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Journal of Virology, December 1998, p. 10246-10250, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Very-Low-Density Lipoprotein Receptor Fragment Shed
from HeLa Cells Inhibits Human Rhinovirus Infection
Thomas C.
Marlovits,
Christina
Abrahamsberg, and
Dieter
Blaas*
Institute of Biochemistry, Medical Faculty,
A-1030 Vienna, Austria
Received 20 May 1998/Accepted 24 August 1998
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ABSTRACT |
The large family of human rhinoviruses, the main causative agents
of the common cold, is divided into the major and the minor group based
on receptor specificity. Major group viruses attach to intercellular
adhesion molecule 1 (ICAM-1), a member of the immunoglobulin
superfamily, whereas minor group viruses use low-density lipoprotein
receptors (LDLR) for cell entry. During early attempts aimed at
isolating the minor group receptor, we discovered that a protein with
virus binding activity was released from HeLa cells upon incubation
with buffer at 37°C (F. Hofer, B. Berger, M. Gruenberger, H. Machat,
R. Dernick, U. Tessmer, E. Kuechler, and D. Blaas, J. Gen. Virol.
73:627-632, 1992). In light of the recent discovery of several new
members of the LDLR family, we reinvestigated the nature of this
protein and present evidence for its being derived from the human
very-low density lipoprotein receptor (VLDLR). A soluble VLDLR fragment
encompassing the eight complement type repeats and representing the
N-terminal part of the receptor was then expressed in the baculovirus
system; both the shed protein and the recombinant soluble VLDLR bind
minor group viruses and inhibit viral infection of HeLa cells in a
concentration-dependent manner.
| |
TEXT |
Since the determination of the
primary structure of the bovine low-density lipoprotein receptor (LDLR)
(37), the number of discovered membrane receptors with
similar structures has been constantly growing. The prototype, LDLR,
the very-low density lipoprotein receptor (VLDLR), the LDLR-related
protein (LRP), and megalin are currently being considered the
most-prominent members of the LDLR gene family (for reviews, see for
example, references 20, 29, and
33). A structural feature common to all of these
membrane proteins includes various numbers of imperfect direct repeats
of about 40 amino acids each, which are located at the extracellular N
terminus and exhibit similarity with the C9 component of complement.
These complement type repeats are involved in the attachment of a
number of different ligands belonging to functionally and structurally
unrelated protein classes. Next to the complement type repeats (or
interspersed with them) are several copies of elements with similarity
to epidermal growth factor precursor. In addition to asparagine-linked
oligosaccharides present within these domains, most of the receptors
also contain a highly O-glycosylated region close to the plasma
membrane. The transmembrane region is followed by a cytoplasmic tail
with amino acid sequence motives characteristic of localization to
coated pits.
The function of the LDLR is the maintenance of cholesterol homeostasis
by internalizing LDL upon binding to apolipoprotein-E and B-100. LRP is
a multiligand receptor which binds ligands as diverse as chylomicron
remnants and various proteinase-proteinase inhibitor complexes (among
other unrelated ligands) and mediates their transport to lysosomes for
degradation. Megalin, which is also termed gp330, is a multiligand
receptor as well and was originally characterized as the Heymann
nephritis antigen in experimental membranous glomerulonephritis in rats
(6, 14). However, the normal physiological function of this
large membrane receptor is still unknown. VLDLR was discovered in
rabbit heart by homology screening (34), shortly followed by
the isolation of its human (28) and mouse homologues
(24). The tissue distribution of this receptor suggested a
function in uptake of triglycerides as energy source (22).
Human rhinoviruses (HRVs) are small, icosahedral, nonenveloped viruses
with an RNA genome of positive (messenger sense) polarity (for a
review, see, for example, reference 27). The large
number of viral serotypes was divided into a major receptor group (91 serotypes) and a minor receptor group (10 serotypes), based on specific
binding to intercellular adhesion molecule 1 (ICAM-1) or to members of
the LDL receptor family. There is one exception; HRV87 binds to an
uncharacterized glycoprotein (35). In attempts to isolate
and characterize proteins which bind to minor receptor group HRVs, we
had previously found that a protein with virus-binding activity was
shed from HeLa cells in a soluble form upon incubation with buffer at
37°C (12). We have reinvestigated the nature of the shed
protein and show that it is an N-terminal fragment of VLDLR. A
recombinant soluble receptor encompassing only the ligand-binding
domain of human VLDLR was then expressed in the baculovirus system. In
the present communication, we demonstrate that the shed material as
well as the recombinant fragment inhibit viral infection in vitro.
A protein shed from HeLa cells binds HRV2.
Rhino HeLa cells
(Flow Laboratories) grown in T flasks (150 cm2) to a
confluency of 80% were washed twice with phosphate-buffered saline
(PBS) and incubated at 37°C in 5 ml of PBS for the times indicated in
Fig. 1. Detached cells were pelleted by a
low-speed centrifugation, and the supernatant was clarified in a type
65Ti rotor (Beckman) at 50,000 rpm for 60 min (S80) and concentrated to
a final volume of 50 µl with a Centricon concentrator (10-kDa cutoff;
Amicon). Aliquots corresponding to 107 cells were then run
under nonreducing conditions on a sodium dodecyl sulfate
(SDS)-polyacrylamide gel, and the separated proteins were
electrophoretically transferred to a polyvinylidene difluoride (PVDF)
membrane (Millipore) in 20 mM Tris-HCl-150 mM glycine (pH 8.8) at 40 mA for 16 h in the cold. The membranes were blocked with 20 mM
Tris-HCl (pH 7.5)-150 mM NaCl-2 mM CaCl2 (1× TBS/Ca) containing 2% Tween 20 (blocking buffer) for 1 h and incubated with 35S-labeled HRV2 (20,000 cpm/ml [23])
as described previously (16, 19). The membranes were dried
and autoradiographed for 24 h on Kodak Biomax MR films (Fig. 1).
In accordance with earlier experiments (12), HRV2 bound to a
protein with a molecular mass of approximately 84 kDa which appeared in
the cell supernatant upon incubation with PBS for 20 min; the binding
activity peaked at 40 min of incubation time, whereupon it declined and
was barely detectable after 80 min of incubation. This points to
inactivation of the protein either by denaturation or by proteolytic
degradation. HeLa cell membranes prepared as described elsewhere
(17) were also analyzed for control purposes. HRV2 binding
to LDLR (120 kDa) was clearly detectable in the cell membranes. Two
additional bands at positions corresponding to approximate
Mrs of 110 and 95 were also apparent.
Previously, these had been tentatively assigned to degradation products
of LDLR (13).
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FIG. 1.
Virus-binding activity is shed from HeLa cells upon
incubation with PBS. HeLa cells grown in T flasks were incubated with
PBS at 37°C. At the times indicated, cell supernatants from
individual flasks were saved and an S80 was prepared and the
supernatant was concentrated to 50 µl. Samples were adjusted to 1×
Laemmli sample buffer without  -mercaptoethanol and run on an 8%
polyacrylamide-SDS gel, and the separated proteins were
electrophoretically transferred onto a PVDF membrane. The membrane was
blocked, incubated with 2 × 105 cpm of
[35S]methionine-labeled HRV2 (19, 23), and
exposed to X-ray film. As a control, a HeLa cell membrane (memb.)
preparation corresponding to 5 × 106 cells was also
used. For the position of LDLR (120 kDa), compare also with Fig. 2.
Positions of marker proteins run on the same gel are indicated.
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The shed protein is derived neither from LDLR nor from LRP.
Rabbit antisera against bovine LDLR (diluted 1:10,000), against rat LRP
(diluted 1:5,000), and monoclonal antibodies IgG-C7 (0.2 µg/ml
[2]) and scFv7 (0.2 µg/ml [11])
were then used to further characterize the nature of the 84-kDa protein
by Western blotting. In order to resolve the 515-kDa 
-chain of LRP,
a polyacrylamide gradient gel was used (Fig.
2A), whereas analysis of smaller proteins was carried out on a linear 10% polyacrylamide gel (Fig. 2B). To allow
for easy comparison, HeLa cell membranes were always analyzed in
parallel to the material released from the cells into the PBS
supernatant. HRV2 attachment was also monitored and revealed binding to
the same proteins shown in Fig. 1. Note, however, that the migration
behavior of the virus-binding proteins somewhat varied in the different
gel systems (compare Fig. 2A and B to Fig. 1). In addition to these
virus-binding proteins, the gradient gel also allowed detection of
binding of HRV2 to the large subunit (515 kDa) of LRP (13).
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FIG. 2.
Characterization of the material shed from HeLa cells by
Western analysis with specific antibodies. Material released from HeLa
cells upon incubation with PBS for 40 min at 37°C was cleared and
concentrated, and proteins were separated on a 4 to 12% polyacrylamide
gradient gel (A) or on a 10% gel (B) under nonreducing conditions and
electrophoretically transferred onto PVDF membranes. The membranes were
incubated with 35S-labeled HRV2, antisera, and monoclonal
antibodies as indicated at the top of the panels. Virus was detected by
autoradiography. Bound antibodies were detected with horseradish
peroxidase (HRP)-conjugated goat anti-rabbit IgG (Promega) diluted
1:5,000; detection of scFv7 was with the anti-myc antibody 9E10
(Stratagene), followed by HRP-conjugated rabbit anti-mouse IgG
(Southern Biotechnology) diluted 1:5,000. IgG-C7 was detected with
HRP-conjugated rabbit anti-mouse IgG (Southern Biotechnology) diluted
1:5,000. HRP activity was revealed with the chemiluminescence substrate
Supersignal (Pierce). Control HeLa cell membranes were prepared as
described previously (17). The positions of marker proteins
run on the same gels are indicated. memb., HeLa membranes; sup.,
material shed from HeLa cells.
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Rabbit antiserum against bovine LDLR, which cross-reacts with the human
homologue, strongly labeled a protein migrating with
an apparent
molecular mass of 120 kDa in HeLa cell membranes;
a minor band of
approximately 110 kDa was also found to be recognized
by the
LDLR-specific antiserum identifying it as a degradation
product or as
an incompletely glycosylated form of LDLR. In the
cell supernatant, a
faint band at the same position was found
to be labeled by anti-LDLR
antibody; this LDLR-related product
is thus clearly different from the
84-kDa virus-binding protein.
As expected, antiserum raised against rat
LRP and cross-reacting
with the human homologue labeled the 515- and
the 85-kDa chain
of LRP, together with receptor-associated protein
(RAP), which
is strongly associated with this receptor (
9,
32). However,
no band was seen in the cell supernatant,
suggesting that the
84-kDa protein was not a degradation product of
LRP.
The monoclonal antibody IgG-C7, which is directed against the
N-terminal complement type repeat of LDLR (
2), was then used
for further identification. As seen in Fig.
2B, this antibody
recognized LDLR present in HeLa cell membranes but failed to bind
to
the material in the cell supernatant. The absence of any reaction
with
the protein in the cell supernatant identified as derived
from LDLR
(with rabbit antiserum; compare to Fig.
2A) might be
explained by
IgG-C7 binding requiring a native conformation for
recognition, whereas
the antiserum also binds to denatured LDLR
(data not
shown).
The human single-chain antibody scFv7 (
11,
25) binds with
high affinity to chicken ovarian VLDLR, cross-reacts with human
LRP
and, to some extent, with human LDLR, and inhibits attachment
of
various ligands including HRV2. As seen in Fig.
2B, scFv7 strongly
labeled a protein present in HeLa cell membranes which migrates
on the
polyacrylamide gel with an apparent molecular mass of 95
kDa and which
is clearly different from LDLR (compare also with
Fig.
2A). Two other
bands with slightly lower mobilities were
also visible. However, none
of them could be unequivocally assigned
to LDLR, as revealed with
anti-LDLR antiserum. In the cell supernatant,
a strong band at 84 kDa
became apparent upon incubation of the
blot with
scFv7.
The protein shed from HeLa cells is a fragment of VLDLR.
The
data from the Western blot experiments made it highly unlikely that the
shed protein was a fragment of LDLR or of LRP. We thus asked whether it
might be related to VLDLR. Plasma membrane preparations from HeLa cells
and, for control purposes, from small oocytes from chicken ovaries
(31) and from CHO cells transformed with a plasmid encoding
the human VLDLR splicing variant which lacks the O-linked sugar domain
(28) were subjected to Western blotting with a rabbit
antiserum raised against recombinant human VLDLR. As seen in Fig.
3, all three membrane preparations
contained a protein which exhibited an apparent molecular mass of 95 kDa under nonreducing conditions. This makes it clear that the short splicing variant of VLDLR is expressed in HeLa cells. Moreover, the
cross-reaction of the VLDLR antiserum with the chicken homologue emphasizes the high similarity between these two proteins. The absence
of any additional bands demonstrates the high specificity of the
antiserum and thus its suitability for identification of the soluble
84-kDa protein in the HeLa cell supernatant as obtained upon
incubation with PBS.
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FIG. 3.
VLDLR is present in HeLa cells. Chicken oocyte membranes
(OM) (30), HeLa cell membrane extract (HM), and membranes
from CHO cells expressing the short splicing variant of human VLDLR
(CHO [28]) were analyzed for the presence of VLDLR by
Western blotting with antiserum raised against recombinant human VLDLR.
Approximately 25 µg of total protein per lane was loaded. Note the
cross-reaction of the VLDLR antiserum with chicken OVR.
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RAP, which was originally found in preparations of LRP (
10),
is considered a specific chaperone (
3,
36) and was shown
to
compete with all ligands for binding to members of the LDLR
family
(
1,
9,
18). Proteins released from HeLa cells upon
incubation in PBS were thus subjected to affinity chromatography
on a
glutathione
S-transferase (GST)-RAP column (
15).
HeLa suspension
cells (8 × 10
8) were washed twice
with PBS and gently stirred at 37°C for 40
min in 20 ml of PBS. The
cell supernatant was centrifuged at 50,000
rpm in a Beckman 65Ti rotor
for 60 min and applied to a GST-RAP
column (0.5 ml) which had been
equilibrated with PBS. GST-RAP
Superose was prepared by coupling 30 mg
of GST-RAP (
9) to 8
ml of CNBr-activated Sepharose
(Pharmacia) as described by the
manufacturer. The column was washed
with 1× Tris-buffered saline
(TBS)-Ca, and bound proteins were eluted
in 0.5-ml fractions with
1 M NH
3 in 0.3× TBS-Ca.
NH
3 was removed, and fractions were concentrated
to a final
volume of 150 µl in a SpeedVac concentrator. Aliquots
(20 µl) from
the cell supernatant, from the flowthrough, and from
the eluted
fractions were applied onto polyacrylamide gels, and
the proteins were
separated under nonreducing conditions and were
electrophoretically
transferred onto PVDF membranes. The membranes
were incubated with
[
35S]methionine-labeled HRV2, an IgY fraction prepared
from eggs
of a chicken immunized with rLDLR
1-7h, a
recombinant LDL
receptor fragment comprising the ligand binding domain
(
17),
at a final concentration of 2 µg/ml and with an
antiserum against
recombinant human VLDLR (diluted 1:10,000). The
respective antibodies
were detected with alkaline phosphatase
(AP)-conjugated goat anti-IgY
(1:5,000) and nitroblue tetrazolium salt
(NBT)-5-bromo-4-chloro-3-indolylphosphate
(BCIP) as a substrate;
horseradish peroxidase (HRP)-conjugated
anti-rabbit IgG was revealed
with chemiluminescence substrate.
As seen in Fig.
4 (left panel), the 84-kDa protein
binding HRV2
was strongly enriched in fractions 2 and 3. A faint band
migrating
with an apparent molecular mass of 110 kDa which also bound
HRV2
appeared in the same fractions. Development of the blot with the
IgY fraction reacting with LDLR showed that this latter band was
clearly related to LDLR (middle panel). Final proof of the identity
of
the 84-kDa virus-binding protein with a fragment of VLDLR was
then
obtained by its reaction with the antiserum raised against
human
recombinant VLDLR (right panel). Coomassie staining of gels
identical
to those used for Western blotting revealed only several
faint bands
(data not shown) which did not correspond to any of
the proteins
revealed with virus or antisera upon Western blotting
(Fig.
4). The
amount of protein shed from approximately 5 × 10
7
HeLa cells is thus certainly less than 100 ng, the approximate
detection limit of Coomassie blue staining.
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FIG. 4.
The virus-binding activity shed from HeLa cells is a
VLDLR fragment. HeLa suspension cells (8 × 108) were
incubated with 20 ml of PBS for 40 min at 37°C. The cells were
pelleted, and the S80 supernatant was applied onto a GST-RAP affinity
column. Fractions (0.5 ml) obtained upon elution with 1 M ammonia were
collected and concentrated to 150 µl, and aliquots of 20 µl
(corresponding to approximately 5 × 107 cells) were
analyzed by polyacrylamide gel electrophoresis on 10% gels, followed
by ligand blotting with [35S]methionine-labeled HRV2,
with LDLR-specific IgY, and with rabbit antiserum against VLDLR,
respectively. A 20-µl aliquot of the original unconcentrated sample
(lanes S) and 20 µl of the flowthrough (lanes F) were also analyzed.
The positions of marker proteins run on the same gels are also
indicated. hVLDLR, human VLDLR.
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Soluble recombinant human VLDLR inhibits HRV infection.
We
have previously shown that the chicken ovarian VLDL receptor (OVR)
binds HRV2 on ligand blots and inhibits HRV2 infection of HeLa cells in
its detergent solubilized form (8). However, HRVs fail
to replicate in birds; therefore, the function of OVR as a viral
receptor is questionable. We thus asked whether human VLDLR would bind
to HRV2 in solution and thereby inhibit viral infection. Since further
purification of the 84-kDa protein appeared tedious and
greater amounts were difficult to obtain, soluble human VLDLR
encompassing only the eight complement type repeats (rVLDLR1-8h) was expressed in insect cells with a
C-terminal hexahistidine tag by using the baculovirus system. A DNA
fragment encoding the eight complement type repeats of human VLDLR
(28) was PCR amplified with pfu polymerase
(Stratagene) by using the two synthetic oligonucleotides (forward,
5'-ATGCGGATCCAGGGAGAAAAGCCAAATGTG-3'; reverse,
5'-GCTGCCCGGGACACTCTTTCAGGGGCTCAT-3')
hybridizing at positions 82 to 100 and 1046 to 1066, respectively, of the coding region of human VLDLR cDNA. Nucleotides
shown in boldface were added to create restriction sites for
BamHI and XmaI (underlined). DNA was denatured at
94°C for 3 min; amplification consisted of 30 cycles at 94 (1 min),
57 (45 s), and 72°C (2 min). The fragment was digested with
BamHI and XmaI, purified by agarose gel
electrophoresis, and ligated into the baculovirus transposition vector
pTM1 (16). Recombinant bacmid DNA was obtained by
transposition in DH10Bac cells according to the manufacturer's
protocol (Life Technologies) and was used for lipofection of Sf9 insect
cells. Recombinant baculovirus (vVLDLR1-8h) was used to
infect Sf9 cells at a multiplicity of infection of 5 for production of
recombinant VLDL receptor fragment rVLDLR1-8h. Cells were
harvested at 80 h postinfection and the secreted protein was
purified from the cell culture supernatant on a Ni-nitriloacetic acid
(NTA) column as described previously (17). The Ni-NTA eluate
was then further purified on a GST-RAP column (data not shown) as
described above, and matched fractions were compared with those of the
84-kDa protein with respect to protection of HeLa cells against
infection with HRV2 essentially as described elsewhere (17).
Briefly, 1.4 µg of Ni-NTA and GST-RAP affinity-purified
rVLDLR1-8h (20 µl) or 20 µl of the 84-kDa protein
(fraction 2 from the GST-RAP column [Fig. 4]) was mixed with 80 µl
of infection medium (minimal essential medium containing 2% fetal calf
serum and 30 mM MgCl2), and serial twofold dilutions were
made in the same medium. To each dilution (50 µl), 100 50% tissue
culture infectious doses (TCID50) of HRV2 (in 50 µl of
infection medium) were added, and the mixtures were incubated for
1.5 h at 34°C. They were then transferred onto monolayers of
HeLa cells in 96-well plates (containing 100 µl of infection medium),
and the plates were incubated for 3 days at 34°C. Remaining cells
attached to the plastic were stained with amido black (0.1% in acetic
acid, methanol, water; 10/40/50 [vol/vol]). Noninfected cells and
cells infected in the absence of receptor were used as positive and
negative controls, respectively. As seen in Fig.
5, both the 84-kDa protein and
rVLDLR1-8h inhibited infection of the cells in a
dose-dependent manner. Whereas the concentration of the 84-kDa protein
in the crude cell supernatant was not sufficient for cell protection,
significant inhibition of cytopathic effect was observed upon partial
purification of the material on the RAP column (Fig. 4, fraction 2). In
contrast, fraction 9, which was used as a negative control, failed to
protect the cells against infection. Under the same conditions, the
purified recombinant soluble receptor present in fraction 2 from the
GST-RAP column (data not shown) strongly inhibited viral infection,
whereas fraction 9 was again negative. Quantitative comparison of the cell protective effect with the enriched 84-kDa protein was, however, not possible due to its small amount and the presence of contaminating proteins.
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FIG. 5.
VLDLR fragments protect HeLa cells against infection
with HRV2. HRV2 (100 TCID50) was incubated for 90 min at
34°C with serial twofold dilutions (left to right) of HeLa cell
supernatant obtained upon incubation with PBS (S), of fraction 2 (F2)
and of fraction 9 (F9) as eluted from the GST-RAP column (see Fig. 4),
and of corresponding F2 and F9 of the GST-RAP column purification of
rVLDLR1-8h. Cells infected with virus preincubated with
plain infection medium (MIM) were used as the negative control. HeLa
cell monolayers were challenged with the mixtures and stained with
amido black after incubation for 3 days at 34°C. MIM and supernatant
from the PBS incubation (S) were also compared in the absence of virus
to exclude any influence on HeLa cell viability.
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Conclusions.
In an attempt to isolate the rhinovirus minor
group receptor, we discovered that a protein with virus binding
activity was released upon incubation of the cells with PBS at 37°C
(12). This was not entirely unexpected, since several other
receptors have been shown to be shed from the cell surface by the
action of membrane-bound specific proteinases (5, 21). We
thus argued that shedding might also occur upon cultivation of the
cells and finally succeeded in purifying a virus-binding activity from
large amounts of spent tissue culture supernatant and unambiguously identified it as human LDLR (13). However, during the
purification process, detergent was used and the protein which was
finally isolated migrated slightly more slowly than that found upon
incubation of HeLa cells with PBS. In the light of the discovery of
novel members of the LDLR family during the last few years, we decided to reexamine the nature of the shed protein and thus to clarify the
discrepancy between its apparent molecular weight (12) with that of LDLR or degradation products and precursors thereof
(13).
Using various specific antisera, we here demonstrate that HeLa cells
express a protein which specifically reacts with antiserum
against
VLDLR. It migrates with an apparent molecular weight corresponding
to
the smaller form of VLDLR lacking the O-linked sugar domain
(
28). In addition to membrane-bound receptor, a soluble
fragment
of this protein is released from the cells upon incubation
with
PBS. However, since identification of this protein as VLDLR relies
only on immunological reagents in the absence of any amino acid
sequence data, it cannot be excluded with absolute certainty that
the
protein has high similarity to VLDLR but is not the VLDLR
proper.
Based on the results from Western blots with various antisera (Fig.
2),
only very minor amounts of soluble LDLR were present
in the HeLa cell
supernatant. LDLR isolated from the spent tissue
culture supernatant
(
13) must thus have originated from membrane
fragments.
Shedding of plasma membrane fragments is a widespread
feature of viable
cells and has previously been reported for HeLa
cells (
4).
Evidence for the presence of soluble LRP
-chain in normal human
serum was recently obtained by affinity chromatography on
methylamine-activated
2-macroglobulin (
26).
The physiological
significance of the soluble LRP is not clear at the
present time.
VLDLR does not bind to
2 macroglobulin,
and, when present in
human serum, it might have escaped detection by
this procedure.
Attempts to isolate soluble VLDLR from spent tissue
culture media
by affinity chromatography on a GST-RAP column indeed
revealed
an HRV2-binding protein migrating with an apparent molecular
mass
of 84 kDa; however, large amounts of immunoglobulins were
unspecifically
retained, preventing efficient purification of VLDLR by
this method
(data not
shown).
The 84-kDa protein released from HeLa cells upon incubation in PBS was
efficiently enriched by affinity chromatography on
a GST-RAP column
(Fig.
4). The eluted material was then assayed
for its capacity to
inhibit the cytopathic effect of HRV2 infection
of HeLa cells. For
control purposes, we also expressed a soluble
N-terminal fragment of
VLDLR encompassing the eight complement
type repeats in Sf9 insect
cells. The shed 84-kDa protein and
the recombinant soluble receptor,
both of which were enriched
on a GST-RAP column, efficiently protected
HeLa cells from infection.
Quantitative comparison was not attempted
because of the difficulties
in obtaining a homogenous preparation of
the 84-kDa
protein.
LDLR fragments composed of various numbers of the ligand binding
repeats have been shown to exhibit antiviral activity toward
HRVs
(
16,
17). An antiviral helper factor, a 28-kDa N-terminal
fragment from LDLR, whose release from various cell types into
the
medium was induced with gamma interferon, has been described
previously. Interestingly, it was active against vesicular stomatitis
virus (VSV) infection (
7), but its activity was not exerted
by interference with virus attachment but probably by an unknown
effect
on virus assembly or budding. Whether the release of the
VLDLR fragment
can also be stimulated by interferon has not been
studied so far.
Further work is required to investigate whether
shedding of the VLDLR
fragment also occurs under normal conditions
in the healthy
organism.
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ACKNOWLEDGMENTS |
This work was supported by grant no. P12189-MOB from the Austrian
Science Foundation.
We thank J. Nimpf, M. Huettinger, and H. Hobbs for the generous gifts
of antisera against LDLR, LRP, and VLDLR, respectively; J. Nimpf for
the VLDLR clone; I. Goesler for excellent tissue culture work; and R. Wandl for the Western blot shown in Fig. 4.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Biochemistry, Medical Faculty, Dr. Bohr Gasse 9/3, A-1030, Vienna,
Austria. Phone: 43 1 4277 61630. Fax: 43 1 4277 9616. E-mail:
dieter.blaas{at}univie.ac.at.
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Journal of Virology, December 1998, p. 10246-10250, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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