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Journal of Virology, April 2000, p. 3353-3365, Vol. 74, No. 7
Graduate Institute of Life Science, National
Defense Medical Center and Institute of Molecular Biology, Academia
Sinica, Taipei, Taiwan, Republic of China,1 and
Australian Animal Health Laboratory, CSIRO Animal Health,
Geelong, Victoria, Australia2
Received 30 September 1999/Accepted 4 January 2000
An immunodominant antigen, p35, is expressed on the envelope of
intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by
the viral late gene H3L, but its role in the virus life cycle is not
known. This report demonstrates that soluble H3L protein binds to
heparan sulfate on the cell surface and competes with the binding of
vaccinia virus, indicating a role for H3L protein in IMV adsorption to
mammalian cells. A mutant virus defective in expression of H3L
(H3L Vaccinia virus is the prototypic
member of the poxvirus family of large DNA viruses. Vaccinia virus
replicates in the cytoplasm of infected cells and produces multiple
forms of infectious particles: intracellular mature virions (IMV),
intracellular enveloped virions (IEV), and extracellular enveloped
virions (EEV) (1, 26, 58). Each form of vaccinia virion
contains different membrane structures and is capable of using
different routes for infection (1, 40, 52).
IMV is the most abundant virion form, and many of its surface envelope
proteins have been identified (30, 57). Some of these
envelope proteins play a role in virus entry into the cell. For
example, A27L protein binds to cell surface heparan sulfate (HS) and is
required for fusion of virus-infected cells (9, 17, 22, 43,
44). In addition, A27L protein is associated with A17L protein
and is important for Golgi membrane wrapping of IMV during formation of
IEV and EEV (46, 48). Inactivation of A27L protein
expression resulted in a small plaque phenotype and reduced EEV
production (44). Another envelope protein, D8L, binds to
cell surface chondroitin sulfate (CS); inactivation of D8L expression
reduces IMV binding to cells in vitro and attenuates virus virulence in
mice (23, 34-36). Another envelope protein, L1R, is
important for virus penetration, since a monoclonal antibody (MAb)
recognizing L1R protein blocked virus entry at a postbinding step
(27, 28, 41, 63).
The product of the H3L gene, p35, is another envelope protein that is
an immunodominant antigen found on vaccinia IMV (66). Strong
immune responses to p35 protein have been detected in mice, sheep,
rabbits, and humans (6, 21, 62). Amino acid sequencing of
p35 purified from the LIVP strain of vaccinia virus revealed that it
was encoded by the H3L gene (50, 66). The H3L gene is
present in the genome of different strains of vaccinia virus as well as
in sheep poxvirus, variola, and orf viruses (6, 16, 20, 50,
53). Despite its wide presence in poxvirus family viruses, the
function of H3L protein in the virus life cycle is not known.
Glycosaminoglycans (GAGs) are ubiquitously expressed in many cell
types. Many viruses, such as herpesvirus, dengue virus, and
foot-and-mouth disease virus, bind to GAGs during virus infections (7, 29, 54). Although vaccinia virus binds to cell surface GAGs through A27L and D8L proteins, other viral envelope proteins may
contribute to virion binding and penetration into various cell types
(9, 22, 23). This study investigates the role(s) of H3L
protein in virus attachment to mammalian cells and in the life cycle of
vaccinia virus. A H3L Reagents and viruses.
Soluble heparin (HP), CS, and
dermantan sulfate (DS) were purchased from Sigma Inc. Spurr resin was
purchased from Electron Microscopy Sciences. Wild-type (WT) vaccinia
virus (WR strain) was grown in BSC40 cells. VMJ360, a recombinant
vaccinia virus that expresses lacZ from a synthetic early
promoter, was obtained from B. Moss (12). IMV were purified
by centrifugation of cell lysates through a 25 to 40% sucrose
gradient, and the pellets were stored for use as virus stocks
(31). EEV were collected from fresh media of the infected
cell cultures and used directly without freezing.
Protein expression and purification.
Full-length recombinant
H3L protein was used as an antigen to generate rabbit antisera. The
full-length H3L gene was amplified by PCR with two primers, as follows:
a 5' primer (5'-ATAGGATCCATGGCGGCGGC-3') and a 3' primer
(5'-TCAAAGCTTAGATAAATGCGGTAACG-3'). The PCR product was
cloned into the pVE02 expression vector fused with a six-histidine tag
at the N terminus. Full-length H3L protein was expressed in Escherichia coli BL21(DE3)/pLysS and was purified in the
presence of Triton X-100 as described previously (20).
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Vaccinia Virus Envelope H3L Protein Binds to Cell Surface
Heparan Sulfate and Is Important for Intracellular Mature Virion
Morphogenesis and Virus Infection In Vitro and In Vivo
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) was constructed; the mutant virus has a small
plaque phenotype and 10-fold lower IMV and extracellular enveloped
virion titers than the wild-type virus. Virion morphogenesis is
severely blocked and intermediate viral structures such as viral
factories and crescents accumulate in cells infected with the
H3L mutant virus. IMV from the H3L mutant
virus are somewhat altered and less infectious than wild-type virions.
However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not
required for cell fusion. Mice inoculated intranasally with wild-type
virus show high mortality and severe weight loss, whereas mice infected
with H3L mutant virus survive and recover faster,
indicating that inactivation of the H3L gene attenuates virus virulence
in vivo. In summary, these data indicate that H3L protein mediates
vaccinia virus adsorption to cell surface heparan sulfate and is
important for vaccinia virus infection in vitro and in vivo. In
addition, H3L protein plays a role in virion assembly.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
mutant virus was constructed and
characterized. The properties of the H3L mutant virus
indicate that H3L protein is important for virus infectivity in vitro
and in vivo as well as for virus morphogenesis.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-D-thiogalactoside (IPTG) for 30 min at 37°C; cells were harvested, resuspended, and lysed by
sonication; the lysate was centrifuged; and the supernatant was loaded
onto a nickel column and purified as described previously (22).
Rabbit antisera and neutralization assays. Two New Zealand White rabbits were inoculated intramuscularly with 250 µg of the purified recombinant full-length H3L protein in Freund's incomplete adjuvant, boosted 3 and 5 weeks after the first inoculation, and bled 1 week after the second boost. The immune response of the animals was checked by Western immunoblot analysis.
For neutralization assays, preimmune or postimmune sera (B&C) at various dilutions were mixed with 200 PFU of WT vaccinia virus at 4°C for 30 min. The mixtures were added to BSC40 cells in 60-mm-diameter dishes in duplicate and incubated at 37°C for 1 h. Cells were washed with phosphate-buffered saline (PBS) and overlaid with 1% agar. Plaque numbers were determined after 3 days. Control plaque numbers were between 100 and 150 plaques per 60-mm-diameter dish. The percent plaque formation was determined by the following calculation: [(number of plaques in the presence of antiserum)/(number of plaques in the absence of antiserum)] × 100.Biotinylation of soluble H3L protein. Biotinylation of soluble ectodomain of H3L protein was performed with an ECL biotinylation system from Amersham Life Science, Inc. In brief, 1 mg of purified recombinant H3L ectodomain protein was mixed with 40 µl of biotinylation reagent N-hydroxysuccinamide ester in 40 mM bicarbonate buffer (pH 8.6) at room temperature for 1 h according to the manufacturer's instructions. The mixture was loaded on a 9-ml Sephadex G25 column previously equilibrated with PBS. Biotinylated H3L protein was collected in 500-µl aliquots from fractions 5 to 9, and the extent of biotinylation was confirmed by Western blot analysis with alkaline phosphatase-conjugated streptavidin, as described previously (9).
Cell binding assays. BSC40 cells (106) were washed with cold PBS and incubated with biotinylated H3L protein (10 µg/100 µl) in staining medium (PBS-4% fetal bovine serum-10 mM HEPES [pH 7.2]). In some experiments, GAGs (50 µg/ml) were also added as competitors. The mixture was incubated at 4°C for 60 min with gentle rotation. Cells were washed with cold staining medium, and phycoerythrin-conjugated streptavidin (1:100) was added for another 60 min at 4°C. Cells were washed three times with cold PBS and analyzed with a fluorescence-activated cell sorter (FACS) (excitation, 488 nm; emission, 578 nm) as described previously (22).
Virion binding assays.
To test if the soluble ectodomain of
H3L protein blocks vaccinia virus infection at the binding step, 4 × 105 BSC40 cells were incubated with various amounts of
soluble H3L protein (0, 1, 10, or 50 µg in 300 µl) at 4°C for 30 min. The cultures were subsequently infected with WT vaccinia virus at a multiplicity of infection (MOI) of 5 PFU per cell at 4°C for 30 min. After washing, cells were immediately harvested for plaque assays
on BSC40 cells. The number of plaques was 100 to 150 per 60-mm-diameter
dish. The percent inhibition was determined by the following
calculation: [1 
(number of plaques in the presence of H3L
protein)/(number of plaques in the absence of H3L protein)] × 100.
-galactosidase
(-gal) assays as described previously (22).
Construction of H3L mutant vaccinia virus.
The
full-length H3L gene was amplified by PCR using a 5' primer
(5'-AATAAGCTTATGGCGGCGGTGAAAACT-3') and a 3' primer
(5'-CTCGGATCCTTAGATAAATGCGGTAAC-3') as described previously
(16). The PCR product was digested with BamHI and
HindIII and cloned into pBluescript KS (Stratagene), resulting in pBS-H3L. A lacZ expression cassette was
obtained from pSC11-5 by restriction digestion with SalI and
PstI (5). The lacZ cassette was
blunt-ended and cloned into an EcoRV site within the H3L
region of pBS-H3L so that the lacZ cassette insertionally inactivated the H3L gene.
-D-glucoronic acid (X-Gal) (150 µg/ml). Pure recombinant H3L virus
expressing -gal was isolated after three rounds of plaque purification. To confirm that the mutant virus contained only a single
insertion, two independent H3L virus isolates were
characterized. Viral DNA was purified from these isolates, and
restriction digestions were performed. The results indicated that the
lacZ gene was inserted into the H3L locus in both isolates
and that both isolates had identical restriction patterns. Subsequent
experiments were carried out with one isolate.
H3L protein expression was examined in IMV (2 × 106
PFU) and cell lysates infected with WT and H3L mutant
viruses. Protein samples were separated by sodium dodecyl sulfate
(SDS)-12% polyacrylamide gel electrophoresis (PAGE) and transferred
for Western blot analysis with antiserum B (1:1,000) against H3L protein.
Cell fusion assay induced by low pH treatment.
BSC40 cells
were infected with WT or H3L mutant virus at an MOI of 5 PFU per cell and incubated at 37°C for 24 h. Cells were washed
three times with PBS (pH 7.2), treated with PBS (pH 4.8) for 3 min at
room temperature, and washed again, and then the PBS was replaced with
normal medium. These cells were incubated for another 3 h and
photographed with a Nikon inverted microscope.
Pulse-chase labeling for viral protein synthesis and proteolytic
processing of P4a and P4b core proteins.
BSC40 cells were infected
with WT or H3L mutant virus at an MOI of 10 PFU per cell,
and the cultures were incubated at 37°C. At 8 h p.i. viral
protein synthesis was monitored with 35S-methionine (50 µCi/ml) for 30 min and chased with normal growth medium for various
times (0, 15, and 30 min and 1, 2, 4, 12, and 24 h). At each time
one set of samples was harvested, lysed in SDS-containing buffer,
separated by SDS-10% PAGE, and analyzed by autoradiograms as
described previously (60).
Electron microscopy of virus morphogenesis and virion particle
determination.
BSC40 cells were seeded on round coverslips and
infected with WT or H3L mutant virus at an MOI of 20 PFU
per cell. These cells were directly fixed on coverslips at 24 h
p.i. in 2.5% glutaraldehyde in 0.1 M sodium phosphate saline buffer
(pH 7.2) at room temperature for 1 h and rinsed in three 15-min
changes of the same buffer. Cells were then treated with 1%
OsO4 in 0.1 M sodium phosphate (pH 7.2) at room temperature
for 60 min and subsequently washed three times in the same buffer.
Cells were dehydrated using an ethanol series, and Spurr's resin was
used for infiltration and embedding, as described previously
(55). After embedding, cells were separated from coverslips
and used for thin sectioning with an Ultracut ultramicrotome. Thin
sections of 90 nm were stained with uranyl acetate and lead citrate and
analyzed with a Zeiss 902 transmission electron microscope
(42).
Assays for virulence. Male BALB/c mice 5 to 6 weeks old were individually weighed on a daily basis for a week prior to infection. They were anesthetized and infected intranasally with 5 µl of diluted IMV for infection with 105, 106, and 107 PFU per mouse. Each day, mice were individually weighed and monitored for signs of illness. The average weight change of the five mice in each group was calculated.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Antisera against H3L protein neutralize vaccinia virus IMV
infection, and soluble H3L protein blocks vaccinia virus IMV adsorption
to cells.
The H3L gene of vaccinia virus encodes a protein of 324 aa with a potential transmembrane region close to the C terminus
(21). The H3L gene is conserved among poxviruses (Fig.
1A). An alignment of known vaccinia H3L
gene and its homologues in other poxviruses is shown in Fig. 1A, which
reveals highly conserved regions. The middle region, from aa 80 to 286, is more conserved than the N-terminal region, from aa 1 to 80. Two
cysteine residues at positions 86 and 90 are identical in all
poxviruses. Furthermore, two short regions, aa 94 to 101 and aa 159 to
164, have sequences similar to the consensus GAG-binding motifs
(X-B-B-B-X-X-B-X and X-B-B-X-B-X, where B is a basic residue and X is a
hydropathic residue) (4). Since H3L protein is expressed on
the IMV envelope, the presence of potential GAG-binding sites suggests
a role for H3L protein in virus adsorption to cells during infection
(20). Experiments described below explore this possibility.
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Soluble H3L protein bound to cell surface HS.
The above
results suggest that H3L protein may bind to the cell surface. This
idea was tested by incubating soluble biotinylated H3L protein with
BSC40 cells and analyzing them with a FACS (Fig. 2). H3L protein bound to BSC40 cells, and
a significant shift in cell surface staining was detected (red lines in
Fig. 2A to C). Soluble GAGs were also added as competitors for H3L
protein binding. HP at concentrations of 10 and 100 µg/ml reduced H3L protein binding to cells and shifted the fluorescence intensity more
than 10-fold (Fig. 2A). Addition of HP at a concentration of 1,000 µg/ml did not reduce the fluorescence staining further, suggesting
that 100 µg of GAGs per ml was adequate to saturate the available HP
binding sites (data not shown). Addition of CS at concentrations of 10 and 100 µg/ml had no effect on H3L protein binding (Fig. 2B), whereas
10 µg of DS per ml (Fig. 2C) did not inhibit binding but 100 µg of
DS per ml caused partial inhibition.
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Inactivation of H3L gene expression affects vaccinia virus growth
in cell culture.
To investigate the biological importance of H3L
protein, an H3L mutant virus was constructed by
homologous recombination, as described in Materials and Methods. The
mutant had a lacZ cassette inserted into the H3L gene so
that H3L protein synthesis was abolished. The H3L mutant
virus was viable, and a pure stock producing blue plaques was isolated
after three rounds of plaque purification on BSC40 cells. Thus, the H3L
gene is not essential for vaccinia virus plaque formation on BSC40
cells. However, the plaques formed by the H3L mutant
virus were smaller than plaques formed by the WT virus (Fig.
3A). Two independent mutant virus clones
were isolated, and their behavior was comparable in cell culture. One
isolate of the H3L mutant virus was used for all
subsequent experiments.
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mutant and
WT virus using Western blot analyses of purified IMV and virus-infected cell lysates (Fig. 3B). A viral protein of 35 kDa was detected in
purified virions and cell lysates from a WT virus infection (Fig. 3B,
lanes 2 and 3), but no protein was detected in samples from an
H3L mutant virus infection (Fig. 3B, lanes 4 and 5),
indicating that expression of H3L protein is completely interrupted in
the H3L mutant virus. Consistent with this fact, the
H3L mutant virus is resistant to neutralization by
antibodies against H3L protein (Fig. 3C, top panel). The acquisition of
resistance to these antibodies was specifically due to the absence of
H3L protein on the virion, since antibody against other envelope
proteins such as L1R neutralized H3L mutant virus and WT
virus to a similar extent (Fig. 3C, lower panel).
Growth of the H3L mutant virus in cell culture was
examined by one-step growth curve analysis (Fig.
4). BSC40 cells were infected with WT or
H3L mutant viruses at an MOI of 5 PFU per cell and
harvested at various times to monitor IMV production. The titer of WT
virus increased more than two log units, reaching 1 × 108 PFU/ml; in contrast, the titers for H3L
mutant virus only increased to 6 × 106 PFU/ml. H3L
protein deficiency apparently reduced the yield of vaccinia virus IMV
more than 10-fold. In addition, the titer of EEV produced during
H3L mutant virus infection was also reduced (Table
1).
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Inactivation of H3L gene expression affects virion
morphogenesis.
There are two possible explanations for reduced
virus titers during infection by the H3L mutant virus.
The H3L mutant virus may be defective at some stage of
the virus life cycle such as during viral gene expression or regulation
or virion assembly. Alternatively, the H3L mutant virus
may grow well but produce virion particles of lower infectivity than WT
virus. Viral gene expression was examined in cells infected by the
H3L mutant virus. BSC40 cells were infected with WT or
H3L mutant virus at an MOI of 5 PFU per cell, and viral
protein synthesis was monitored at various times p.i. with
35S-methionine. Shutoff of host protein synthesis was
evident at 8 h p.i., and the overall pattern of viral protein
synthesis was comparable up to 24 h p.i. for H3L
mutant and WT virus (data not shown). This result indicates that the
H3L gene is not important for viral gene expression or regulation.
mutant virus
contained mostly intermediate viral structures such as crescents
associated with viral factories and immature virions (IV) (Fig. 5A and
E) in the cytoplasm, with only a slight difference in the assembly
process, i.e., cells infected by the mutant virus seemed to accumulate
more nucleoprotein mass. However, at 24 h p.i., progression of
virion assembly for WT and H3L mutant virus was
dramatically different. In cells infected by WT virus, IMV and some IV
were present (Fig. 5B); in cells infected by mutant virus, there were
more viral crescents associated with nucleoprotein mass and unclosed
viral membrane structures were predominant (Fig. 5F). This difference
became more pronounced at 48 h p.i., when most cells infected by
WT virus contained more IMV (Fig. 5C), but the number of IMV in cells
infected by mutant virus was greatly reduced (Fig. 5G). Few cells
infected by WT virus still contained IV at 48 h p.i. (Fig. 5D),
but most cells infected with H3L mutant virus contained
many viral crescents and IV (Fig. 5H). These data indicate that
deficiency in H3L protein interrupts the IMV assembly process, most
likely affecting IV conversion to IMV.
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mutant virus, BSC40 cells
were infected by each virus, pulsed at 8 h p.i., and chased for
periods of up to 24 h p.i. (Fig. 6). In cells infected by WT virus, the processing of P4a and P4b was initiated after 60 min of chasing time. Most of the precursor processing was accomplished after a 4-h chasing period (Fig. 6A). In
cells infected by H3L mutant virus, little processing was
observed within the first 2-h chasing period. Cleavages of P4a and P4b
proteins were observed after 4 h of chasing time, although the
processing was only partial even after 24 h of chasing (Fig. 5B).
Thus, in the absence of H3L protein the vaccinia virus life cycle
proceeds beyond the core protein processing step and is arrested at a
step downstream of where it is a arrested in F18R,
I7L, and L1R mutants.
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mutant. Blockage of the
morphogenesis pathway might also alter accumulation of other virions
forms such as EEV and IEV. EEV are derived from Golgi wrapping of IMV
particles. A decrease in EEV production was observed for the
H3L mutant, as shown in Table 1. While the amount of IEV
in cells is difficult to quantitate, its importance for cell-to-cell
transmission in plaque formation has been demonstrated (51).
The fact that the H3L mutant virus formed small plaques
implies that the number of IEV with actin tails is also affected
(10, 11).
In summary, deficiency in H3L protein expression significantly blocks
IV maturation to IMV particles in cells; consequently, the production
of IEV and EEV were also affected in the H3L mutant.
Previously, two IMV envelope proteins, A27L and D8L, were shown to bind
to GAGs during vaccinia virus entry into cells. However, neither of
these two proteins is required for IMV assembly and only A27L is
essential for formation of EEV (46). Thus, H3L protein is
unique in that it plays dual roles in the vaccinia virus life cycle;
H3L protein is a GAG-binding protein during virus entry, and it
mediates the virion assembly process.
H3L mutant IMV exhibit structural alterations and
have low infectivity compared to WT virus.
As indicated above, it
is possible that H3L mutant particles are less infectious
than WT virus particles. Since H3L protein also mediates virion binding
to cell surface HS, the absence of H3L protein on the IMV envelope may
result in defective virion adsorption. To test if H3L
mutant virus is as infectious as WT virus, the particle-to-PFU ratio
(i.e., the specific infectivity) of both IMV virions was determined.
Purified WT and H3L mutant virions were counted under the
electron microscope, and biological titer assays were performed as
described previously. The particle-to-PFU ratio was 4.5 ± 2.7 for
WT virions and was 28 ± 6.9 for the H3L mutant
(values are means ± standard deviations). This result demonstrates that the H3L mutant virion has sixfold lower
infectivity than WT.
mutant virus to bind to cells. To test
this idea, WT and H3L mutant virions were incubated with
BSC40 cells at 4°C for various times, cells were harvested, and the
number of cell-associated viruses were determined by plaque assays.
Binding of WT virus to cells increased with incubation time and was
saturated after 4 to 5 h. However, binding of H3L
mutant IMV to cells was lower than that of WT IMV even after a 5-h
incubation (data not shown). These data are consistent with the soluble
protein competition experiments and indicate that H3L protein acts to
help IMV adsorption to HS on the cell surface. In addition, other
variables may also contribute to the low infectivity of
H3L mutant virus. For example, H3L protein may play a
structural role to help maintain of the shape of IMV and its absence
could have a pleiotropic effect on the virion structure. Indeed, by electron microscopy, the purified H3L mutant virus
appeared slightly bigger than WT virus, with approximately a 10%
increase in length in both dimensions (300 ± 13 nm by 235 ± 11 nm versus 271 ± 11 nm by 211 ± 7 nm) (Fig.
7A). It thus appears that H3L protein is
important for virion rigidity and that H3L mutant virions
either are loose in native structure or are more vulnerable to
environmental alterations such as osmotic change and become swollen
during virion preparation or the staining process.
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mutant virions did not change the ability of antibody 2D5 to neutralize L1R protein on these mutant virions (Fig. 3C), it did not exclude the
possibility that other envelope proteins are affected. It is
conceivable that if the above-mentioned virion structural alterations indirectly influence the conformation of other GAG-binding proteins, such as A27L and D8L proteins, the virion infectivity could be affected
as well. Western blot analysis confirmed that both A27L and D8L
proteins are still present on purified H3L mutant virions
(data not shown). To analyze the native structures of A27L and D8L
proteins on IMV we compared H3L mutant virus and WT virus
for their sensitivity to neutralization by anti-A27L and anti-D8L
antisera, respectively. As shown in Fig. 7B, anti-A27L serum E5, at a
1:24,000 dilution, neutralized 50% of WT virus plaque formation,
whereas a lower dilution of 1:6,000 was required to inhibit
H3L mutant virus to the same extent, indicating that
H3L mutant became more resistant to the anti-A27L serum.
In contrast, anti-D8L serum exerted an opposite effect on these two
viruses, since a dilution of 1:200 led to 50% inhibition of the
plaques produced by WT virus, whereas a higher dilution of 1:1,600 was needed for H3L mutant virus (Fig. 7C).
Taken together, the above data revealed a possible role of H3L protein
for virion architecture. Consequently, the lower infectivity of
H3L mutant virus may not be due to the lack of H3L
protein per se. Instead, the side effects on other viral GAG-binding
proteins due to H3L deletion need to be considered as well.
H3L protein is not required for fusion of virus-infected cells
under acidic treatment.
Cells infected by vaccinia virus undergo
cell fusion when they are briefly incubated in acidic buffer with a pH
level below 6 (13, 17). Previously, it was shown that IMV
envelope protein A27L is required for fusion of infected cells and the
N-terminal HS-binding region is necessary for the fusion activity
(17, 22, 44). Although parameters mediating virus and cell
fusion are not necessarily identical with those for fusion of infected cells, the cell fusion assay has been widely used to investigate virus
and cell fusion. Since H3L protein also binds to HS, its potential role
in cell fusion was examined (Fig. 8).
BSC40 cells infected with WT virus and maintained at a neutral pH level
did not develop cell fusion at 24 h p.i. (Fig. 8B). However, when these cells were briefly treated with acidic buffer they developed cell
fusion (Fig. 8D). Cell fusion progressed to form giant cells containing
multiple nuclei which ultimately floated up and died. Similarly, cells
infected by H3L mutant virus rounded up (Fig. 8C) and
fused together when exposed to acidic buffer (Fig. 8E). Although for
these cells the kinetics of fusion was slow and the extent of cell
fusion was less complete compared to those of cells infected by WT
virus, we conclude that H3L protein is not required for cell fusion.
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H3L mutant virus has attenuated virulence in
vivo.
Since the H3L mutant virus is defective in
cell culture growth in vitro, the biological importance of H3L protein
was determined during vaccinia virus infection of mice. BALB/c mice
were infected with WT and H3L mutant viruses
intranasally, and body weight was measured on a daily basis (56,
59). As shown in Fig. 9, at the
highest dosage of 107 PFU per animal all the mice infected
with WT virus lost a significant amount of weight and died after 10 days (Fig. 9A). In contrast, mice infected with H3L
mutant virus lost weight initially, but then they regained weight gradually and recovered after 2 weeks. At the lower dosage of 106 or 105 PFU per animal, the body weight loss
of mice infected with WT virus was more severe than that of mice
infected by H3L mutant virus (Fig. 9B and C). These data
provide direct evidence that H3L protein is important for vaccinia
virus virulence in vivo.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Vaccinia virus has a wide host range and binds to cell surface GAGs during virus entry (9). Previously, two envelope proteins on IMV, A27L and D8L, were shown to mediate virus-GAG interactions with different specificity (9, 22, 23). A27L protein binds to cell surface HS, whereas D8L protein recognizes cell surface CS (9, 23). Mechanistically, D8L protein plays a role in IMV adsorption to cells, whereas A27L protein is more involved in virus penetration (23, 45, 47).
Since GAGs are widely expressed on cells, these studies indicate that GAG recognition helps recruit vaccinia virus to interact with a wide variety of cell types. However, mutant viruses defective for A27L and/or D8L gene expression have been constructed and were viable in cell culture (23, 38, 46, 49). One possible explanation is that other viral proteins substitute for the GAG-binding function in these mutant viruses. Here, we report the identification of a third IMV envelope protein, H3L, that binds to cell surface HS during virus entry. We show that soluble H3L protein binds to cell surface HS and blocks IMV adsorption to cells. In addition, inactivation of the H3L gene results in a sixfold reduction of virion infectivity in vitro and significant attenuation of virulence in vivo. Although H3L, like A27L protein, binds to HS, our data suggest that it mediates the initial adsorption of IMV to cells rather than the penetration stage (9, 22, 46).
From a biochemical point of view, both A27L and H3L proteins may bind to HS through similar motifs. Short hexapeptide sequences rich in basic amino acids have been identified as consensus GAG-binding sites; A27L protein has one such sequence and H3L protein has at least two (4). The consensus GAG-binding site on A27L protein has been shown to be critical for A27L protein binding to cell surface HS and cell fusion (22). Therefore, the two putative GAG-binding sites on H3L protein may be responsible for HS binding. Future studies with various mutant H3L protein constructs are needed to explore this issue.
In addition to its role as a GAG-binding protein during virus entry,
H3L protein also participates in virion morphogenesis, since deficiency
of H3L protein severely interrupts assembly of IMV in cells. The
assembly defect is not due to a polar effect on neighboring gene
expression, since H4L mutant was reported with a
different phenotype (32, 64). Furthermore, a revertant virus
expressing H3L protein was constructed from the H3L mutant virus. In
cells infected by the revertant virus, both H3L protein expression and
virion morphogenesis was restored, supporting the idea that H3L protein
has a specific role in virion assembly (data not shown). Our data with
core protein processing further indicate that H3L protein is not
required for P4a and P4b processing but is required for IV conversion
to IMV during virion morphogenesis.
The electron microscopic images of H3L mutant-infected
cells revealed unusually large amounts of dense nucleoprotein mass with
unclosed crescents. After literature searches we found that mutations
of two other vaccinia virus genes such as A8L and D6R resulted in a
similar phenotype (24, 25). Vaccinia virus A8L and D6R
proteins are subunits of viral early transcription factors of 82 and 70 kDa and are assembled into mature virions. Cells infected by
A8L or D6R mutant virus accumulated
immature particles and granular masses associated with viral crescents,
reminiscent of what was observed for H3L mutant virus.
Why would the mutations of early transcription factors result in
morphogenesis blockage similar to that observed with H3L mutation? One
possibility is that encapsidation of A8L and D6R proteins somehow
requires H3L protein. Alternatively, these early transcription factors
may regulate certain late genes such as H3L that are required for
morphogenesis. If the first hypothesis is correct, we would expect that
the specific activity of permeabilized H3L mutant virions
in transcription in vitro would be lower than WT virions. If the second
hypothesis is right, we would expect that no H3L expression occurs in
cells infected by A8L or D6R mutant virus.
These interesting possibilities will be sorted out in the future.
The blockage of IMV formation due to H3L mutation also affects
subsequent formation of IEV and EEV indirectly. Such pleiotropic effects on all three forms of virions may explain the phenotype of the
H3L mutant virus. For example, the titer of EEV in cells
infected by the H3L mutant was reduced to 10% of the WT
level. In addition, the plaque size was previously shown to be
correlated with the ability for actin tail formation on the tips of IEV
(10, 11). The small plaque phenotype of H3L
mutant virus may reflect the fact that fewer IEV are formed in cells
(37, 51).
Although mature H3L mutant IMV were greatly reduced in
number, they were not completely eliminated in the infected cells. The
reason for incomplete interruption of virion morphogenesis is not
understood, but it is not likely that the interruption is due to
low-level expression of H3L protein expression in cells infected by the
mutant virus. Western blot analyses and neutralization assays indicate
that no detectable level of H3L protein is expressed by the mutant virus.
Finally, H3L protein may also be important for virion structure
maintenance, based on electron microscopy and neutralization analyses.
When purified H3L IMV were examined by electron
microscopy, they still appeared brick-shaped but were slightly bigger
than WT virions. At first we assumed that the lack of H3L protein did
not cause gross structure abnormality in IMV, since both WT and the
mutant IMV are equally sensitive to 2D5, a neutralizing MAb against L1R
protein (39). However, the mutant virus exhibits an altered
sensitivity to neutralization effects of antibodies against A27L and
D8L proteins, indicating possible structural alterations of these two
GAG-binding proteins on H3L mutant virions. Thus, the
interpretation of lower infectivity of H3L mutant virions
needs to be done very carefully, for it may not be a direct consequence
of the lack of H3L protein per se.
The attenuation of H3L mutant virulence in vivo could be
due to several factors. The mutant IMV were less efficient in
initiating infections in new hosts since binding to GAGs may not be
optimal. In addition, the infected cells produced fewer progeny,
including IMV and EEV. Consequently, the severity of secondary
infections transmitted to distant organs through the bloodstream in
infected animals may be reduced.
In summary, this study indicates that vaccinia virus uses multiple envelope proteins to bind to cell surface GAGs. This may be an adaptation in cell culture due to extended passages in vitro. It appears that vaccinia virus possesses a battery of GAG-binding proteins to expand its ability to infect different cells and to maximize its binding capacity by recognizing different GAG-containing structures on the cell surface. For example, if a cell expresses both HS and CS on the surface, vaccinia virus could simultaneously recognize both structures and effectively engage virus adsorption onto the cells. On the other hand, some cells may only express a particular type of GAG. In that case, the presence of arrays of GAG-binding proteins on the virion may ensure that vaccinia virus binds to diverse cell populations. The interactions of envelope protein with GAGs only initiate the first step in virus entry. Further investigation will be required for understanding additional steps of vaccinia virus entry into cells.
| |
ACKNOWLEDGMENTS |
|---|
We thank Sue-Ping Lee for excellent techniques in electron microscopy. We also thank F. Tufaro for L, gro2C, and sog9 cells and R. Condit for suggestions regarding the manuscript.
This work was supported by grants from Academia Sinica and the National Science Council (NSC89-2311-B-001-080) of the Republic of China.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan 11529, Republic of China. Phone: 886-2-2789-9230. Fax: 886-2-2782-6085. E-mail: mbwen{at}ccvax.sinica.edu.tw.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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[Abstract] [Full Text] -
Davies, D. H., McCausland, M. M., Valdez, C., Huynh, D., Hernandez, J. E., Mu, Y., Hirst, S., Villarreal, L., Felgner, P. L., Crotty, S.
(2005). Vaccinia Virus H3L Envelope Protein Is a Major Target of Neutralizing Antibodies in Humans and Elicits Protection against Lethal Challenge in Mice. J. Virol.
79: 11724-11733
[Abstract] [Full Text] -
Townsley, A. C., Senkevich, T. G., Moss, B.
(2005). The Product of the Vaccinia Virus L5R Gene Is a Fourth Membrane Protein Encoded by All Poxviruses That Is Required for Cell Entry and Cell-Cell Fusion. J. Virol.
79: 10988-10998
[Abstract] [Full Text] -
Chahroudi, A., Chavan, R., Koyzr, N., Waller, E. K., Silvestri, G., Feinberg, M. B.
(2005). Vaccinia Virus Tropism for Primary Hematolymphoid Cells Is Determined by Restricted Expression of a Unique Virus Receptor. J. Virol.
79: 10397-10407
[Abstract] [Full Text] -
Townsley, A. C., Senkevich, T. G., Moss, B.
(2005). Vaccinia Virus A21 Virion Membrane Protein Is Required for Cell Entry and Fusion. J. Virol.
79: 9458-9469
[Abstract] [Full Text] -
Carter, G. C., Law, M., Hollinshead, M., Smith, G. L.
(2005). Entry of the vaccinia virus intracellular mature virion and its interactions with glycosaminoglycans. J. Gen. Virol.
86: 1279-1290
[Abstract] [Full Text] -
Senkevich, T. G., Moss, B.
(2005). Vaccinia Virus H2 Protein Is an Essential Component of a Complex Involved in Virus Entry and Cell-Cell Fusion. J. Virol.
79: 4744-4754
[Abstract] [Full Text] -
Chung, C.-S., Huang, C.-Y., Chang, W.
(2005). Vaccinia Virus Penetration Requires Cholesterol and Results in Specific Viral Envelope Proteins Associated with Lipid Rafts. J. Virol.
79: 1623-1634
[Abstract] [Full Text] -
Fogg, C., Lustig, S., Whitbeck, J. C., Eisenberg, R. J., Cohen, G. H., Moss, B.
(2004). Protective Immunity to Vaccinia Virus Induced by Vaccination with Multiple Recombinant Outer Membrane Proteins of Intracellular and Extracellular Virions. J. Virol.
78: 10230-10237
[Abstract] [Full Text] -
Senkevich, T. G., Ward, B. M., Moss, B.
(2004). Vaccinia Virus Entry into Cells Is Dependent on a Virion Surface Protein Encoded by the A28L Gene. J. Virol.
78: 2357-2366
[Abstract] [Full Text] -
Hung, J.-J., Hsieh, M.-T., Young, M.-J., Kao, C.-L., King, C.-C., Chang, W.
(2004). An External Loop Region of Domain III of Dengue Virus Type 2 Envelope Protein Is Involved in Serotype-Specific Binding to Mosquito but Not Mammalian Cells. J. Virol.
78: 378-388
[Abstract] [Full Text] -
Crotty, S., Felgner, P., Davies, H., Glidewell, J., Villarreal, L., Ahmed, R.
(2003). Cutting Edge: Long-Term B Cell Memory in Humans after Smallpox Vaccination. J. Immunol.
171: 4969-4973
[Abstract] [Full Text] -
Belyakov, I. M., Earl, P., Dzutsev, A., Kuznetsov, V. A., Lemon, M., Wyatt, L. S., Snyder, J. T., Ahlers, J. D., Franchini, G., Moss, B., Berzofsky, J. A.
(2003). Shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses. Proc. Natl. Acad. Sci. USA
100: 9458-9463
[Abstract] [Full Text] -
Smith, G. L., Vanderplasschen, A., Law, M.
(2002). The formation and function of extracellular enveloped vaccinia virus. J. Gen. Virol.
83: 2915-2931
[Abstract] [Full Text] -
Smit, J. M., Waarts, B.-L., Kimata, K., Klimstra, W. B., Bittman, R., Wilschut, J.
(2002). Adaptation of Alphaviruses to Heparan Sulfate: Interaction of Sindbis and Semliki Forest Viruses with Liposomes Containing Lipid-Conjugated Heparin. J. Virol.
76: 10128-10137
[Abstract] [Full Text] -
Chiu, W.-L., Chang, W.
(2002). Vaccinia Virus J1R Protein: a Viral Membrane Protein That Is Essential for Virion Morphogenesis. J. Virol.
76: 9575-9587
[Abstract] [Full Text] -
Boulanger, D., Green, P., Jones, B., Henriquet, G., Hunt, L. G., Laidlaw, S. M., Monaghan, P., Skinner, M. A.
(2002). Identification and Characterization of Three Immunodominant Structural Proteins of Fowlpox Virus. J. Virol.
76: 9844-9855
[Abstract] [Full Text] -
Hung, J.-J., Chung, C.-S., Chang, W.
(2002). Molecular Chaperone Hsp90 Is Important for Vaccinia Virus Growth in Cells. J. Virol.
76: 1379-1390
[Abstract] [Full Text] -
Mandl, C. W., Kroschewski, H., Allison, S. L., Kofler, R., Holzmann, H., Meixner, T., Heinz, F. X.
(2001). Adaptation of Tick-Borne Encephalitis Virus to BHK-21 Cells Results in the Formation of Multiple Heparan Sulfate Binding Sites in the Envelope Protein and Attenuation In Vivo. J. Virol.
75: 5627-5637
[Abstract] [Full Text] -
da Fonseca, F. G., Wolffe, E. J., Weisberg, A., Moss, B.
(2000). Characterization of the Vaccinia Virus H3L Envelope Protein: Topology and Posttranslational Membrane Insertion via the C-Terminal Hydrophobic Tail. J. Virol.
74: 7508-7517
[Abstract] [Full Text] -
da Fonseca, F. G., Wolffe, E. J., Weisberg, A., Moss, B.
(2000). Effects of Deletion or Stringent Repression of the H3L Envelope Gene on Vaccinia Virus Replication. J. Virol.
74: 7518-7528
[Abstract] [Full Text] -
Seet, B. T., Barrett, J., Robichaud, J., Shilton, B., Singh, R., McFadden, G.
(2001). Glycosaminoglycan Binding Properties of the Myxoma Virus CC-chemokine Inhibitor, M-T1. J. Biol. Chem.
276: 30504-30513
[Abstract] [Full Text]
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