG. 2.
Purification of wild-type VLPs and their dissociation to
VP1 pentamers in vitro. (A) Purification of VLPs by cesium chloride
density gradient centrifugation. Aliquots of fractions after
ultracentrifugation were separated by SDS-PAGE and stained with CBB. M,
molecular mass standards; Inp, input. VP1 protein was detected in
fractions 5 and 6. (B) Negatively stained electron micrograph of
purified VLPs. Scale bar, 50 nm. (C) Size exclusion chromatography. The
purified VLPs were incubated for 1 h at 37°C with EGTA and/or
DTT as indicated and loaded on a Superdex 200 gel filtration
chromatography column (Pharmacia). The minus sign indicates the VLP
preparation prior to incubation. Fractions were separated by SDS-PAGE
and stained with CBB. Molecular mass standards are thyroglobulin (670 kDa) and bovine gamma globulin (158 kDa). (D) Electron micrograph of
the 200-kDa fraction of the bottom chromatogram in panel C. Scale bar,
50 nm.
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For in vitro dissociation analysis, purified VLPs were incubated at
37°C for 1 h with EGTA and/or DTT. Size exclusion chromatography
showed that VP1 protein in the form of VLPs was detected in the
void
volume (Fig.
2C). Following addition of both DTT and EGTA,
approximately 80% of VP1 was detected in fractions corresponding
to a
molecular mass of approximately 200 kDa, in which VP1 pentamers
would
be expected, suggesting efficient dissociation of VLPs to
VP1
pentamers. No VP1 proteins were detected in fractions corresponding
in
size to VP1 monomer (data not shown). Consistent with these
observations, electron microscopic observation of the 200-kDa
fraction
revealed pentamer-like structures but not VLPs (Fig.
2D). Addition of
either DTT or EGTA alone resulted in only partial
dissociation (Fig.
2C).
These results demonstrate that the recombinant VP1
assembled into VLPs in the nucleus and dissociated to VP1 pentamers
following
treatment with DTT and EGTA in vitro. These characteristics
are
similar to those of wild-type SV40 particles, indicating that
VLPs
composed of recombinant VP1 can be used as a model of the
association
and dissociation of SV40 capsid. Using this system,
we analyzed the
roles of disulfide linkage and calcium ion-mediated
interactions in the
assembly and disassembly of VLPs by introducing
amino acid
substitutions at cysteine residues and putative calcium
ion binding
sites.
Cysteine mutants.
The VP1 cysteine residue mutants used in the
present study are shown schematically in Fig.
3A. Of the seven cysteine residues in
SV40 VP1, Cys-9, Cys-104, and Cys-254 are conserved between SV40 and
murine polyomavirus (20, 31), suggesting that these residues may be of particular functional importance. These three cysteine residues were mutated singly in mt1, mt2, and mt4 and in
combination in mt5 and mt6. We included a mutation at Cys-207 (mt3) as
a control. The mutated VP1 constructs were introduced into baculovirus
vectors, which were used to infect Sf9 cells. SDS-PAGE analysis of
whole-cell extracts of mutant baculovirus-infected Sf9 cells showed
that soluble proteins of about 50 kDa were specifically expressed at a
level comparable to the expression of wild-type VP1 (Fig.
4A). Western blotting analysis with
anti-VP1 antibody confirmed that these ~50-kDa proteins were VP1
(Fig. 4B).
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FIG. 3.
Schematic representation of SV40 VP1 mutants. (A)
Cysteine mutants. (B) Calcium ion binding site mutants. Amino acid
residues are represented in the one-letter code and numbered as in
references 20 and 30.
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FIG. 4.
Expression and VLP formation in Sf9 cells of VP1
cysteine mutants. (A) SDS-PAGE analysis of whole-cell extracts of Sf9
cells infected with baculoviruses expressing cysteine mutants. The
soluble fractions of whole-cell lysates at 3 days p.i. were
separated by SDS-PAGE and stained with CBB. WT, wild type; ctl
(control), lysate of cells infected with a baculovirus encoding no
exogenous gene; mock, mock-infected cell lysate. (B) Detection of
mutant VP1 proteins by immunoblotting with anti-VP1 antibody. (C)
Analysis of VLP formation in Sf9 cells. Whole-cell lysates were
separated by 10 to 30% sucrose gradient centrifugation and
fractionated from the tops of the tubes. The fractions were separated
by SDS-PAGE and subjected to Western blotting with anti-VP1 antibody
(wild type [WT] and mt1 to mt6) or stained with CBB (VLP and
pentamer). VLP and pentamer, purified wild-type VLPs and VP1 pentamer,
respectively. (D) Electron micrograph of the material from the middle
of the gradient. Fractions 4 and 5 of wild-type VP1 were further
purified by cesium chloride density gradient ultracentrifugation, and
VP1-containing fractions were negatively stained and observed by
electron microscopy. Scale bar, 50 nm.
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To analyze the ability of the mutant VP1 proteins to form VLPs in
insect cells, the cell lysates were subjected to sucrose
gradient
centrifugation followed by immunoblotting with anti-VP1
antibody (Fig.
4C). Purified VLPs composed of wild-type VP1 were
detected in fractions
8 to 12 (Fig.
4C, VLP), and pentamers remained
at the top of the
gradient (Fig.
4C, pentamer). The majority of
wild-type VP1 was
detected in VLP fractions but not in fractions
corresponding to
pentamer (or monomer, which has a smaller sedimentation
coefficient and
also remains at the top of the gradient), indicating
that VLPs were
efficiently formed in insect cells. VP1 protein
was also detected in
intermediate fractions between VLP and pentamer
(fractions 4 and 5).
Further purification of VP1 from these fractions
followed by electron
microscopic observation revealed that the
protein was present in
particles approximately 20 nm in diameter
(Fig.
4D; see Fig.
2B for
comparison). Particles considerably
smaller than virus capsid are also
formed from recombinant polyomavirus
VP1 pentamers under certain in
vitro conditions (
28). These
small particles probably form
as a result of inaccurate interactions
between pentamers during capsid
assembly. All of the cysteine
mutants were detected as a peak in
fractions 8 to 12 (Fig.
4C),
indicating that these mutants formed VLPs
in insect cells. mt2,
mt3, and mt4 also showed a peak in fractions 4 and 5, similar
to wild-type VP1. However, mt1, mt5, and mt6, which all
contain
a mutation at Cys-9, were detected in fractions 2, 3, and 4, suggesting
inefficient or unstable interpentameric
assembly.
The observation that all of these mutants formed VLPs prompted us to
purify the VLPs by cesium chloride density gradient
ultracentrifugation.
SDS-PAGE and silver staining demonstrated that all
of the VLPs
were purified to near homogeneity (Fig.
5A). Electron microscopic
observation
showed that all of the mutant VLPs were morphologically
indistinguishable from wild-type VLPs (Fig.
5B). These results
indicate
that disulfide linkages at the mutated cysteines are
not essential for
the formation of VLPs in insect cells. However,
the final yields of
mt1, mt5, and mt6 VLPs were 1/10 to 1/40 of
the yield of wild-type
VLPs, because of dissociation or degradation
during the purification
process (data not shown and see below).
Together with the results of
sucrose gradient centrifugation analysis
(Fig.
4C), these observations
suggest that Cys-9, probably in
a disulfide linkage, may be important
in the stabilization of
VLPs.
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FIG. 5.
Purification and morphological analysis of cysteine
mutant VLPs. (A) Silver staining of purified mutant VP1 proteins.
Cysteine mutant VLPs were purified by cesium chloride density gradient
centrifugation. VLP fractions were separated by SDS-PAGE, and proteins
were visualized by silver staining. (B) Electron micrographs. Purified
VLPs were negatively stained and observed by electron microscopy. Scale
bar, 50 nm.
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To determine which cysteines form intermolecular disulfide linkages in
VLPs, cysteine mutant VLPs were analyzed by SDS-PAGE
under nonreducing
or reducing conditions and then immunoblotted
with anti-VP1 antibody.
Under nonreducing conditions, wild-type
VLPs dissociated to multimers
(more than 200 kDa in size) and
dimers (approximately 100 kDa) as well
as monomer (50 kDa) after
denaturation by boiling in SDS (Fig.
6A, lane
1). These oligomers
were completely
dissociated to monomer by the addition of DTT
(lane 8). These results
showed that intermolecular disulfide linkages
were formed in wild-type
VLPs. mt3 and mt4 VLPs exhibited the
same patterns as wild-type VLPs
(lanes 4 and 5), indicating that
Cys-207 and Cys-254 are not involved
in intermolecular disulfide
linkage. In contrast, mt1 and mt2 VLPs were
dissociated to dimer
and monomer but did not appear as multimers (lanes
2 and 3). The
different electrophoretic mobilities of the dimers of
these mutants
may be due to different conformations (see Discussion).
Since
mt5 and mt6, which carry mutations on both Cys-9 and Cys-104,
formed VLPs which completely dissociated to monomer in the absence
of
DTT (lanes 6 and 7), it appears that both Cys-9 and Cys-104
form
intermolecular disulfide linkages and that no disulfide linkages
which
do not involve these cysteines are formed in VLPs. The partner
cysteines forming disulfide linkages with Cys-9 and Cys-104 are
discussed below. The observation that even mt5 and mt6, which
no longer
formed intermolecular disulfide linkages, formed VLPs
(Fig.
5B) clearly
demonstrates that intermolecular disulfide linkages
are not essential
for VLP formation.
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FIG. 6.
Analysis of intermolecular disulfide linkages and
sensitivity to protease digestion in cysteine mutant VLPs. (A) SDS-PAGE
analysis under reducing or nonreducing conditions. Purified VLPs
assembled from the indicated mutant VP1 proteins were added to SDS
sample loading buffer without -mercaptoethanol. After the mixture
was boiled, DTT (lanes 8 to 14) or nothing (lanes 1 to 7) was added and
the samples were boiled again. The samples were separated by SDS-PAGE
(8% polyacrylamide) and immunoblotted with anti-VP1 antibody. The
circle, diamond, and box indicate the VP1 multimer, dimer, and monomer,
respectively. WT, wild type. (B) Analysis of VLP sensitivity to
proteinase K digestion. Purified VLPs were incubated with 0.1 ng (lanes
2, 6, 10, 14, 18, 22, and 26), 0.3 ng (lanes 3, 7, 11, 15, 19, 23, and
27) or 1 ng (lanes 4, 8, 12, 16, 20, 24, and 28) of proteinase K, or
without proteinase K (lanes 1, 5, 9, 13, 17, 21, and 25), at 37°C for
15 min. The samples were separated by SDS-PAGE and immunoblotted with
anti-VP1 antibody. The lower panel shows the relative intensity of
bands representing intact VP1 protein.
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To test the possibility that disulfide linkages help to stabilize VLPs,
we analyzed the sensitivity of the VLPs to protease
digestion. Purified
VLPs were treated with increasing amounts
of proteinase K, and the
remaining VP1 proteins were detected
by Western blotting with anti-VP1
antibody (Fig.
6B, upper panel).
The relative intensities of the bands
representing intact VP1
proteins are also indicated in the figure (Fig.
6B, lower panel).
mt1, mt2, mt5, and mt6 were more sensitive to
protease than was
wild-type VP1, while mt3 and mt4 showed wild-type
protease resistance.
These results indicate that intermolecular
disulfide linkages
at Cys-9 and Cys-104 contribute to the resistance of
VLPs to
protease.
We also tested the effect of each disulfide linkage on dissociation
caused by DTT and EGTA treatment in vitro. Purified VLPs
were treated
with DTT in either the presence (Fig.
7A)
or absence
(Fig.
7B) of calcium ions and were separated by native
agarose
gel electrophoresis. In the presence of calcium ions, all
mutants
remained in the VLP form (Fig.
7A, lanes 1 to 7), and DTT had
no effect (lanes 8 to 14), demonstrating that the reducing agent
did
not affect VLP dissociation in the presence of calcium ions.
In
contrast, in the absence of calcium ions (note that chelating
agent was
not added), all VLPs dissociated to pentamers following
the addition of
DTT (Fig.
7B, lanes 8 to 15). When no DTT was
added, mt2, mt3, mt4 and
wild-type VP1 remained in VLP form (lanes
1, 3, 4, and 5). However,
VLPs formed from mt1, mt5, and mt6,
all of which carry a mutation at
Cys-9, dissociated to pentamers
(lanes 2, 6, and 7). These results
indicate that in the absence
of calcium ions, VLP dissociation to
pentamers depends on the
reduction of a disulfide linkage at Cys-9. The
results also suggest
that the interpentamer disulfide linkage at Cys-9
prevents VP1
protein from releasing bound calcium ions, which results
in the
stabilization of VLPs through calcium ion-mediated interactions.
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|
FIG. 7.
Analysis of the dissociation of cysteine mutant VLPs to
pentamers in vitro. (A) Native agarose gel electrophoresis in the
presence of calcium ions. Purified VLPs assembled from the indicated
mutants were incubated for 1 h with (lanes 8 to 14) or without
(lanes 1 to 7) 30 mM DTT in the presence of 2 mM CaCl2 and
were separated on a 0.8% agarose gel containing 2 mM CaCl2
in both gel and electrophoresis buffer. Proteins were transferred to a
polyvinylidene difluoride membrane and immunoblotted with anti-VP1
antibody. The circle indicates VLPs. WT, wild type. (B) Native agarose
gel electrophoresis in the absence of calcium ions. Purified VLPs were
processed as in panel A except that no CaCl2 was added to
the samples, gel, or electrophoresis buffer. No chelating agent was
added. Purified wild-type VP1 pentamer was loaded as a control (lane
15). The circle and box indicate VLPs and VP1 pentamer, respectively.
|
|
This idea was further supported by our observation that mt1, mt5, and
mt6 were more susceptible to dissociation or degradation
during the
purification process. Moreover, modification of the
purification
procedure (use of nuclear extracts instead of sonicated
whole-cell
lysates of infected Sf9 cells as starting material,
and addition of 2 mM CaCl
2 in all purification steps) resulted
in an increase
in the final yields of purified mt1, mt5, and mt6
VLPs to levels
comparable to wild-type VLP yields (data not
shown).
Calcium ion binding site mutants.
X-ray
analyses of SV40 virions have revealed that two calcium ions are bound
per VP1 molecule (calcium ions 1 and 2), and the amino acid residues
interacting with these calcium ions have been identified. Three VP1
molecules are linked by these two calcium ions. Calcium ion 1 interacts
with Ser-213 and Glu-216 of the first VP1 molecule, Glu-46 and Glu-48
of the second VP1 in the same pentamer, and Glu-330 of the third VP1 in
a neighboring pentamer. Calcium ion 2 interacts with Glu-157, Glu-160,
Lys-214, and Glu-216 of the first VP1 and Asp-345 of the third VP1
(20, 30). To elucidate the functional roles of these
interactions, we introduced mutations at these residues, as shown
schematically in Fig. 3B. Amino acids which bind to calcium ion 2 (Glu-157, Glu-160, and Asp-345) were mutated singly (mtA, mtB, and mtC)
or in combination (mtD and mtE). The carboxy terminus of VP1 invades
and links to VP1 in the neighboring pentamer through calcium ions 1 and
2; residues implicated in this interaction (Glu-330 and Asp-345) were
substituted in mtF. In this mutant, we also substituted Glu-329 to
avoid the possibility that calcium ion binding to this adjacent residue
might substitute for binding to Glu-330. mtG was mutated at amino acids
near the amino terminus which interact with VP1 molecules in the same
pentamer (Glu-46 and Glu-48). In mtH, all of the amino acid residues
involved in calcium ion binding were mutated. All of these mutants were
introduced into baculovirus vectors and successfully expressed in Sf9
cells at levels comparable to wild-type VP1 expression (Fig. 8A and
B). mtF and mtH showed slightly higher
electrophoretic mobility (apparent molecular mass, approximately 45kDa)
than did the other mutants or wild-type VP1 (approximately 50 kDa),
possibly because of changes in electric charge caused by the mutations.
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FIG. 8.
Expression and VLP formation in Sf9 cells of VP1 calcium
binding site mutants. (A) Analysis of expression in insect cells. Sf9
cells infected with baculoviruses expressing calcium binding site
mutants were lysed at 3 days p.i., and the soluble fractions of
whole-cell lysates were separated by SDS-PAGE and stained with CBB. WT,
wild type. (B) Immunoblotting with anti-VP1 antibody of an identical
gel to that shown in panel A. (C) Analysis of VLP formation in insect
cells. Whole-cell lysates were subjected to 10 to 30% sucrose gradient
centrifugation, and fractions were separated by SDS-PAGE and
immunoblotted with anti-VP1 antibody. The percentages of total VP1
protein represented by the material observed in fractions 8 to 12 are
shown on the right. Inp, input.
|
|
To analyze VLP formation by these mutant proteins in insect cells,
whole-cell lysates of infected cells were subjected to
sucrose gradient
centrifugation (Fig.
8C). Peaks of all mutants
except for mtH were
detected in fractions 8 to 12, indicating
that they formed VLPs
efficiently in insect cells, whereas mtH
VLP formation was relatively
inefficient. More mtF VP1 was detected
in fractions 11 and 12 than in
fractions 9 and 10, suggesting
that this mutant formed particles larger
than normal VLPs. Interestingly,
all of the mutants except mtH showed a
lower tendency to form
the small particles detected in fractions 4 and
5 than did wild-type
VP1; mtD, mtE, and mtF formed particularly low
levels of the small
particles. Densitometric quantification also showed
that a greater
proportion of total VP1 was detected in fractions 8 to
12 in the
analysis of the mutants (except mtH) than in the analysis of
wild-type
VP1. These results indicate that the mutant VP1 proteins
formed
intact VLPs more efficiently than wild-type VP1
did.
All mutant VLPs except for those assembled from mtH were purified to
near homogeneity by cesium chloride density gradient
ultracentrifugation (Fig.
9A). mtH could
not be purified in quantities
sufficient for further analysis even by
the modified purification
procedures described above. Electron
microscopic analysis showed
that all of the purified mutant VLPs were
morphologically indistinguishable
from wild-type VLPs (Fig.
9B). mtF
was unique in that it frequently
formed tubular particles, which may
have a larger sedimentation
coefficient than VLPs, as well as intact
VLPs (Fig.
9B). These
results indicate that the carboxy-terminal
calcium ion binding
sites of VP1 protein affect the accuracy of viral
assembly. No
homogeneous particles, not even the small particles, were
observed
in mtH preparations (data not shown). The inability of this
mutant
to form VLPs indicates that calcium ion-mediated interactions
are essential for VLP formation, although there remains the alternative
possibility that mtH VP1 could no longer achieve the necessary
conformation because of the (calcium-independent) effects of its
many
amino acid substitutions.
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FIG. 9.
Purification and morphological analysis of calcium
binding site mutant VLPs. (A) Purification of mutant VLPs by cesium
chloride density gradient centrifugation. Fractions containing VLPs
were separated by SDS-PAGE, and proteins were visualized by silver
staining. WT, wild type. (B) Electron micrographs. Purified VLPs were
negatively stained and observed by electron microscopy. Scale bar, 50 nm.
|
|
We next analyzed the dissociation properties of these mutants under
various conditions by native agarose gel electrophoresis
(Fig.
10). In the presence of calcium ions
(Fig.
10A), mtA, mtB,
mtC, mtF, mtG, and wild-type VP1 remained in VLP
form in the absence
or the presence of DTT (Fig.
10A, lanes 1 to 4, 7, 8, 9 to 12,
15, and 16). mtG VLPs migrated faster than the other VLPs
(lane
8) despite being indistinguishable from the other VLPs in shape
or size. The difference in migration rate may be due to changes
in
electric charge on the surface of the mtG VLP. The relative
mobilities
of mtD and mtE VLPs were reduced whether or not DTT
was added (lanes 5, 6, 13, and 14), indicating that these mutant
VLPs formed aggregates in
the presence of calcium ions.
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FIG. 10.
Dissociation of calcium ion binding site mutant VLPs to
pentamers in vitro. Purified VLPs assembled from the indicated mutants
were incubated for 1 h with (lanes 9 to 16) or without (lanes 1 to
8) 30 mM DTT in the presence of 2 mM CaCl2 (A), in the
absence of either calcium ion or chelating agent (B), or in the
presence of BAPTA, an efficient calcium ion-chelating agent (C). The
samples were separated on 0.8% native agarose gels with (A) or without
(B and C) 2 mM CaCl2 in both gel and electrophoresis
buffer. VP1 proteins were detected by immunoblotting with anti-VP1
antibody. WT, wild type; pentamer, purified wild-type VP1 pentamer
(panel C, lane 17). The circle and box indicate VLPs and pentamer,
respectively.
|
|
In the absence of calcium ions (Fig.
10B), all mutants as well as
wild-type VP1 remained in VLP form in the absence of DTT
(Fig.
10B,
lanes 1 to 8). Again, mtG VLPs migrated a little faster
than the other
VLPs did. Under these conditions, VLPs of mtD and
mtE did not
aggregate. All of the calcium binding site mutants
behaved differently
from wild-type VP1 in the presence of DTT
(lanes 9 to 16). Under
reducing conditions, wild-type VLPs dissociated
to pentamers (Fig.
7B,
lanes 8 and 15) whereas mtA, mtD, and mtE
VLPs did not dissociate at
all (Fig.
10B, lanes 10, 13, and 14).
mtB, mtC, mtF, and mtG VLPs
dissociated, but the mobility of the
dissociated products was lower
than that of VP1 pentamer, indicating
incomplete dissociation (lanes
11, 12, 15, and 16). In addition,
a fraction of mtB and mtC VLPs
remained intact (lanes 11 and 12).
These results were remarkable
because all of the mutant VLPs were
more resistant to dissociation than
were wild-type VLPs, suggesting
that calcium ion-interacting amino
acids, especially Glu-157,
contribute to the flexibility of
VLPs.
We further analyzed the behavior of these mutant VLPs in the presence
of BAPTA, an efficient calcium ion-chelating agent.
Under these
conditions, addition of DTT caused all VLPs to dissociate
completely to
VP1 pentamers (Fig.
10C, lanes 9 to 17). However,
in the absence of
DTT, none of the mutant VLPs dissociated at
all, while wild-type VLPs
dissociated only partially (lanes 1
to 8). This observation further
indicates that the calcium binding
site mutant VLPs were more rigid
than the wild type and that the
high stability of the mutants was due
to stronger calcium ion-mediated
interactions. These results, taken
together, demonstrate that
the conformation of calcium ion binding
sites moderates the affinity
of VLPs for calcium ions and may confer a
greater tendency to
dissociation on the SV40
capsid.
| |
DISCUSSION |
Utility of the baculovirus expression system for the study of viral
capsid assembly and disassembly.
The advantages of baculovirus
expression systems are that relatively large amounts of recombinant
protein can be expressed in a eukaryotic environment, and folding of
the protein is expected to be similar to that of native protein
(24). To date, recombinant viral capsid proteins from
polyomaviruses (6, 10, 11, 21, 25), papillomaviruses
(17, 18, 26), and adeno-associated virus (12,
13) have been shown to assemble into VLPs in insect cells. We
also utilized this system and demonstrated that VP1 of SV40 formed VLPs
in the nucleus (in which location the capsid of wild-type SV40 is
naturally formed) of insect cells (Fig. 1); that the VLPs were
morphologically indistinguishable from wild-type SV40 particles (Fig.
2B) (19); and that, like wild-type SV40 particles
(7), the VLPs dissociated to VP1 pentamers following treatment with DTT and EGTA in vitro (Fig. 2). Thus, this expression system offers a powerful and convenient system for the study of assembly and disassembly of viral capsid proteins.
Disulfide linkages between cysteine residues stabilize VLPs.
Although a study of SV40 VP1 cysteine mutants using a rabbit
reticulocyte translation system has been reported (15),
this system could produce only pentamers and pentamer-oligomers but not
VLPs due to the low yield of VP1. Thus, it was not suitable for
analysis of particle formation. In the present study, using a
baculovirus expression system, we demonstrated that both Cys-9 and
Cys-104 formed intermolecular disulfide linkages in VLPs, that these
disulfide linkages were not essential for the formation of VLPs in
vivo, and that these disulfide linkages increased the resistance of
VLPs to protease. We also showed that a disulfide linkage at Cys-9 was
critical for maintenance of VLPs at low calcium ion concentrations and
contributed to the stabilization of VLPs against dissociation to
pentamers by preventing the release of calcium ion.
Previous studies of VP1 cysteine mutants using a cell-free expression
system have shown that Cys-9, Cys-104, and Cys-207 are
necessary and
sufficient for disulfide linkage between pentamers
(
15).
In contrast, our present results show that Cys-207 did
not form any
intermolecular disulfide linkage in VLPs (Fig.
6A).
This inconsistency
may result from differences in the protein
expression system. Proteins
synthesized in a cell-free system
may be exposed to air oxidization,
which allows nonspecific disulfide
linkages. Crystallographic analyses
have shown that Cys-207 is
located inside the VP1 molecule in pentamers
(
20,
30), so
that formation of intermolecular disulfide
linkages at Cys-207
seems unlikely. Thus, we propose that Cys-207 does
not form intermolecular
disulfide linkages in the viral
capsid.
On the other hand, our observation that Cys-104 formed a disulfide
linkage agrees with the results from the cell-free system
(
15). Crystallographic analyses of the SV40 virion have
shown
that Cys-104 is located in a loop of VP1 that is very close to
another Cys-104 loop on a neighboring pentamer (
20), and a
disulfide
linkage involving Cys-104 has been identified in
-
'
interpentamer
interaction (
30). Thus, the VP1 dimer shown
in Fig.
6A, lane
2, is probably the result of a disulfide linkage
between Cys-104
and Cys-104.
Our observation that Cys-9 formed a disulfide linkage is also
consistent with the results from the cell-free system
(
15).
The structure of the amino-terminal 13 amino acid
residues of
VP1, a region which includes Cys9, has not been
crystallographically
determined, probably because it is located inside
the capsid (
20,
30). However, since crystallographic
analysis has shown that
no cysteines other than Cys-9 are located
inside the virion, the
most likely disulfide bonding partner of Cys-9
is Cys-9. Thus,
the VP1 dimer shown in Fig.
6A, lane 3, whose mobility
is different
from that of the dimer linked by Cys-104, is probably due
to a
disulfide linkage between Cys-9 and Cys-9. Although the structures
of the amino termini of SV40 and polyomavirus VP1 proteins in
the
capsid have yet to be determined (
20,
30,
31), two
functional
domains have been localized to this region. The first is a
nonspecific
DNA binding domain (
5,
22; P. P. Li, C.-K. Sun, A. Miyao,
and H. Kasamatsu, Abstr. 8th Annu. Meet. Am.
Soc. Virol., p. 107,
1997), which supports the idea that the VP1 amino
terminus extends
inward to interact with viral DNA. The second is a
nuclear localization
signal (
14,
23). In addition to these
functions, we demonstrated
that the amino terminus of VP1 plays a novel
functional role in
strengthening the calcium ion binding affinity by
forming an interpentamer
disulfide bond at Cys-9, which results in
stabilization of
VLPs.
Multimers of mt3, mt4, and wild-type VP1, formed by disulfide linkages,
were observed in nonreducing SDS-PAGE (Fig.
6A). Thus,
the partner VP1
molecule forming disulfide linkages with Cys-9
and Cys-104 appears to
be different in some VP1 molecules, suggesting
that disulfide linkages
form an extensive covalent network throughout
the VLP and contribute to
its
stability.
Calcium ion-mediated interactions of VP1 proteins affect viral
assembly and stability.
Studies of the assembly and disassembly of
SV40 and murine polyomavirus capsids have indicated that calcium ions
are important for capsid formation (2-4, 7). In addition,
the assembly into VLPs of recombinant polyomavirus VP1 expressed in
Escherichia coli was driven by calcium ions at physiological
pH and ionic strength in vitro (27, 28). Further evidence
of the importance of calcium ions in capsid formation is provided by
the observation that the increase in calcium ion concentration induced
by treatment with the calcium ionophore ionomycin causes recombinant
polyomavirus VP1 to assemble into VLPs not only in the nucleus but also
in the cytoplasm of insect cells, at which location VLPs are not formed
under normal physiological conditions (21).
Crystallographic studies of SV40 VP1 have identified three amino acid
residues (Glu-157, Glu-160, and Asp-345) which bind
to calcium ion 2 (
20). More refined crystallographic analysis
has shown
that Lys-214 and Glu-216 are also involved in binding
to calcium ion 2 and that calcium ion 1 interacts with Glu-46,
Glu-48, Ser-213, Glu-216,
and Glu-330 (
30). These studies have
revealed that calcium
ion-mediated interactions form bridges between
a carboxy-terminal arm
extended from one pentamer and the internal
loops of a neighboring
pentamer. In the present study, we analyzed
the functional roles of the
amino acids involved in calcium ion
binding
sites.
We demonstrated that calcium ion binding sites on the carboxy-terminal
arm affect the association of VP1. Unexpectedly, we
found that
mutations at any of the calcium ion-interacting amino
acids tested,
especially Glu-157, resulted in the formation of
more rigid VLPs than
were formed from wild-type VP1. We also showed
that VP1 mutated at all
of these amino acids (mtH) was deficient
for VLP formation, indicating
that calcium ion-mediated interactions
are essential for capsid
assembly, although there remains the
possibility that the large number
of amino acid substitutions
in mtH VP1 resulted in sufficient
conformational change to inhibit
assembly.
The VP1 carboxy terminus plays an important role in
interpentameric interactions. Crystallographic studies of SV40
showed
that in the fully assembled virus shell, most pentamers
contact
each other only through the carboxy-terminal 49 amino acid
residues
(residues 313 to 361) protruding into an adjacent pentamer
(
20,
30). In addition, deletion of the corresponding
region of recombinant
polyomavirus VP1 led to deficient VLP assembly in
vitro, despite
the ability of the mutant VP1 to form pentamers
(
9). It is
interesting that mtF, which has mutations in
the carboxy-terminal
arm, frequently formed tubular particles as well
as intact VLPs
in insect cells. It has been shown that wild-type
polyomavirus
forms such tubular particles at low frequency in vivo, a
phenomenon
which is considered to result from incorrect selection among
three
possible configurations of interpentameric interactions
(
1).
These observations suggest that the carboxy-terminal
calcium ion
binding sites affect the specificity of interpentameric
interactions
during capsid
assembly.
We found that amino acid substitutions at any of the calcium ion
binding sites, especially Glu-157, resulted in the formation
of VLPs
which were more rigid than wild-type VLPs in the absence
of calcium
ions. It thus appears that these calcium ion binding
sites moderate
calcium ion binding affinity, which provides flexibility
to the viral
capsid and promotes dissociation during viral
uncoating.
We also note that mtD and mtE formed VLPs more efficiently than did
wild-type VP1 in insect cells and that these VLPs were
the most rigid
of the mutant VLPs, suggesting that these mutants
might be useful as
capsules to package DNA for gene
therapy.
Biological significance.
Taken together, our present
observations provide a preliminary model of the molecular mechanisms of
SV40 capsid assembly and disassembly in vivo. The concentration of
calcium ions is much higher in blood (approximately 2 mM) than in cells
(approximately 0.05 to 0.3 µM). That VLPs are much more stable and
resistant to reducing agents in the presence of calcium ions indicates
the reason for SV40 virion survival in extracellular environments. Studies of early events in SV40 infection have indicated that for the
transportation of its genomic DNA into the nucleus, where replication
and transcription occur, the capsid must dissociate at least partially
in the cytoplasm, because the capsid is too large to pass through the
nuclear pore complex (reviewed in reference 16).
Thus, it is thought that after binding to cellular receptors and
endocytotic internalization, the low calcium ion concentration and
reducing conditions in the cytoplasmic environment lead to viral
uncoating. During this step, reduction of the Cys-9-to-Cys-9 disulfide linkage may trigger dissociation, and the calcium ion binding
sites probably facilitate the reaction. In the late phase of infection,
newly synthesized VP1 proteins are translocated and assembled into
capsids in the nucleus. At this stage, calcium ion-mediated
interactions at the VP1 carboxy terminus are important whereas
intermolecular disulfide linkages between cysteines are not essential.
Studies of the behavior in mammalian cells of recombinant SV40 carrying
the VP1 mutations presented here may help to further
clarify the
precise molecular mechanisms of capsid assembly and
disassembly.
| |
ACKNOWLEDGMENTS |
This work was supported by a Research Grant from Core Research
for Evolutional Science and Technology (CREST) of Japan Science and
Technology Corporation (JST); a Grant-in-Aid for Scientific Research
from the Ministry of Education, Science, Sports and Culture; and a
grant for research and development projects in Cooperation with
Academic Institutions from New Energy and Industrial Technology Development Organization (NEDO).
We are grateful to Peggy Li and Harumi Kasamatsu (University of
California, Los Angeles, Calif.) for helpful suggestions. We thank
Kenji Suzuki (National Institute of Infectious Diseases, Tokyo, Japan)
for help with electron microscopy.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Frontier
Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan. Phone:
81-45-924-5872. Fax: 81-45-924-5145. E-mail:
hhanda{at}bio.titech.ac.jp.
| |
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Journal of Virology, January 2001, p. 61-72, Vol. 75, No. 1
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.1.61-72.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
