FEMS Microbiology Letters 168 (1998) 251^258

Putative dengue virus receptors from mosquito cells

Mar|a de Lourdes Munoz *, Alejandro Cisneros, Jorge Cruz, Pradeep Das,
Rosalinda Tovar, Arturo Ortega
Departamento de Genetica y Biolog|a Molecular, CINVESTAV-IPN, Apartado Postal 14-740, Mexico D.F. 07000, Mexico

Received 7 March 1998 ; received in revised form 3 August 1998; accepted 11 September 1998

Abstract

Dengue viruses are arthropod-borne, single-stranded RNA viruses. Aedes aegypti and Aedes albopictus are the principal
vectors. In order to understand the molecular basis of dengue virus infections we explored the biochemical identity of dengue-2
(DEN-2) virus receptors in the Aedes albopictus-derived cell line C6/36. We show here that DEN-2 interacts with two major
polypeptides of 80 and 67 kDa. Polyclonal anti-C6/36 membrane antibodies block DEN-2 binding to intact C6/36 monolayers
as well as to membrane extracts. Our results strongly suggest that the identified polypeptides are part of the DEN-2
receptors. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords : Dengue virus; Receptor; C6/36 cell

1. Introduction which are used for initial binding by varicella-zoster

The interactions between specic viral surface pro-
teins (cell attachment proteins) and receptors on tar-
get cells play an important role in viral tropism. Any
constituent of the cell membrane, including carbohy-
drates, lipids, and proteins, may be a viral receptor,
depending on the virus. These cell membrane com-
ponents serve normal cellular functions and are used
by viruses for attachment and entry. A number of
cellular molecules have been identied as viral recep-
tors, e.g., the epidermal growth factor receptor for
vaccinia virus [1], complement receptor type 2 for
Epstein-Barr viruses [2], the L-adrenergic receptor
for reovirus type 3 [3], among others. Some mole-
cules, like mannose 6-phosphate and heparan sulfate,
* Corresponding author. Fax: +52 (5) 747-7100;
E-mail: lmunoz@gene.cinvestav.mx
and herpes simplex viruses and probably by DEN-2
[4^6], are present in many types of cells. However, in
view of the tissue tropism of these viruses, virus
entry cannot be controlled by these ubiquitous recep-
tor molecules alone. The role of the broblast
growth factor receptor as a more specic receptor
for herpes simplex virus type I is an example where
heparan sulfate also serves as an accessory molecule
[7].
The four serotypes of DEN virus form a distinct
antigenic subgroup of some 70 members of the ar-
thropod-borne, serologically related aviviruses, now
classied within the Flaviviridae family [8]. They
contain a single and positive-stranded RNA of ap-
proximately 11 kb in length as their genome. The
genomic RNA presents a long open reading frame
which encodes three structural proteins: a major gly-
cosylated envelope protein E (51^59 kDa), a small

0378-1097 / 98 / $19.00 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 4 3 4 - 0

FEMSLE 8437 5-11-98

252 M. de Lourdes Munoz et al. / FEMS Microbiology Letters 168 (1998) 251^258
non-glycosylated envelope protein M (8 kDa) and a
non-glycosylated nucleocapsid protein C (14 kDa),
followed by at least seven non-structural proteins.
During translation, a putative polyprotein of about
3400 amino acid residues is synthesized and cleaved
by cellular and/or viral proteases [9]. Monoclonal
antibodies to the envelope E glycoprotein protect
mice against lethal dengue infection, suggesting that
this protein is involved in the recognition of the tar-
get cell [10]. Furthermore, binding of DEN-4 E pro-
tein to cell surface receptors correlates with cell sus-
ceptibility to virus infection [11]. More recently, it
has been demonstrated that E protein mediates the
binding of virus to cells lacking Fc receptors [12].
Mutations in E protein also aect the virulence of
virus variants [13].
DEN viruses are transmitted in a human-mosqui-
to-human cycle, with Aedes aegypti and Aedes albo-
pictus as the principal arthropod vectors. Data sug-
gest that distribution and incidence of DEN virus
infections have steadily increased since the 1950s,
and major epidemics of dengue involving hundreds
of thousands of cases now occur. Dengue infection
typically results in an acute febrile illness. A severe
manifestation, known as DEN hemorrhagic fever-
shock syndrome, has likewise increased throughout
the world [14].
A strategy to tackle this health problem is to mod-
ify the opportunity to transmit the viral infection to
mosquitoes. For example, if viral entry to the vec-
tor's cells is blocked, no transmission will take place
to the vector. To date no denitive evidence for the
identity of the cellular DEN receptors has been re-
ported [15^17]. As a preliminary step to an alterna-
tive way to control DEN infection, we report here
the biochemical characterization of DEN-2 virus re-
ceptors expressed in the mosquito-derived cell line
C6/36. The results are discussed in terms of the re-
cent characterization of DEN-4 binding sites ex-
pressed in this cell line and the role of heparan sul-
fate as an initial binding site for DEN [16,17].
2. Materials and methods
2.1. Viruses, purication and biotinylation
DEN-2 virus (NGC strain) was kindly supplied by
FEMSLE 8437 5-11-98
Dr. Gubler (CDC, Fort Collins, CO). The virus was
amplied in rhesus monkey (Macaca mulatta) kidney
cells (LLC-MK2). Viruses were puried as previously
described [18]. Briey, DEN-2 virus was passaged in
LLC-MK2 cells and claried from cell cultures. The
infected cells were collected and centrifuged at
20 000Ug for 15 min. The supernatant was incu-
bated overnight with 7% polyethylene glycol 6000
(PEG-6000) in 0.5 M NaCl2 at 4C and the suspen-
sion was centrifuged for 30 min at 10 000Ug. The
pellet was resuspended in PBS and the PEG elimi-
nated with chloroform. The aqueous phase was lay-
ered on top of a 20^50% discontinuous sucrose gra-
dient in TGEN buer (50 mM Tris-HCl, 200 mM
glycine, 1 mM EDTA, 100 mM NaCl, pH 7.5) and
centrifuged for 3 h at 100 000Ug. The virus was
recovered from the interface of the gradient and
the stock was kept frozen at 370C until use. The
titer of the viral stock was 6U106 PFU ml31 . Puri-
ed viruses were biotinylated by resuspension of the
viral pellet (approximately 1 mg of protein) in 1 M
NaHCO3 pH 9.5 and incubated overnight with 1 mg
ml31 of N-hydroxy-succimido-biotin, diluted in
DMSO. Centrifugation at 10 000Ug for 1 h was
done after the incubation and the pellet that con-
tained the biotinylated virus was resuspended in
PBS. The degree of biotinylation was determined
by dot blot, and the viral stock was kept at 370C
until use.
2.2. Cell culture and infection
C6/36 cells were cultured at 32C in minimal es-
sential medium (MEM) containing 5% fetal bovine
serum, 2 mM glutamine, 100 mM non-essential ami-
no acids, 100 U of penicillin and streptomycin (100
Wg ml31 ) [18]. LLC-MK2 cells were grown at 37C in
a humidied atmosphere of 5% CO2 in MEM con-
taining 5% fetal bovine serum, 100 U of penicillin
and streptomycin (100 Wg ml31 ). Conuent mono-
layers of C6/36 cells were exposed to 0.2 ml of
DEN-2 virus inoculum with an input MO1 ranging
from 200 to 600 PFU. These cell monolayers were
also exposed to DEN-2 virus preincubated with ex-
tracts of puried membranes of C6/36 cells to deplete
the viral stock. Neutralizing assays were performed
with cells incubated for 40 min with the appropriate
antibodies and exposed to DEN-2 virus.
 M. de Lourdes Munoz et al. / FEMS Microbiology Letters 168 (1998) 251^258
2.3. Radiolabeling of C6/36 cell membrane proteins
Cell surface iodination of conuent monolayers
was performed by the lactoperoxidase method as de-
scribed elsewhere [19]. Briey, 1 mCi of 125 I (Amer-
sham Pharmacia Biotech) and lactoperoxidase (100
Wg ml31 ; Sigma-Aldrich Quimica, Mexico) in 4 ml of
phosphate-buered saline (PBS) were added to a
monolayer of conuent cells (2^5U107 ). The labeling
reaction was continued for 3 min, with H2 O2 at 4C.
The labeled cells were washed with PBS containing
10 mM KCl to remove free 125 INa. Membrane pro-
teins were solubilized with PBS containing 0.5% Tri-
ton X-100, 0.25% Nonidet P-40, 5 mM p-hydroxy-
methylbenzoate (PHMB), 5 mM N-ethylmaleimide
(NEM) and 1 mM phenylmethylsulfonyl uoride
(PMSF) for 30 min on ice. The debris was cleared
by centrifugation at 35 000Ug for 60 min. The solu-
bilized membrane proteins were used immediately or
stored at 370C.
2.4. Membrane preparation
C6/36 cell membranes were prepared essentially as
described elsewhere [20] by scraping the cells from
conuent plates in the presence of PBS. The suspen-
sion was washed three times by centrifugation at
600Ug for 10 min. The resulting pellet was resus-
pended in 1 M Tris-HCl pH 7.2 containing 5 mM
PHMB, 5 mM NEM and 1 mM PMSF after vigo-
rous agitation for 5 min, and subjected to three
cycles of freezing and thawing. An aliquot of 0.4
ml was layered on top of a freshly prepared glycerol
gradient consisting of ve 0.4-ml layers of 60%, 50%,
40%, 30% and 20% glycerol (v/v) in 50 mM MgCl2 .
The tubes were placed in a SW 50.1 rotor and cen-
trifuged at 4C for 40 min at 40 000Ug in a Beckman
ultracentrifuge. The 20% and the 60% interfaces were
separated and centrifuged for 20 min at 60 000Ug.
The nal pellet was resuspended in 0.1 ml of a pro-
tease inhibitor cocktail containing 5 mM PHMB, 5
mM NEM and 1 mM PMSF and 5 mM EDTA pH
8.
2.5. Preparation of anti-membrane antibodies
The C6/36 cell membrane preparation was used as
the antigen to produce polyclonal antibodies in mice.
FEMSLE 8437 5-11-98
253
BALB/c mice were subcutaneously injected with 70
Wg of membrane proteins in 20 Wl of Titer-Max
(CytRx Corporation, Georgia) diluted 1:1 with
PBS. Fifteen days later, the mice were intraperito-
neally inoculated with 70 Wg of antigen in PBS and
30 days later the serum was obtained.
2.6. Electrophoresis and virus overlay protein binding
assay (VOPBA)
Proteins were rst separated by SDS-PAGE using
7.5% polyacrylamide gels [21]. In order to use the
membrane proteins as targets for virus binding, a
standard Western blotting procedure was performed
[22]. The electrophoretic blots were incubated in PBS
containing 5% dried skimmed milk for 1 h to block
excess protein binding sites. Filters were then incu-
bated overnight with 6.1U105 PFU of biotinylated
virus, and the polypeptides that reacted with the
probe were revealed using streptavidin-phosphatase
according to the manufacturer's protocol (Bio-Rad
kit, catalog numbers 170-6532 and 170-6539). Inhib-
ition of virus binding to immobilized C6/36 mem-
brane proteins was performed by incubation of the
blots with anti-membrane antiserum or preimmune
mouse serum prior to the exposure to biotinylated
virus. Protein electroelution was performed accord-
ing to Hunkapiller et al. [23].
2.7. Staining procedures
Cell staining with anti-membrane antisera or anti-
DEN-2 monoclonal antibodies was performed. C6/
36 cells grown and infected as described above were
xed for 15 min with 1.85% formaldehyde and
0.125% glutaraldehyde at 37C, washed with PBS,
and incubated with 1 M glycine for 15 min at
37C. The cells were then permeabilized with 0.3%
Triton X-100 in 15 mM phosphate buer (pH 7.2)
containing 180 mM NaCl and washed three times
with the same solution and incubated with 0.0025%
Evans blue for 20 min at 37C. Exposure to anti-
membrane polyclonal antibodies (1 mg ml31 , diluted
1:20) or the anti-DEN-2 mAbs (ATCC number:
HB-46) (diluted 1:50) was carried out at room tem-
perature for 1 h. After washing the preparations
were mounted in 50% glycerol and observed by uo-
rescence microscopy.
254 M. de Lourdes Munoz et al. / FEMS Microbiology Letters 168 (1998) 251^258

the virus during 1 h to infect cells as mentioned

above. After washing, cells were xed and stained
with the specic antibody anti-DEN-2 as indicated
in Section 2.7 for the indicated time periods. The
uorometric measurements were performed in a LS
50B Perkin Elmer uorometer.

3. Results and discussion

The identication of viral receptors represents a

critical step in the designing of possible targets for
preventing viral entry in cells. Possible approaches to
intervention are mAbs blocking the receptor, and
synthetic peptides mimicking the viral receptor and
thus competing with the host receptor for the attach-
ment of the virus. Dengue viruses have pathogenic
capacities that are not yet fully understood. The
identication and characterization of its receptors
Fig. 1. Virus overlay of puried membrane preparations of C6/36
cells. The putative receptors were revealed after incubation with
could aid in the design of novel strategies for the
biotynylated DEN-2 and peroxidase-linked streptoavidin. Lane
A, nitrocellulose membranes exposed to the streptavidin-peroxi-
dase conjugate. Lane B, membranes exposed to DEN-2 and
streptavidin-peroxidase. The size of the molecular weight markers
is shown on the left side.

2.8. Enzymatic treatment of C6/36 cells

Cell suspensions (approximately 5U106 cells) were

incubated with 2 U of neuraminidase (Calbiochem-
Novabiochem International, California) in serum-
free culture medium at 37C for 20 min. The cell
suspension was washed twice with PBS and a mem-
brane preparation was prepared as detailed above.
For trypsin treatment, cell monolayers were treated
with a solution of 0.5 mg ml31 of trypsin 0.25%
(Gibco, Cat. no. 15050-065) for 5 min at 37C.

2.9. Fluorometric quantitation of DEN-2 virus

infection
Approximately 3U104 C6/36 cells were grown on
tissue culture multiwell plates (model 76-003-05 96
at-bottomed wells, diameter 0.6 cm, Linbro, Chem-
ical Co., Hamden, CT) for 1 day. Cells were then
incubated with preimmune or anti-C6/36 membrane
antibodies (1 mg ml31 , diluted 1:100) for 40 min at
30C. After washing the cells were incubated with
Fig. 2. Anti-membrane Abs inhibit DEN-2 binding to immobi-
lized putative receptors. Lane A, membranes exposed to the
streptavidin-peroxidase conjugate. Lane B, membranes exposed
to DEN-2 and streptavidin-peroxidase. Lane C, membranes ex-
posed to normal mouse serum prior to DEN-2 and streptavidin-
peroxidase. Lane D, membranes exposed to pre-immune anti-
membrane serum prior to DEN-2 and streptavidin-peroxidase.
Lane E, membranes exposed to anti-membrane antisera prior to
DEN-2 and streptavidin-peroxidase. The size of the molecular
weight markers is shown on the left side.

FEMSLE 8437 5-11-98

 M. de Lourdes Munoz et al. / FEMS Microbiology Letters 168 (1998) 251^258 255

virus (Fig. 3, lanes 3 and 5). In addition, two major

proteins were identied by radiolabeling of the apical
surface of C6/36 cells with apparent molecular

Fig. 3. Radiolabeling of C6/36 cell surface proteins and electro-

elution of the 67 kDa polypeptide from C6/36 membrane prepa-
rations. Lane 1, radiolabeling of the apical surface of C6/36 cells.
Lanes 2 and 4, control without biotynylated virus. Lanes 3 and
5, membranes exposed to DEN-2 and streptavidin-peroxidase.
The size of the molecular weight markers is shown on the left
side.

control and prevention of dengue. Published data

have shown that the viral E protein is involved in
the recognition of target cells [10,12] and that anti-E
antibodies prevent viral infection [10]. Recently
Ludwing et al. [24], using an overlay assay, identied
a 32-kDa polypeptide as the putative receptor for
Venezuelan equine encephalitis virus in C6/36 cells.

3.1. Identication of putative DEN-2 receptors

When immobilized C6/36 membrane preparations

were incubated with biotinylated DEN-2 virus ve
polypeptides were recognized (Fig. 1). These in-
cluded two prominent polypeptides with apparent
molecular masses of 80 and 67 kDa, respectively.
The specicity of the reactions was determined by
using anti-C6/36 membrane antisera. The results
are shown in Fig. 2. Pre-exposure of the biotinylated
virus to these sera inhibited the recognition of the Fig. 4. Anti-membrane Abs inhibit DEN-2 binding to C6/36
67-kDa as well as a faint band of 19 kDa (Fib. 2, monolayers. Panel A: Cells infected for 3 days and detection of
lane E). As expected, treatment of the immobilized the virus with anti DEN-2 mAbs followed by uorescein-labeled
membranes with normal or preimmune sera does not anti mouse IgGs. Panel B: Cells preexposed to pre-immune anti
block the viral binding (Fig. 2, lanes C and D re- membrane serum and infected with DEN-2, the detection of the
virus after 3 days of infection was done as detailed. Panel C:
spectively). When the 67-kDa polypeptide was elec- Monolayers pre-treated with anti-membrane polyclonal Abs.
troeluted from the gels and re-analyzed via the over- Note the signicant reduction in uorescence. The size of the
lay assay, it was recognized by biotinylated DEN-2 molecular weight markers is shown on the left side.

FEMSLE 8437 5-11-98

256 M. de Lourdes Munoz et al. / FEMS Microbiology Letters 168 (1998) 251^258

uorescein-labeled anti-mouse IgGs. As depicted in

Fig. 4C, anti-membrane antiserum inhibited the
binding of DEN-2 in C6/36 monolayers cultured
for 3 days. By contrast, when preimmune serum
was used prior to the incubation with virus, a strong
signal of anti-DEN-2 mAbs antibody binding was
revealed (Fig. 4B). A quantication of the anti-
DEN-2 antibody binding signal at 0, 1, and 3 days
post-infection is presented in Fig. 5. These results
demonstrate that a membrane component interacts
with the virus, and this is shown by the fact that
puried membrane preparations depleted the viral

Fig. 5. Fluorometric quantication of the inhibition of DEN-2

binding to intact C6/36 cells. The experiments were performed as
detailed in Fig. 4 except that uorescence was measured at 0, 1
or 3 days of infection. The average of the uorometric quanti-
cation of two independent experiments is shown.

masses of 80 and 67 kDa (Fig. 3, lane 1). Previously,

DEN-4 virus has been reported to interact with 40-
and 45-kDa polypeptides from total C6/36 extracts
[17]. These results are in contrast with the ones pre-
sented here. A plausible explanation for this discrep-
ancy is the protein source used for VOPBA assays.
While in the present study a puried membrane
preparation was used, the study by Salas and Del
Angel [17] used a total protein extract, in which
the amount of the 67-kDa band might be minimal.
Although we detected more than one polypeptide
with the biotinylated virus and with the radioactive
labeling, we failed to detect 40- and 45-kDa proteins,
suggesting that these are absent in puried C6/36
membranes. In fact, no conclusive evidence about
the subcellular distribution of the putative DEN-4
receptors is available at this moment [17]. Since in
the overlay assay the putative receptors are linear-
ized due to the SDS-PAGE procedure, one could
argue that the polypeptides revealed with the bioti-
nylated virus are not the membranal receptors that
bind the virus. To investigate this issue, we decided
to test if anti-membrane antisera could block viral
binding to C6/36 monolayers. Cells were exposed to
PBS, or to preimmune serum, or to anti-membrane
Fig. 6. Puried C6/36 membrane preparations neutralize DEN-2.
antisera for 45 min at 30C. After one brief wash, the Conuent C6/36 monolayers were infected with DEN-2 virus
cells were infected with virus. After 0, 1 or 3 days the treated either with puried membrane preparations (B) or 1 mg/
cells were exposed to anti-DEN-2 mAbs followed by ml of BSA (C). Panel A: mock-infected cells.

FEMSLE 8437 5-11-98

 M. de Lourdes Munoz et al. / FEMS Microbiology Letters 168 (1998) 251^258 257

Fig. 7. Trypsin treatment of C6/36 cells blocks DEN-2 infection of C6/36 cells. Cultured cells were treated with trypsin as detailed in
``Methods'' and infected with DEN-2. Panel A, control cells. Cells infected for 0 (Panel B), 8 (Panel C) and 15 (Panel D) days.

stock and therefore a signicant reduction in the
characteristic cytopathic eect in conuent mono-
layers was obtained (Fig. 6B). The eect is specic
since exposure of the viral preparation to BSA did
not aect the viral infection (Fig. 6C).
3.2. Biochemical nature of the putative DEN-2
receptor
Although the receptor is likely to be a protein as
postulated in the case of Venezuelan equine encepha-
litis virus [24], the overlay assay and the inhibition
experiments described above do not rule out the pos-
sibility that the virus could be binding to other com-
ponents, such as carbohydrates. In fact, Chen and
co-workers have demonstrated that recombinant
DEN-2 E protein binds to a highly sulfated type of
heparan sulfate [6]. To explore the biochemical na-
ture of the putative DEN-2 virus receptors, two dif-
ferent approaches were undertaken. The rst one
consisted of treatment of a suspension of C6/36 cells
(approximately 5U106 cells) with 2 U of neuramini-
dase for 20 min. Using untreated cells as a control,
membrane preparations were obtained and analyzed
for the presence of putative DEN-2 virus binding
sites using the virus overlay assay. The virus-recog-
nized polypeptides were not aected by the enzy-
matic treatment, suggesting that sialic acid is not a
component of the putative receptors.
The second approach was to explore the results of
trypsin treatment of C6/36 monolayers to a subse-
quent DEN-2 virus infection. Cells were treated with
trypsin prior to infection with the virus. The results
demonstrate that trypsin treatment abolished viral
infection, strongly suggesting that DEN-2 virus re-
ceptors involve proteins in C6/36 cells (Fig. 7).
In summary, we have demonstrated that DEN-2
interacts with protein receptor(s) including one with
an apparent molecular mass of 67 kDa.
Acknowledgments
This work was supported by a grant from CON-
ACYT-Mexico (F547-N-9303). We thank the sta of
the Electron Microscopy Unit, CINVESTAV, IPN,
for use of their facilities. We also thank Mr. Israel
for the drawing work.

FEMSLE 8437 5-11-98

258 M. de Lourdes Munoz et al. / FEMS Microbiology Letters 168 (1998) 251^258

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