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Journal of Virology, August 2001, p. 7727-7731, Vol. 75, No. 16
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.16.7727-7731.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
DNA-Induced Structural Changes in the
Papillomavirus Capsid
Claudia
Fligge,
Frank
Schäfer,
Hans-Christoph
Selinka,
Cornelia
Sapp, and
Martin
Sapp*
Institute for Medical Microbiology and
Hygiene, University of Mainz, D-55101 Mainz, Germany
Received 27 March 2001/Accepted 10 May 2001
 |
ABSTRACT |
Human papillomavirus capsid assembly requires intercapsomeric
disulfide bonds between molecules of the major capsid protein L1.
Virions isolated from naturally occurring lesions have a higher degree
of cross-linking than virus-like particles (VLPs), which have been
generated in eukaryotic expression systems. Here we show that DNA
encapsidation into VLPs leads to increased cross-linking between L1
molecules comparable to that seen in virions. A higher trypsin
resistance, indicating a tighter association of capsomeres through DNA
interaction, accompanies this structural change.
| |
TEXT |
Human papillomaviruses (HPV) are
nonenveloped DNA viruses harboring a double-stranded DNA genome of
approximately 8,000 bp. They exclusively infect epithelial cells of
skin and mucosa, inducing benign and malignant lesions
(32). The spherical viral capsid with T=7 icosahedral
symmetry (5) is composed of 72 pentameric capsomers
containing 360 copies of the major capsid protein L1 (1).
Sixty of these capsomeres are hexavalent, i.e., have six nearest
neighbors, whereas 12 capsomeres are pentavalent. It is believed that
in addition to L1, 12 copies of the minor capsid protein L2 are
associated with the pentavalent capsomeres (27).
Since the productive life cycle of HPV requires differentiated tissue,
it is difficult to produce significant amounts of virions in vitro.
Therefore DNA-free virus-like particles (VLPs) were generated for the
study of structural and immunological aspects of the capsid and for the
study of virus-cell interactions using eukaryotic expression systems
(10, 14, 21, 29, 31). L1 alone is sufficient for VLP
formation, but L2 is incorporated at the expected molar ratio when
present. Electron microscopic analyses revealed that VLPs are
structurally indistinguishable from virions isolated from naturally
occurring lesions (11). In addition, they induce
neutralizing antibodies (2, 3, 6, 19, 20, 25) and compete
with virions for binding to the cellular receptor (18),
suggestive of a high structural similarity. Recently, systems were
developed that allowed incorporation of marker plasmids into VLPs in
vitro (12, 26) and in vivo (24, 28), yielding
pseudovirions. Pseudovirions are helpful tools for the detection of
neutralizing antibodies (6, 9, 13, 19, 28) as well as for
the study of very early events in infection, such as binding and uptake
of virions (8).
Disulfide bonds between adjacent capsomeres stabilize HPV capsids
(23). Recently, we showed that papillomavirus assembly requires two conserved cysteines to connect capsomeres, resulting in
the formation of L1 trimers (22). In VLPs, about 50% of
L1 proteins are cross-linked by disulfide bonds, whereas the L1
proteins of virions are completely cross-linked. To investigate which
structural differences between VLPs and virions underlie this
observation, we compared DNA-free VLPs and pseudovirions with regard to
disulfide bonding and trypsin sensitivity.
HPV-33 VLPs encapsidate DNA upon long-term infection of insect
cells.
VLPs of HPV type 33 (HPV-33) found in supernatants of
insect cells infected with baculoviruses recombinant for HPV-33L1
(bac33L1) and HPV-33L2 (bac33L2) and cultivated for 2 to 3 weeks in
serum-free Sf900II medium (Life Technologies) were subjected to cesium
chloride density gradient centrifugation. Interestingly, they banded in two broad peaks corresponding to buoyant densities of 1.33 and 1.30 g/cm3 (Fig. 1A). Two peaks
with similar densities were also observed when nuclear extracts from
HPV-induced hand warts were analyzed (Fig. 1B). The corresponding peak
fractions of HPV-33L1/L2 obtained from insect cell supernatants were
further characterized by electron microscopy. As expected, VLPs were
found in the light fraction but interestingly also in the heavy
fractions (Fig. 1C). We therefore designated these fractions light VLPs
(L-VLPs) and heavy VLPs (H-VLPs). Assembly of L1 into particles
with these characteristic buoyant densities has also been reported
during generation of pseudovirions in COS-7 cells, and it was shown
previously that the packaged marker plasmid was exclusively present in
H-VLP fractions (28). These observations suggested that
the H-VLPs obtained from long-term expression of recombinant HPV-33 L1
and L2 proteins in insect cells contained DNA. To verify this
assumption, we isolated DNA from L-VLPs and H-VLPs. Pooled fractions
were extensively dialyzed against phosphate-buffered saline (PBS).
Magnesium chloride (10 mM) and DNase I (250 µg/ml) were added to
digest contaminating unpackaged DNA for 2.5 h at 37°C. The
reaction was stopped with EDTA (200 mM), and VLPs were digested with
proteinase K for 12 h. Subsequently, encapsidated DNA was
extracted by phenol-chloroform, precipitated with ethanol, and labeled
with [32P]dATP, using the Klenow fragment of
Escherichia coli DNA polymerase I. The resulting products
were analyzed by agarose gel electrophoresis and visualized by
autoradiography (Fig. 1D). A DNA smear ranging from 1.5 to 8 kb was
exclusively found in the H-VLP fraction. In addition to the DNA
encapsidated by H-VLPs, a high-molecular-weight DNA larger than 20 kb,
most likely DNA extracted from copurifying baculoviruses, was present
in both L-VLPs and H-VLPs.
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FIG. 1.
Analysis of VLPs from long-term infections. Supernatants
of long-term cultures of insect cell infected by baculoviruses bac33L1
and bac33L2 (A) and a nuclear extract from an HPV-induced hand wart (B)
were subjected to cesium chloride density gradient centrifugation.
Fractions were analyzed by SDS-PAGE, and L1 proteins were detected by
immunoblotting using MAb 33L1-7. The apparent molecular masses of
marker proteins are indicated in kilodaltons. The buoyant densities of
the peak fractions, as determined by refractometry, are indicated by
arrows. (C) Electron micrograph of 1.33-g/cm3 H-VLPs from
insect cell supernatants. (D) Autoradiography of
32P-labeled DNA extracted from L-VLPs (L) and H-VLPs (H)
and subjected to agarose gel electrophoresis. Sizes of marker DNA
fragments are indicated in kilobases.
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Our results demonstrate that DNA encapsidation into papillomavirus-like
particles occurred after long-term infection of insect
cells with
baculovirus-expressed HPV-33 L1 and L2. This could
be observed in
long-term infections only, and H-VLPs were detected
exclusively in the
supernatants of infected cells. We therefore
assume that DNA packaging
occurs late in infection, since DNA
degradation induced by the
baculovirus infection is required to
generate DNA fragments small
enough to be incorporated into VLPs
(
7,
16). It is highly
likely that H-VLPs harbor chromatin
since eukaryotic cells do not
contain naked DNA. In H-VLPs, the
size of encapsidated DNA is rather
heterogenous, with an upper
size limit in the range of 8 kb, which is
also the size of the
viral genome. The fact that the capsid does not
discriminate against
smaller DNA molecules argues against a minimal
size requirement
for incorporation into the viral capsid. This is in
accordance
with DNA incorporation into pseudovirions containing
plasmids
of variable lengths in the range from 5.4 to 8 kb (
12,
24,
26,
28,
30).
DNA packaging into VLPs induces a high degree of disulfide
cross-linking.
In virions, L1 molecules are completely
cross-linked by intercapsomeric disulfide bonds (4, 22),
whereas only 50% of the L1 protein found in VLPs is covalently
connected (22, 29). The differences in disulfide bonding
may possibly be due to different redox potentials in the differentiated
keratinocytes, where HPV virions assemble, and in the cell lines used
for VLP production. Alternatively, DNA encapsidation might induce a
tighter packaging of capsid proteins and thus closer contacts between
cysteines. If DNA encapsidation induces a higher degree of
cross-linking, L-VLPs and H-VLPs prepared from the same cell line
should display differences in disulfide cross-linking. As shown in Fig.
2, about 50% of the L1 molecules found
in L-VLPs from COS-7 cells were disulfide bonded, forming trimers of
150 to 160 kDa as observed previously (22, 29). In H-VLPs,
more than 90% of L1 molecules formed trimers. In addition, when H-VLPs
from the supernatants of insect cells were analyzed under these
conditions, complete cross-linking of L1 was observed. These
observations support the hypothesis that DNA encapsidation allows the
formation of additional disulfide bonds, possibly due to tighter
packaging of capsid proteins.
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FIG. 2.
Disulfide cross-linking of L1 proteins in VLPs and
pseudovirions. L-VLPs (L) and pseudovirions (PV), prepared from COS-7
cells, and H-VLPs (H), prepared from supernatants of long-term-infected
insect cells, were examined by SDS-PAGE under nonreducing conditions.
L1 protein was visualized by immunoblotting using MAb 33L1-7. Apparent
molecular masses of marker proteins are indicated in kilodaltons.
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|
DNA encapsidation renders capsids less sensitive to trypsin
digestion.
We noticed that the L1 protein present in high-density
VLPs and pseudovirions was always less degraded than that in
low-density VLP (Fig. 1A). Similar observations were made when wart
extracts were analyzed in buoyant density gradients (Fig. 1B). This
suggests that DNA encapsidation renders the L1 protein less sensitive
to protease digestion. To further investigate this hypothesis, we carried out trypsin digestions of H-VLPs and pseudovirions
encapsidating cellular and plasmid DNA, respectively, and DNA-free
L-VLPs. Pseudovirions and L-VLPs generated in COS-7 cells were
incubated at 37°C in a total volume of 50 µl of PBS-0.025%
trypsin (Gibco BRL) for up to 24 h. Digestion was terminated by
addition of trypsin inhibitor (Gibco BRL). Since H-VLPs prepared from
supernatants of insect cells were less concentrated, digestion was
carried out in a total volume of 500 µl of PBS-0.025% trypsin,
followed by trichloroacetic acid precipitation. Samples were than
resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) followed by Western blot analysis. Blots were stained using
monoclonal antibody (MAb 33L1-7) or the polyclonal rabbit antiserum
Rb890, respectively. Rb890 was raised against the carboxy-terminal 15 amino acids of HPV-33 L1 (8). As shown in Fig. 3A and
B, pseudovirions and H-VLPs were less
sensitive to trypsin than L-VLPs. After digestion with trypsin for more
than 4 h, full-length L1 protein was still detected in Western
blots of pseudovirions and H-VLPs. In contrast, no full-length L1
protein was present in L-VLP fractions treated with trypsin for only 30 min (Fig. 3A). The main degradation products were fragments with
apparent molecular masses of about 53 and 45 kDa. As shown in Fig. 3C,
using the polyclonal antiserum Rb890 directed toward the C terminus of
L1, only full-length protein was detected. After 1 h of treatment
with trypsin, full-length L1 protein was still present in preparations
of pseudovirions but not in L-VLPs. Obviously, DNA packaging not only
induces a higher degree of cross-linking of L1 proteins via disulfide
bonds but also renders the carboxy terminus of L1 less sensitive to proteases.
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FIG. 3.
Trypsin digestion of pseudovirions, L-VLPs, and H-VLPs.
Pseudovirions (PV) and L-VLPs (L), purified from COS-7 cells infected
with recombinant viruses vac33L1 and vac33L2, and H-VLPs, prepared from
supernatants of long-term bac33L1- and bac33L2-infected insect cells,
were digested with trypsin at 37°C for the indicated periods of time
(hours). Samples were subjected to SDS-PAGE followed by immunoblotting
using MAb 33L1-7 (A and B) or polyclonal rabbit antiserum Rb890 (C).
The apparent molecular masses of marker proteins are indicated in
kilodaltons.
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|
Li et al. (
15) have shown that dithiothreitol (DTT)
treatment of bovine papillomavirus type 1 virions renders the L1
carboxy
terminus trypsin sensitive without complete disruption of the
capsid structure. To investigate if similar observations can be
made
with pseudovirions, we carried out trypsin digestions and
sucrose
gradient sedimentations of pseudovirions treated with
20 mM DTT for 90 min at room temperature. As depicted in Fig.
4A, pseudovirions became sensitive to
trypsin in a manner comparable
to that seen for untreated L-VLPs (Fig.
3A). However, they partially
retained their high sedimentation
velocity, in contrast to L-VLPs,
which completely dissociated into
capsomeres under these conditions
(Fig.
4B). Identical results were
obtained for H- and L-VLPs generated
in insect cells (data not shown).
These data further demonstrate
the similarity of pseudovirions and
H-VLPs with authentic virions.
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FIG. 4.
Trypsin digestion and sucrose gradient analysis of
DTT-treated pseudovirions. (A) DTT-treated pseudovirions (PV) were
digested with trypsin (trp) at 37°C for the indicated periods of
time. Samples were subjected to SDS-PAGE followed by immunoblotting
using MAb 33L1-7. (B) DTT-treated pseudovirions and L-VLPs were loaded
onto a 20 to 40% sucrose gradient and spun for 2.5 h at 36,000 rpm and 4°C in a Beckman SW40 rotor. Eighteen fractions were
collected from the top; proteins were precipitated with trichloroacetic
acid and analyzed by immunoblotting. Analysis of the upper nine
fractions is shown. The apparent molecular masses of marker proteins
are indicated in kilodaltons.
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Trypsin-treated pseudovirions remain infectious.
We were
further interested to assay the infectivity of pseudovirions after
trypsin digestion. Pseudovirions harboring a GFP expression cassette
were further purified by a sucrose step gradient (28) and
treated with trypsin as described above. After addition of trypsin
inhibitor, 6.6 × 104 COS-7 cells (resuspended in
PBS-100 µg of bovine serum albumin/ml [pH 6.8]) were added.
Samples were incubated at 4°C under constant agitation, subsequently
seeded into 24-well plates, and cultivated for 72 h at 37°C. To score
the infection events, medium was removed and wells were screened for
GFP-expressing cells in a fluorescence microscope. Surprisingly,
trypsin treatment for 2 to 3 h resulted in a more than twofold
increase in infectivity, as shown in Fig. 5, whereas longer digestions yielded a
gradual decrease in infectivity as expected. Compared to untreated
pseudovirions, a reduction in infectivity by trypsin treatment was
observed only after overnight (24-h) incubations.
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FIG. 5.
Infectivity assay of trypsin-digested pseudovirions.
Digestions were carried out at 37°C for the indicated periods of time
and terminated by addition of trypsin inhibitor. Subsequently COS-7
cells were added, and infected cells were counted after cultivation for
72 h.
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The increase in infectivity after trypsin digestion may be due to the
removal of noninfectious L-VLPs competing with intact
pseudovirions for
receptor binding. We have observed that only
a fraction of
pseudovirions present in our preparations bind to
cells, suggesting
that VLP binding sites on the cells are saturated
(data not shown).
Trypsin digestion may destroy the less stable
particles, thus
increasing the probability of specific uptake
of pseudovirions.
Alternatively, the increase of infectivity may
be due to activation
of pseudovirions after treatment with proteases.
Similar observations
have been made for poliovirus uptake (
17).
Activation
might be achieved by a conformational change in the
capsid structure
resulting in facilitated binding, uptake, or
uncoating. For bovine
papillomavirus, Li et al. have shown that
such an alteration in the
capsid structure may be important for
virus uncoating
(
15). According to their model, disulfide bridges
in the
capsid structure become cleaved in the reducing environment
of the
cytoplasm. This leads to a swelling of the capsid structure
whereby the
C terminus becomes accessible to proteolytic cleavage
resulting in the
release of
DNA.
In this report we have demonstrated for the first time that DNA
encapsidation into papillomavirus-like particles leads to
the formation
of additional disulfide bonds in addition to those
shown previously to
be essential for VLP assembly (
22). The
increase of
disulfide bonding from about 50 to 100% may be due
to a conformational
change in L1 molecules, which allows a tighter
association of
capsomeres. The decrease in trypsin sensitivity
of pseudovirions versus
VLPs supports this hypothesis. These data
are in line with the
observation of Li et al. that reduction of
disulfide bonds results in a
relaxation of capsid structures (
15).
The increase of
disulfide bonding between L1 molecules caused
by DNA encapsidation does
not significantly affect the overall
outward capsid structure: no
differences between VLPs and virions
were detected by cryoelectron
microscopy (
11), and the antigenic
properties of virions
and VLPs are similar (
2,
3,
20,
25). Since DNA-free
particles can also be extracted from naturally
occurring lesions in
significant amounts (Fig.
1B) (
5), the
higher proteolytic
sensitivity of empty capsids may be one way
by which papillomaviruses
avoid the competition between virions
and VLPs for binding to the
cellular
receptor.
| |
ACKNOWLEDGMENTS |
We are grateful to R. E. Streeck for critical reading of the
manuscript and helpful suggestions throughout the work.
This work was supported by grants to M.S. from the Deutsche
Forschungsgemeinschaft and the Stiftung Rheinland-Pfalz für
Innovation. F.S. received a grant from the Graduiertenkolleg 194.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute for
Medical Microbiology and Hygiene, Johannes-Gutenberg-Universität
Mainz, Hochhaus am Augustusplatz, D-55101 Mainz, Germany. Phone:
49-6131-393-0211. Fax: 49-6131-393-2359. E-mail:
sapp{at}mail.uni-mainz.de.
| |
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Journal of Virology, August 2001, p. 7727-7731, Vol. 75, No. 16
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.16.7727-7731.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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