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Journal of Virology, February 2000, p. 1477-1485, Vol. 74, No. 3
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Sialylation of the Host Receptor May Modulate Entry
of Demyelinating Persistent Theiler's Virus
Lan
Zhou,1,2
Yu
Luo,2
Yan
Wu,1
Jun
Tsao,1,2 and
Ming
Luo1,2,*
Department of
Microbiology1 and Center for
Macromolecular Crystallography,2 University
of Alabama at Birmingham, Birmingham, Alabama 35294
Received 16 August 1999/Accepted 19 October 1999
 |
ABSTRACT |
Theiler's murine encephalomyelitis virus (TMEV) is a
picornavirus of the Cardiovirus genus. Certain strains of
TMEV may cause a chronic demyelinating disease, which is very similar
to multiple sclerosis in humans, associated with a persistent viral
infection in the mouse central nervous system (CNS). Other strains of
TMEV only cause an acute infection without persistence in the CNS. It
has been shown that sialic acid is a receptor moiety only for the
persistent TMEV strains and not for the nonpersistent strains. We
report the effect of sialylation on cell surface on entry and the
complex structure of DA virus, a persistent TMEV, and the receptor
moiety mimic, sialyllactose, refined to a resolution of 3.0 Å. The
ligand binds to a pocket on the viral surface, composed mainly of the
amino acid residues from capsid protein VP2 puff B, in the vicinity of
the VP1 loop and VP3 C terminus. The interaction of the receptor moiety
with the persistent DA strain provides new understanding for the
demyelinating persistent infection in the mouse CNS by TMEV.
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INTRODUCTION |
Theiler's murine
encephalomyelitis virus (TMEV) belongs to the
Cardiovirus genus of the family Picornaviridae
based on sequence analysis (34). Based on the
characteristics of the disease they cause, TMEV strains are divided
into two groups. Viruses of one group, including strain GDVII and FA,
are highly neurovirulent, causing a rapid destruction of the neuron and
killing their host in a matter of days. These viruses are unable to
persist and to induce demyelination in those rare survivors
(22). The other group, including strains DA, BeAn, WW, TO4,
and Yale, are referred to as the Theiler's original (TO) group
(23). Viruses of the TO group are much less virulent than
GDVII and FA and cause a biphasic disease with mild central nervous
system (CNS) damage. The first phase, or early onset, is acute
encephalitis that occurs during the initial few days following
intracranial inoculation. At that time, the virus is also found in
neurons of the gray matter in the brain and spinal cord. However, the
number of infected cells is small and most of the animals survive. Soon
after, the neurons are cleared of the virus and the disease enters a
second phase, or late onset, during which the virus is found to be
persistent in the white matter of the spinal cord. At this stage of the
viral persistence, patchy inflammation and demyelination develops in the spinal cord at the sites of infection (7, 23). The
symptoms of the demyelination disease of mice caused by TMEV
persistence infection in the CNS resemble those of multiple sclerosis
(MS), a chronic demyelinating disease of the human CNS. Among the
animal models of virus-induced demyelination in the last two decades, TMEV infection has emerged as one of the best models for studying MS.
Although these two groups of TMEV strains have both been shown to
replicate well in BHK-21 cells and share high amino acid sequence
homology (31, 34-36), they apparently display different pathogenesis characteristics in vivo. Many efforts have been taken to
map a determinant for virus persistence, and it has been elucidated that the capsid that carries the host receptor recognition site and the
antigenic sites is responsible for viral persistence (4, 11). The capsid of TMEV shares common features of the
picornaviruses, a family of small RNA viruses that have three major
capsid proteins (VP1, VP2, and VP3) assembled into an icosahedral
shell. When the three-dimensional structure of human rhinovirus 14, a
picornavirus, was determined, it was hypothesized that the depression
surrounding the fivefold vertices, the canyon, was the site for
receptor attachment (38). Subsequently, host receptors were
identified for several picornaviruses, including rhinovirus
(40), poliovirus (28), cardiovirus
(14), foot-and-mouth disease virus (FMDV) (2), coxsackievirus B (1), and coxsackievirus A9 (37).
The site of receptor recognition for rhinovirus 16 was later shown to
be in the surface depression, as demonstrated by cryoelectron
microscopy and mutagenesis (32). For FMDV, however, the
receptor recognition peptide sequence RGD that interacts with the virus
receptor integrin is located on a protruding loop in the virus capsid
(24). The binding site for an oligosaccharide receptor of a
cell culture adopted FMDV strain was also located on a separate region
on the surface of the capsid (10). Despite of the failure to
clearly identify the host receptor for TMEV at this time, there is some evidence that the host receptor may be a glycoprotein (20). The sites that influence the capability of TMEV demyelinating persistence in the mouse CNS are located around the depression, a
putative receptor recognition site, observed in the crystal structure
(12, 25, 26). These results imply that virus entry may be
related to viral persistence in the mouse CNS by TMEV.
Efforts to identify the host receptor for Theiler's virus have been
reported (9). When membrane proteins of susceptible cells
were separated by gel electrophoresis, radiolabeled TMEV was found to
bind predominantly to a 34-kDa glycoprotein (20). Wheat germ
agglutinin that binds sialic acid specifically could inhibit the
infectivity of TMEV or block its attachment to the protein. This
suggests that sialic acid may be involved in the virus attachment,
which was further approved by an experiment in which removing sialic
acid molecules from the cell surface by sialidase treatment could
reduce the infectivity of BeAn virus by 90% (9). More
evidence was provided by our recent studies on the effects of the
sialyl moiety of oligosaccharides on virus attachment and replication
with the BeAn virus (44). It was shown that the infection of
a demyelinating persistent TMEV strain, BeAn, was reduced by
103-fold when 9 mM sialyllactose was included in the
culture medium. In addition, it was demonstrated that the inhibition of
the infection of the BeAn virus strain is due to prevention of virus
attachment to the cell surface (44). 35S-labeled
BeAn virus was used to measure virus attachment to solubilized BHK-21
cells, and sialyllactose was shown to be effective in blocking the
virus attachment to BHK-21 cells. There were no observed effects by
sialic acids on the infectivity and the receptor attachment of the
nonpersistent strain, GDVII, when the experiments of the 3-sialyllactose inhibition and sialidase treatment were performed under
the same conditions (9, 44).
Like other picornaviruses, TMEV capsid is composed of 60 copies each of
four polypeptides (VP1, VP2, VP3, and VP4), arranged with a pseudo T=3
icosahedral symmetry. Although not obviously related in sequence, VP1,
VP2, and VP3 have a similar folded structure and share an antiparallel
eight-stranded 
-barrel. Among the capsid proteins, only VP1, VP2,
and VP3 are exposed on the viral surface and are presumably responsible
for cell receptor recognition. Atomic structures of TMEV strains from
each group have been determined by X-ray crystallography for strains
BeAn, DA, and GDVII (12, 25, 26). Sequence alignment of
three major capsid proteins of these two groups of TMEVs shows that the
amino acids that are identical in BeAn and DA strains but are different
in GDVII are clustered on the outside of the capsid in the loops and
corners connecting the strands. Structure comparisons between GDVII and BeAn or DA revealed that structure differences between these two
groups of TMEVs are small and local. Logically, these structural differences may play a functional role in differentiating these phenotypically different viruses by altering the way in which virus
binds to its cellular receptor.
In this report, we describe our recent findings for the involvement of
sialic acid in the entry of Theiler's virus and the crystal structure
of a persistent Theiler's virus (strain DA) in complex with
sialyllactose. It appears that sialic acid is a receptor moiety
required only for the attachment of the persistent Theiler's viruses.
This might have implications for the mechanism of in vivo persistence
in the mouse CNS by the TO group of the Theiler's virus.
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MATERIALS AND METHODS |
Virus propagation, purification, and crystallization.
The
TMEV DA strain, DAFL3, was kindly provided by R. P. Roos, at the
University of Chicago Medical Center. BHK-21 cells were routinely
maintained in Dulbecco modified Eagle medium DMEM medium supplemented
with 7% fetal bovine serum, 100 U of penicillin per ml, and 100 µg
of streptomycin per ml in a 37°C incubator with 5% CO2.
For production, virus was propagated by infection of confluent BHK-21
cells at a multiplicity of infection (MOI) of approximately 10. The
infection was allowed to proceed until extensive cytopathic effect
(CPE) was observed (ca. 2 days). The virus titer and CPE assays for
sialyllactose effects and sialidase treatment were carried out as
described previously (9, 44).
To purify the DA virus, infected cells were scraped off and clarified
by centrifugation at 11,300 × g for 10 min at 4°C.
The pellet was resuspended in 20 mM Tris buffer with 150 mM NaCl and frozen at 
80°C overnight. After thawing and sonication, the virus material was treated with 0.3% sodium dodecyl sulfate at 37°C to
dissociate virus from the membranes. The virus was then loaded on the
top of a 15 to 30% sucrose step gradient, followed by centrifugation at 72,100 × g for 20 h at 25°C. After
centrifugation, a pellet appeared at the bottom of the tube and was
treated with DNase and 2% Triton X-100. The viruses were subsequently
banded through a 20 to 70% sucrose linear gradient and a
Cs2SO4 equilibrium gradient. The purified
viruses were dialyzed extensively against 20 mM Tris buffer (pH 8.5)
before they were concentrated to a final concentration of ca. 15 mg/ml
for crystallization.
The hanging-drop vapor diffusion method was used to crystallize the
virus. The precipitant solution in the reservoir contained
1 to 3%
polyethylene glycol (PEG)-monomethyl ether (mme) 5,000
and 0.1 M sodium
phosphate buffer at pH 6.5. The drop contained
3 µl each of the virus
solution and the reservoir solution. The
diamond-shaped crystals grew
to a maximum size ranging from 0.02
to 0.5 mm in 3 days at 20°C. The
suitable crystals were harvested
in 7% PEG-mme 5,000 in the same
phosphate buffer before they were
soaked in the harvest buffer with 30 mM 3'-sialyllactose.
Data collection and structure determination and refinement.
Soaked crystals were transferred to a solution containing 5% PEG-mme
5,000, 35% PEG 400, 30 mM sialyllactose, and 0.1 M sodium phosphate
buffer (pH 6.5). Oscillation data were collected at 0.3° per frame at
SSRL (Stanford Synchrotron Radiation Laboratory) beam line 7-1. A data
set of a total number of 128 images measured from a frozen crystal was
obtained. The diffraction data were then processed by using the HKL
package (29, 33) data, with a completeness of ca. 72% to a
3.0-Å resolution. The statistics of the data set are listed in Table
1. The crystal symmetry is P21212, the same as the reported DA crystal
(12) except that the frozen crystal's unit cells had shrunk
ca. 6.5 to 7 Å from each direction.
The frozen crystal shares the same packing scheme with the reported DA
structure (
12). One virus particle sits on the 2-fold
crystallographic symmetry axis, which gives rise to the 30-fold
noncrystallographic redundancy. Rotation and translation function
calculations indicated a particle orientation of 3.97° around
the
twofold (
z) axis relative to a standard icosahedral
orientation
and the particle center to be close to
z = 0.2501. Rigid-body
refinement with X-PLOR (
3) with the
DA native coordinate yielded
an R factor of 0.32 for data between
resolutions of 20 and 3.0
Å. Phases were improved by 30-fold
noncrystallographic symmetry
electron density averaging. An averaged
difference map (see Fig.
3a) with CCP4 (
6) and RAVE
(
21) between the observed data
and DA native structure
located unambiguously the position of
the sialyllactose. An omit map
from the improved phases also showed
very clear electron density
contributed by the bound 3'-sialyllactose.
The galactose has
recognizable density, while the glucose is completely
disordered. The
complex structure was then refined by one round
of positional
refinement by using X-PLOR to yield an R factor
of 29.1% for
resolution data from 6.0 to 3.0 Å. No water molecules
were included
except for the one coordinating with the sialic
acid
moiety.
| |
RESULTS |
Inhibition of DA virus growth by sialyllactose.
Sialyllactose
was used in this experiment to present the sialic acid molecule in the
-configuration that is mostly observed in the natural
glycoconjugates. Free sialic acid molecules would mostly assume the
-configuration in aqueous solutions. To verify that a second
demyelinating persistent strain, DA, has the same characteristics in
receptor attachment as the BeAn virus, the same experiment was
performed by including sialyllactose in the growth medium. As clearly
demonstrated in Fig. 1, soluble
sialyllactose effectively reduced the CPE caused by DA virus infection
in BHK-21 cells. The titer of the DA virus was reduced by at least 3 orders of magnitude when 9 mM sialyllactose was included in the growth medium, which was a degree of inhibition similar to that observed with
BeAn virus. The results appear to support the notion that sialic acid
is a critical part of the host receptor.
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FIG. 1.
Effects of the sialyllactose in medium on the
infectivity of the DA virus. The virus was purified as described by
Zhou et al. (44). (A) At 48 h postinfection, DA virus
was able to infect and induce CPE in BHK-21 cells at an MOI of 10 in
the presence of 0.0, 1.1, and 2.3 mM sialyllactose, while 5.4 and 9.0 mM sialyllactose prevented CPE. The virus yield titer under the same
conditions was then determined by plaque assay in six-well plates
containing confluent monolayers of BHK-21 cells. In the presence of 3.6 mM sialyllactose, plaques were observed. (B) The virus yield titer was
reduced by roughly 10-fold at each increment of sialyllactose
concentration, with 103 reduction at 9.0 mM.
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Effect of sialylation on DA virus infection.
To confirm that
the dependence of sialylation for entry is universal for all persistent
strains of TMEV, the similar experiment previously performed with BeAn
virus was repeated with the DA virus. Monolayers of BHK-21 cells were
treated with bacterial sialidase (S. typhimurium, prepared
in the lab of M.L. for unrelated crystallization experiments) prior to
DA virus infection. Bacterial sialidase effectively removes the
terminal sialic acid moieties that are linked to the oligosaccharides
by 2,3 or 2,6 linkages. Confluent monolayers of BHK-21 cells were
incubated with 1 ml of a sialidase solution diluted in DMEM (1 µg/liter) for 10 min. The sialidase solution was then removed. The
bacterial sialidase-treated cells were then infected with an equal
amount of the DA virus as with the untreated BHK-21 cells. As shown in
Fig. 2, the titer of the DA virus was
reduced by 3 orders of magnitudes when the BHK-21 cells were treated
with bacterial sialidase. Reduced CPE that has the same appearance as
that of DA virus infection in the presence of 9.0 mM sialyllactose in
the medium was also observed with the bacterial sialidase treated cells
(Fig. 2). Apparently, the DA virus lost its ability to attach
efficiently to the host receptor for entry when sialic acids were
removed from the receptor.
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FIG. 2.
Effects of sialidase (SA) treatment on the infectivity
of the DA virus. Confluent monolayers of BHK-21 cells were incubated
with 1 ml of a sialidase solution diluted in DMEM (1 µg/liter) for 10 min. The sialidase solution was then removed. Purified DA virus stock
has a concentration of 0.1 mg/ml and was diluted 100 times with DMEM
medium. One milliliter of the diluted virus was added to each flask of
monolayer cells, which were treated with bacterial sialidase and
incubated at 37°C in an incubator with 5% CO2 for 2 h. Unattached virus was removed by washing with phosphate-buffered
saline. Then, 15 ml of DMEM containing the same amount of antibiotics
and bacterial sialidase but only 1% fetal bovine serum was added to
the flask and incubated for 48 h at 37°C in an incubator with
5% CO2. Parallel controls were included by using untreated
monolayer cells. (A) CPE was easily observed on untreated cells during
DA infection, while CPE was significantly reduced when the cells were
treated with bacterial sialidase. (B) The virus yield titer was
determined for the two infection batches.
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Binding site of the sialic acid on DA virus.
Based on the
structural analyses with phenotypically different parental as well as
chimeric TMEVs, we predicted that, when carried only on the persistent
strains, a surface structural component next to the picornavirus
putative receptor binding site may influence the ability of TMEV to
persist through the differential interaction with the cellular
receptor, such as binding to sialic acid (44).
To clearly identify the binding site for sialic acid and to map the
three-dimensional interactions between the DA virus and
this receptor
moiety, the complex structure of DA virus crystals
prepared by soaking
in a solution containing 30 mM sialyllactose
was determined and refined
to a resolution of 3 Å. The binding
site (Fig.
3a and
4a) for sialic acid is
located at the interface
between VP1 and VP2, close to the C terminus
of VP3. The center
of the putative receptor binding site, the
"pit," is about 15
Å from the sialic acid binding site. The sialic
acid molecule
is accommodated in a positively charged depression formed
by a
gigantic loop, puff B of VP2 (Fig.
3b and
4b).
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FIG. 3.
(a) Averaged (Fo-Fc) electron density map (>5 )
corresponding to the sialic acid moiety in the complex of DA virus and
sialyllactose. The galactose moiety has a recognizable density, while
the glucose moiety is completely disordered. The complex structure was
refined by using X-PLOR to yield an R factor of 29.1% for 6.0- to
3.0-Å resolution data. (b) Stick-and-ball drawing illustrating the
detailed interactions of the sialic acid with the DA virus. The
hydrogen bonds are indicated by yellow sticks.
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FIG. 4.
(a) Ribbon drawing for the protomer (VP1 in blue, VP2 in
green, and VP3 in red) with the bound sialyllactose (magenta)
(5). The protomer location on the icosahedral lattice is
shown on the left. (b) Electrostatic potential calculated by using the
coordinates of a protomer (VP1, VP2, and VP3) and the program GRASP
(30). The sialic acid moiety is situated in a largely
negatively charged depression near the putative receptor binding site.
The areas on the side of the protomer will not be exposed in the intact
icosahedral capsid. (c) Superposition of residues of GDVII virus
(magenta) with those of DA virus involved in binding sialic acid. Amino
acid sequence variations in GDVII virus compared to DA virus are
presented in parentheses. VP2 Gly2174 in DA virus forms a hydrogen bond
with the sialic acid N-acetyl carbonyl group through its
main chain nitrogen. The expanded conformation of this region in GDVII
virus does not allow the formation of the same hydrogen bond while
maintaining the interactions with other functional groups of sialic
acid. The side chain orientation of Gln2161 in GDVII does not favor a
similar hydrogen bond formation with the carboxyl group of the sialic
acid either. However, the distance from the amide group of Gln2161 in
GDVII virus to the carboxyl group of the sialic acid is still within
hydrogen bond distance.
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Interaction of sialic acid with the capsid of DA virus.
The
pyranose ring of the sialic acid assumes the usual chair conformation
with all constituent groups at equatorial positions except for the
characteristic carboxylate. In several cases, the negatively charged
carboxyl group of sialic acid interacts with positively charged side
chains when binding to proteins (16, 27, 41). In the DA
virus, however, a hydrogen bond between the carboxylate and the side
chain of VP2 Gln2161 was found (Fig. 3b). The carbonyl oxygen of the
N-acetyl group is stabilized by a hydrogen bond with the
main chain amide of VP2 Gly2174. There is no hydrogen bond with the
amino nitrogen. The glycerol group is held extended by a hydrogen bond
between the 9-hydroxyl group and the carbonyl oxygen of VP2 Ala2163. In
addition, the C terminus of VP3 is positioned in the vicinity of the
7-hydroxyl group of the glycerol. The interaction between the
7-hydroxyl group and the VP3 C terminus is mediated by a well-ordered
water molecule. No salt bridge is formed with the sialic acid
carboxylate. The surface electrostatic potential showed that the sialic
acid binding site is within a capsid surface depression with a
significant positive potential (Fig. 4b). In addition to the specific
hydrogen bonds, the shape complementarity and long-range charge-charge interactions may also play important roles in sialic acid recognition by the DA virus. No contacts have been observed between the capsid proteins and the last two sugar residues in the sialyllactose. No
intramolecular hydrogen bond has been observed between sialic acid and
either galactose or glucose.
| |
DISCUSSION |
Entry is the first interaction of the virus with its host cell. It
has been shown that viruses can use proteins or oligosaccharides as
host receptors, such as influenza virus that uses sialic acids as the
receptor (13, 42) or poliovirus that uses a glycoprotein (28). A virus could use two separate receptors during entry, as shown by human immunodeficiency virus that uses CD4 as the initial
receptor and the chemokine receptor, CCR5, as the second receptor
(8). In rhinoviruses, two types of receptors are used by
different members of the same virus family that are divided into major
and minor groups (40). TMEV presents a new scenario in which
the same family of viruses binds to the same glycoprotein receptor but
in different ways. The binding of the demyelinating persistent group is
dependent on the interaction with both a sialyl moiety and the protein
surface of the receptor, while the nonpersistent group is only
dependent on the interaction with the protein surface of the receptor,
even though the two groups bind to the same receptor competitively
(20).
The structure of the sialic acid-DA complex clearly identified the
binding site for a receptor moiety that binds at the rim of the pit,
which has been postulated as the receptor binding site (25).
Mutations in the pit alter the receptor binding of Theiler's virus (S. Hertzler, M. Luo, and H. L. Lipton, unpublished data.) In
demyelinating persistent strains of TMEV, the sialic acid moiety is an
essential part for sufficient virus attachment to the host receptor
that results in virus entry. Without the presence of sialic acid
molecules on the cell surface after treatment with sialidase or with
the sialic acid binding site of the virus occupied by sialyllactose,
the virus can no longer bind to its host receptor tightly enough to
induce endocytosis. The location of the sialic acid binding site is
consistent with the positions of residues which impact virus
replication and its ability to induce a demyelinating persistent
infection in the mouse CNS. For instance, the major sequence variation
between persistence and nonpersistent virus strains were located at VP2
position 2162 and VP2 positions 2171 to 2173, both of which are on the
VP2 puffB. In DA and BeAn strains, VP2 position 2162 is Gly, and VP2
positions 2171 to 2173 are Ser-Arg-Thr. In the nonpersistent GDVII
strain, however, the corresponding residues are Ser and Arg-Gln-Ala,
respectively. These variable residues are not part of the sialic acid
binding site (Fig. 4b and c), but they could induce changes in the
residues that directly form hydrogen bonds with sialic acid, as was
observed in the GDVII structure (26). The position of the
amide nitrogen of Gly2174 was moved away from the sialic acid binding
site in GDVII, probably due to the conformation changes induced by
residues 2171 to 2173 (Arg-Gln-Ala) (Fig. 4c). The movement of the
nitrogen atom of Gly2174 in GDVII is likely to abolish its hydrogen
bonding capability to sialic acid. In other examples, mutations in the capsid proteins modified the ability of TMEV's persistence. When VP1
Thr1101 was mutated to Ile or Ala or when VP2 Thr2173 was mutated to
Phe, the persistent DA virus either lost or had a reduced ability to
persist and demyelinate (39; Y. Wada, M. L. Pierce, and R. S. Fujinami, Abstr. 12th Gen. Meet. Am. Soc.
Microbiol. 1993, abstr. A37, 1993). Thr2173 is directly over the bound
sialic acid moiety. A mutation to Phe will introduce a steric hindrance to the sialic acid binding site. In another instance, chimeric viruses
between DA and GDVII could persist only when residue 2141 in VP2 is Lys
in the parental DA virus (17, 18). These surface residues
may have changed the interaction of the virus with the host CNS, most
likely at the virus entry stage. Their locations suggest a role in
sialic acid affinity, even though there is no direct evidence that they
reduce the binding of DA virus to sialic acid.
More-direct evidence for the involvement of some of those residues in
virus entry is provided by the adaptation of DA viruses from BHK-21
cells to L929 cells. The wild-type DA virus was shown to have a
defective entry when infecting L929 cells, which results in a slow
replication rate of DA virus in L929 cells. The KJ6 DA variant that had
a more rapid CPE in L929 cells was generated by multiple passages in
L929 cells (19). This adaptation appears to be related to an
enhanced viral entry rather than to an increased genome replication
rate. Interestingly, the mutations responsible for the adaptation were
found at Gly1100
Asp and Thr1102Ala in VP1, Gly2162Ser,
Ser2171Gly, and Thr2173Ile in VP2. All of these mutational sites
are located around the sialic acid binding site (Fig.
5). These residues overlap with the
mutations described above that change the virus persistence in the CNS.
KJ6 does not show a significant persistence in the mouse CNS. It is
possible that these mutations alter the sialic acid binding of the DA
virus by causing conformational changes in the VP2 puffB, a finding which is consistent to what we have suggested for the nonpersistent mutants.
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FIG. 5.
Loop structures surrounding the sialic acid binding
site. Residues that alter the capability of a demyelinating persistent
infection by the DA virus are marked by spheres. The purple spheres
represent those from the nonpersistent mutants. The orange spheres are
those from the adaptation variant KJ6. The pink sphere represents
both.
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The persistence by the TO group of TMEV in the CNS is strictly an in
vivo phenomenon. All strains of TMEV go through a lytic infection cycle
in tissue culture. Since the capability of recognizing the sialic acid
moiety is the unique feature of the demyelinating persistent group of
TMEV, it may be speculated that this special interaction of the virus
with a major surface component, the sialyl moiety of the gangliosides
and other glycoconjugates, on the glial cells is one of the
determinants of virus persistence and demyelination. There is some
circumstantial evidence that might support this idea. It has been
reported that administration of sialyl gangliosides to mice at the
later phase of the TMEV demyelinating persistent infection could
suppress the activation of pathogenic Th1 cells and did suppress the
TMEV-induced demyelinating disease both clinically and histologically
(15). Sialyl gangliosides could either inhibit virus
infection in the CNS as that by sialyllacose in tissue culture or else
act as decoys to prevent the interactions of TMEV with gangliosides on
the glial cells. In the CNS, the virus particles may adhere on the
surface of the glial cells by binding to nonspecific sialyl conjugates.
This nonproductive attachment to the sialylated surface molecules could
reduce the effective infectivity of persistent TMEV (an increased ratio
of particle/PFU). It could also prevent the association of several key
proteins required for myelination, such as myelin-associated
glycoproteins (MAG). MAG mediates the association of myelinating glial
cells with neuronal axon. It has been shown that the MAG-mediated
cell-cell interactions occur through the recognition and attachment of
MAG to sialyl gangliosides, as demonstrated by Yang et al.
(43). A recent study on the interaction of the
foot-and-mouth disease with heparan sulfate also revealed a receptor
binding site for an oligosaccharide moiety on the viral surface
(10).
| |
ACKNOWLEDGMENTS |
L.Z. and Y.L. contributed equally to this work.
We are very grateful to Michael Soltis and the staff for their help at
SSRL beamline 7-1. We also appreciate the help from Bindong Sha in data
collection. We thank Ray P. Roos, University of Chicago Medical Center,
for providing the DAFL3 virus stock. We also thank L. Andrew Ball for
critical reading of the manuscript.
L. Zhou was supported by a National Research Service Award from the
National Institutes of Health.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Center for Macromolecular Crystallography, University of Alabama at Birmingham, 260 Basic Health Sciences Bldg., 1918 University Blvd., Box 79 THT, Birmingham, AL 35294-0005. Phone: (205) 934-4259. Fax: (205) 975-9578. E-mail: ming{at}cmc.uab.edu.
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Journal of Virology, February 2000, p. 1477-1485, Vol. 74, No. 3
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
