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Journal of Virology, April 2001, p. 4002-4007, Vol. 75, No. 8
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.8.4002-4007.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Biophysical Characterization and Vector-Specific
Antagonist Activity of Domain III of the Tick-Borne Flavivirus
Envelope Protein
S.
Bhardwaj,1
M.
Holbrook,2
R. E.
Shope,2
A. D. T.
Barrett,2 and
S. J.
Watowich1,*
Department of Human Biological Chemistry & Genetics and Sealy Center for Structural
Biology1 and Department of Pathology
and Center for Tropical Diseases,2 University of
Texas Medical Branch, Galveston, Texas 77555
Received 30 October 2000/Accepted 23 January 2001
 |
ABSTRACT |
The molecular determinants responsible for flavivirus host cell
binding and tissue tropism are largely unknown, although domain III of
the envelope protein has been implicated in these functions. We
examined the solution properties and antagonist activity of Langat
virus domain III. Our results suggest that domain III adopts a stably
folded structure that can mediate binding of tick-borne flaviviruses
but not mosquito-borne flaviviruses to their target cells. Three
clusters of phylogenetically conserved residues are identified that may
be responsible for the vector-specific antagonist activity of domain III.
| |
TEXT |
Flaviviruses (family
Flaviviridae, genus Flavivirus) are organized
into distinct vector-specific classes and serocomplex-specific subgroups (6), although little is known of the molecular
determinants that dictate vector-specific pathogenesis, host cell
specificity, and tissue tropism. Both mosquito-borne and tick-borne
flaviviruses are responsible for epidemics throughout the developing
world and pose serious public health threats in developed countries (7, 9, 10). Vaccines are only available to help control infection by yellow fever, Japanese encephalitis, Central European tick-borne encephalitis (TBE), and louping ill flaviviruses (1, 2, 16, 17). No antiviral therapy is available to treat any
flavivirus infection.
Flavivirus infection requires attachment and entry into a target cell,
mediated by binding of the viral envelope (E) proteins to cell surface
receptors. The host cell receptor (or receptors) and the region (or
regions) of the E protein responsible for flavivirus attachment are
unknown, although heparan sulfate has been suggested as one factor
mediating the interaction between dengue 2 virus and its target cells
(3). It is unknown whether different cell surface
receptors and/or envelope-receptor interactions are responsible for
flavivirus pathogenesis, host range, and tissue tropism.
The structure of the E protein ectodomain from TBE virus has been
determined (13). This ectodomain forms a homodimer, with each dimer subunit organized into three domains, designated I, II, and
III. Comparisons of E proteins from wild-type viruses with those of
attenuated or escape mutant viruses have identified a number of
residues in domain III that may be responsible for receptor recognition
(4, 8, 12-14). We examined the solution properties of
domain III of the E protein from Langat virus (a tick-borne flavivirus)
and the ability of this domain to function as an antagonist for virus
infectivity. We demonstrate that recombinant domain III adopts a highly
stable folded structure in solution and shows reduced infectivity in
tick-borne flaviviruses but not mosquito-borne flaviviruses. This
suggests that interactions between the envelope protein and its host
cell receptor may be vector specific, although it is likely that other
factors also influence flavivirus vector preference. Sequence and
structure analyses identified a small number of residues that may be
responsible for vector-specific receptor binding.
Cloning and expression of domain III of Langat virus E
protein.
cDNA encoding domain III of the Langat virus E protein (E
protein residues 300 to 395; LgtE-D3) was amplified by PCR. Template DNA for the PCR was plasmid pUC18 containing cDNA encoding Langat virus
structural proteins. The PCR product was subcloned into pGEX-2T
expression vector (Pharmacia), and the fidelity of the cloned sequence
was confirmed by DNA sequencing (T. Woods, unpublished data).
Escherichia coli DH5
cells were transformed with pGEX-2T expressing LgtE-D3 and grown at 37°C in 2xYT medium. The cultures were chilled to 16°C, and expression of a glutathione
S-transferase (GST)-LgtE-D3 fusion protein was induced by
the addition of 1 mM isopropyl-1-
-D-galactopyranoside.
Cultures were maintained at 16°C for 6 to 12 h, pelleted, and
frozen. Pellets were resuspended in ice-cold PBST buffer (10 mM sodium
phosphate, 2 mM potassium phosphate [pH 7.4], 140 mM NaCl, 3 mM KCl,
0.1% [vol/vol] Tween 20) and lysed with mild sonication.
Glutathione-agarose beads were added to the supernatant and mixed
gently at 4°C for 2 to 4 h. The beads were washed with PBST buffer
and high-salt PBST buffer (PBST with 500 mM NaCl) and the GST-LgtE-D3
fusion protein was eluted from the beads with Tris-glutathione buffer
(50 mM Tris [pH 8.1], 20 mM glutathione). Eluted GST-LgtE-D3 protein was cleaved by treatment with thrombin at room temperature for 12 to
16 h. Alternatively, GST-LgtE-D3 protein could be cleaved on the
glutathione-agarose beads and LgtE-D3 could be eluted (Fig. 1). The supernatant was concentrated and
applied to a size exclusion column (Biosep SEC-S3000 column;
Phenomenex). Recombinant LgtE-D3 was >98% pure, as determined by
Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, and immunogenic (data not shown).
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FIG. 1.
Coomassie blue-stained composite sodium dodecyl
sulfate-polyacrylamide gel showing expression and purification of
recombinant LgtE-D3. Lane 1, GST-LgtE-D3 fusion protein bound to
glutathione-conjugated agarose beads. Lane 2, total protein at the
initiation of thrombin cleavage of bound GST-LgtE-D3. Lane 3, soluble
protein after thrombin cleavage of bound GST-LgtE-D3. Lanes 4 to 6, soluble protein recovered after sequential washes of
glutathione-agarose beads with low-salt buffer. Lane 7, protein bound
to glutathione-agarose beads after thrombin cleavage. Lane 8, Western
immunoblot of purified LgtE-D3 probed with rabbit polyclonal antibody
generated against recombinant LgtE-D3 (Alpha Diagnostics, Inc.). The
GST-LgtE-D3 fusion protein migrates as a single band at ~37 kDa.
Recombinant LgtE-D3 migrates as a single band at ~10.7 kDa.
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Solution properties of LgtE-D3.
LgtE-D3 was highly soluble and
could be concentrated to >40 mg/ml in Tris buffer. Its circular
dichroism (CD) spectra showed a local minimum at around 215 to 220 nm
(Fig. 2), characteristic of proteins
composed primarily of a -sheet secondary structure (19). Deconvolution of the CD spectra into component
secondary-structure basis spectra indicated that LgtE-D3 contained
~40% -sheet and ~60% random coil elements. This composition
was consistent with the secondary structural elements observed in the
crystal structure of TBE virus envelope protein, where ~55% of
domain III formed -sheets (13). The CD spectra were
unchanged in 2 and 4 M urea (Fig. 2), indicating that domain III is
highly stable and resistant to denaturation even in the presence of
high urea concentrations. However, in 8 M urea, the CD spectra were
similar to those obtained for proteins composed entirely of a random
coil structure (19), demonstrating that domain III was
largely unfolded in 8 M urea.
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FIG. 2.
Far-UV CD spectra of recombinant LgtE-D3 incubated with
increasing amounts of denaturant. Spectra from LgtE-D3 in 0 M urea
(solid curve, solid circles), 2 M urea (dotted curve, open circles), 4 M urea (dotted curve, open squares), and 8 M urea (dashed curve, open
triangles) are shown. The LgtE-D3 concentration was 0.3 mg/ml in Tris
buffer (pH 7.4). Spectra were collected on an Aviv 62 DS circular
dichrometer operating with a 0.5-nm step increment and a 1-s interval.
Cylindrical quartz cuvettes with a 0.1-cm path length were used for all
measurements. All sample spectra were recorded five times, averaged,
and corrected for buffer contributions. Measurements were considered
unreliable when the instrument dynode voltage exceeded 410 V and were
not included in subsequent analyses.
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Sedimentation velocity experiments performed at 23°C with a Beckman
Optima XL-A analytical ultracentrifuge showed that LgtE-D3
was a single
low-molecular-weight species with a sedimentation
coefficient
(
15) of 1.3S in physiological buffer at room temperature
(data not shown). Analysis of equilibrium velocity measurements
in
physiological buffer gave a mean molecular mass of 10.7 kDa
for
LgtE-D3. This molecular mass was within 0.5% of the theoretical
molecular mass calculated from the LgtE-D3 amino acid sequence.
The model of LgtE-D3 as a noninteracting monomer in solution
provided
a very good fit to the experimental equilibrium velocity data
(data not
shown).
Antagonist activity of LgtE-D3.
The ability of LgtE-D3 to
protect cells from infection with tick-borne and mosquito-borne
flaviviruses was tested. LgtE-D3 significantly reduced the amount of
observed cytopathic effect (CPE) in Vero cells infected with the two
tick-borne viruses but not with the mosquito-borne viruses (Table
1). In addition, there was a significant
delay in the appearance of CPE in cells challenged with tick-borne
viruses and protected with LgtE-D3 relative to unprotected cells (data
not shown). The protection afforded Vero cells by LgtE-D3 was
quantitated by examining virus titers produced in cells challenged with
tick-borne viruses. Approximately 10-fold less tick-borne virus was
produced from cells protected with LgtE-D3 than from unprotected cells
(Table 2). These observations suggest that domain III functioned as a vector-specific antagonist to interfere
with flavivirus binding to host cell receptors. This is the first
direct experimental evidence that domain III is involved in host cell
receptor binding. Recent immunofluorescence studies support the
conclusion that LgtE-D3 binds to a membrane-associated host cell
receptor (M. Holbrook, personal communication). The observed
vector-specific differences could result from the tick-borne and
mosquito-borne flaviviruses' interacting with an identical receptor
binding site via different intermolecular contacts, with the
mosquito-borne flaviviruses binding to this site with greater affinity
than the tick-borne flaviviruses. Alternatively, vector specificity
could result from flaviviruses' recognizing different cell surface
receptors or different binding sites on the same cell surface receptor.
Molecular basis of vector-specific antagonist activity.
Since
LgtE-D3 displays vector-specific antagonist activity, an analysis of
vector-based invariant residues was performed to identify domain III
regions that mediate host cell binding (Fig. 3). Analogous sequence comparisons in the
hemagglutinin receptor-binding site of influenza A viruses have
identified phylogenetically conserved residues that determine receptor
specificity (11, 18). Domain III amino acid sequences
showed ~80 to 95% sequence identity among tick-borne flaviviruses,
~50 to 90% identity among mosquito-borne flaviviruses, and ~50%
identity among two non-vector-borne flaviviruses. Twelve residues were
invariant, and nine residues were highly conserved (>93%) in the
sequences examined. Six amino acids, at positions 324, 355, 381, 382, 384, and 394 (TBE virus numbering), were completely conserved within a
flavivirus vector class but differed between the tick-borne and
mosquito-borne viruses. In addition, vector-specific insertions and
deletions occurred at positions 309, 369, 376, 378, and 386, either at
loops between -sheets or in extended coils (Fig.
4).
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FIG. 3.
Amino acid sequence alignment of domain III of
flavivirus E proteins. Residues that are conserved in at least 15 of
the 16 sequences are indicated by a star above the alignment. Shaded
boxes indicate positions of conserved vector-specific residues and
insertions. Amino acid numbering refers to TBE virus residue positions.
The virus and vector classes are indicated along the left-hand margin.
Protein sequences were obtained from Swiss-Protein database entries and
correspond to Langat virus (LGT; strain V, accession number P29837),
Powassan virus (POW; accession number Q04538), Central European TBE
virus (accession number Q01299), louping ill virus (LI; accession
number P22338), yellow fever virus (YF; strain 1899/81, accession
number P29165), Murray Valley encephalitis virus (MV; accession number
P05769), Japanese encephalitis virus (JE; strain Nakayama, accession
number P27395), St. Louis encephalitis virus (SLE; accession number
P09732, strain MS1-7), Kunjin virus (KUN; strain MRM61C, accession
number P14335), West Nile virus (WN; strain RO97-50, accession number
Q9WHD1), dengue virus type 1 (DEN1; strain AHF82-80, accession number
P27912), dengue virus type 2 (DEN2; isolate Malaysia M2, accession
number P14338), dengue virus type 3 (DEN3; accession number P27915),
dengue virus type 4 (DEN4; accession number P09866), Rio Bravo virus
(RIO; accession number AF144692), and Apoi virus (accession number
AF160193).
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FIG. 4.
Structure of TBE virus envelope protein. (A) Top view of
the E protein homodimer. (B) Side view of the E protein homodimer.
Orientation nomenclature is adapted from that of Rey et al.
(13), where top suggests a view towards and normal to the
virus surface, and side suggests a view tangent to the virus surface,
with the viral membrane below the protein. Each monomer of the
homodimer is shaded differently, and the dashed lines delineate the
domain III boundaries. (C) Isolated domain III viewed approximately
perpendicular to the twofold axis of the E protein homodimer,
corresponding to the solvent-exposed lateral surface of domain III.
Positions of conserved vector-specific residues and insertions in
domain III are indicated with spheres. Amino acid numbering refers to
the TBE virus residue positions. Domain III structures were displayed
using Swiss-Pdb Viewer (5; http://www.expasy.ch/spdbv).
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The 11 vector-specific positions were clustered in three spatially
distinct regions in domain III (Fig.
4), which may be responsible
for
the vector-specific antagonist activity of domain III. Region
1, formed
by residues 309, 384, and 386, was located on the solvent-exposed
lateral face of domain III. These residues were flanked by invariant
residues, and at residue 386 three homologous residues were inserted
in
mosquito-borne flaviviruses relative to tick-borne flaviviruses.
Thus,
the structural and chemical properties of region 1 may be
similar among
vector-specific flaviviruses, allowing this region
to form
vector-specific interactions with cell surface receptors.
Recent
studies showed that mutations in region 1 were associated
with virus
attenuation (
8,
13,
14). Region 2, formed by
residues 324, 355, 376, 378, 381, 382, and 394, made extensive
contacts between the
-sheet termini of domain III (Fig.
4). The
majority of these
residues were partially buried within the domain
III structure and thus
likely shielded from intermolecular interactions.
Region 3, containing
residue 369, was part of a solvent-exposed
loop (Fig.
4). Between
residues 369 and 370 of the mosquito-borne
flaviviruses, one
additional residue was inserted in the dengue
and yellow fever viruses
and three additional residues were inserted
in the non-dengue and
non-yellow fever viruses relative to the
tick-borne
flaviviruses. Invariant residues were not adjacent
to residue 369, implying that the tertiary structure and chemical
properties of region
3 are different for different mosquito-borne
flaviviruses.
Vector-specific residues in the non-vector-borne flaviviruses had
similarities to both the tick- and mosquito-borne flaviviruses.
The
non-vector-borne and mosquito-borne flaviviruses had the same
residues
at positions 324, 355, and 381 and had a conserved deletion
at position
309. The non-vector-borne and tick-borne flaviviruses
had similar
residues at positions 376 and 394 and conserved deletions
at positions
378 and 386. The lysine residue at position 384 in
the non-vector-borne
flaviviruses was distinct from the conserved
residue found in the
mosquito- and tick-borne
flaviviruses.
Domain III mutants can be used to delineate E protein receptor
interaction surfaces and test if different E protein interaction
surfaces are responsible for tissue tropism. Domain III and its
derivatives may be useful first-generation vector-specific antagonists
and as targets against which to generate antiviral agents that
prevent
binding of flavivirus to its host cell receptor. In addition,
recombinant domain III may be valuable in the development of flavivirus
vaccines and diagnostic
reagents.
| |
ACKNOWLEDGMENTS |
This work was supported by grant N65236-97-1-5811 (R.E.S.) from the
Defense Advanced Research Projects Agency, a fellowship from the
McLaughlin Foundation (M.H.), and the Sealy Center for Structural Biology.
We thank J. Lee and C. Chan for assistance with the analytical
ultracentrifugation experiments, L. Lee for assistance with the CD
experiments, and D. Konkel for critically reading the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of Human
Biological Chemistry & Genetics, University of Texas Medical Branch, Galveston, TX 77555-0645. Phone: (409) 747-4749. Fax: (409) 747-4745. E-mail: watowich{at}bloch.utmb.edu.
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Journal of Virology, April 2001, p. 4002-4007, Vol. 75, No. 8
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.8.4002-4007.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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