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Differential inhibition of dengue virus infection in
mammalian and mosquito cells by iota-carrageenan
Laura B. Talarico,
1
3Miguel D. Noseda,
2
Diogo R. B. Ducatti,
2
Maria E. R. Duarte
2
and Elsa B. Damonte
1
Correspondence
Elsa B. Damonte
edamonte@qb.fcen.uba.ar
Received 8 November 2010
Accepted 14 February 2011
1
Laboratorio de Virologı´a, Departamento de Quı´mica Biolo
´gica, Facultad de Ciencias Exactas
y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina
2
Departamento de Bioquı´mica e Biologia Molecular, Universidade Federal do Parana
´, PO Box
19046, Curitiba, Parana
´, Brazil
The antiviral activity against dengue virus-2 (DENV-2) of carrageenans reported here has shown a
differential susceptibility of C6/36 HT and Vero cells, taken as models of mosquito and
mammalian cells, depending on the structural class of polysaccharides: all polysaccharides
blocked DENV-2 infection in monkey Vero cells, but only iota-carrageenans were virus inhibitors in
mosquito cells. However, iota-carrageenans were less effective in mosquito cells in comparison
with mammalian cells with effective concentration 50 % (EC
50
) values in C6/36 HT cells
4.9–17.5-fold higher than in Vero cells, as determined by virus yield reduction assay. The mode of
action of iota-carrageenan in both cell types was strikingly different: in Vero cells the inhibitory
activity was exerted only at the initiation of the cycle, affecting virion binding, whereas in mosquito
cells DENV-2 adsorption was not affected and comparable levels of inhibition were obtained if the
compound was added to cells together with the virus, after 8 h of infection or by cell pre-
treatment before infection. Furthermore, iota-carrageenans induced a subtle alteration in mosquito
cells, detected by cell proliferation and protein synthesis analyses, suggesting that a probable
cellular target may be responsible for the refractory state of mosquito cells to DENV-2 infection
produced by this class of polysulfates. The failure of iota-carrageenan to block DENV-2
adsorption to mosquito cells appeared to be related to the low presence of adequate heparan
sulfate (HS) in C6/36 HT cell surface and is indicative of a differential participation of HS residues
for DENV-2 entry in both types of cells.
INTRODUCTION
Dengue virus (DENV), a member of the genus Flavivirus in
the family Flaviviridae, is the most widespread arbovirus
and re-emerged as a global health problem in the last
decades (Gubler, 2002). There are four serotypes (DENV-
1–4) which can cause a mild illness known as dengue fever
or the severe dengue haemorrhagic fever/dengue shock
syndrome in human. In nature, DENV is transmitted to its
vertebrate host through the bite of an infected mosquito
from the species Aedes aegypti and Aedes albopictus, and the
continuous occurrence of mosquito-vertebrate-mosquito
transmission cycles allows virus maintenance in the
environment.
The virion is an enveloped particle containing a positive-
sense RNA that is translated as a polyprotein then cleaved
into three structural proteins and seven non-structural
proteins. DENV primary infection is initiated by the
binding of the envelope E glycoprotein to a cell surface
receptor. Although the precise nature of DENV receptor is
still controversial, several investigators demonstrated that
heparan sulfate (HS) is involved in the first interaction
with E glycoprotein to initiate DENV multiplication cycle
in different types of vertebrate cells (Chen et al., 1997;
Germi et al., 2002; Hilgard & Stockert, 2000; Hung et al.,
1999). HS is a member of highly sulfated glycosaminogly-
cans (GAGs) which is very abundant on the surface of most
mammalian cells and serves as a receptor for many
microbial agents including bacteria, parasites and viruses
(Rostand & Esko, 1997; Spillmann, 2001). For DENV, the
interaction with HS is unusual owing to its specificity for a
highly sulfated form of HS (Chen et al., 1997).
Based on the proposed role of HS for DENV entry, diverse
HS-like molecules were evaluated as antiviral agents against
flaviviruses. Sulfated polysaccharides including heparin,
galactans, fucoidans, glucans, mannans and carrageenans
were found to be very potent and selective inhibitors of
DENV-2 multiplication in mammalian cells (Hidari et al.,
2008; Lee et al., 2006; Lin et al., 2002; Marks et al., 2001;
3Present address: Fundacio
´n Infant, Gavila
´n 94, 1406 Buenos Aires,
Argentina.
Journal of General Virology (2011), 92, 1332–1342 DOI 10.1099/vir.0.028522-0
1332 028522 G2011 SGM Printed in Great Britain
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Ono et al., 2003; Pujol et al., 2002; Qiu et al., 2007; Talarico
& Damonte, 2007). Surprisingly, no inhibitory activity was
observed when some polysulfates were evaluated in
mosquito cells (Talarico et al., 2005; Thaisomboonsuk
et al., 2005).
Here, we report the antiviral activity against DENV-2 in
mosquito cells of a particular type of sulfated polysaccha-
ride, the iota-carrageenans, that show a different behaviour
respect to other classes of polysulfates and an unusual
mode of inhibition in comparison to mammalian cells.
RESULTS
Antiviral activity of carrageenans against DENV-2
multiplication in mosquito and Vero cells
Three structural types of carrageenans and the reference
polysaccharide heparin were assayed for anti-DENV-2
activity in Vero and C6/36 HT cells, as model systems of
mammalian and mosquito cells, respectively, by a virus
yield inhibition assay. As previously reported in plaque
reduction antiviral test (Talarico et al., 2007), the three
carrageenans were inhibitors of DENV-2 multiplication in
Vero cells, with lambda- and iota-carrageenans as the most
active compounds (Fig. 1a). By contrast, only the iota-
carrageenan was an inhibitor of DENV-2 multiplication in
mosquito cells (Fig. 1b). Heparin was also able to inhibit
DENV-2 multiplication in Vero cells but was inactive in
C6/36 HT cells. From data in Fig. 1(a, b) the values of
effective concentration 50 % (EC
50
) calculated for iota-
carrageenan in Vero and C6/36 HT cells were 0.4±0.1 and
7.0±0.7 mgml
21
, respectively.
Since the commercial iota-carrageenan was the only type of
tested polysaccharide able to affect DENV-2 multiplication
in C6/36 HT cells, we decided to assay another iota-
carrageenan, obtained from a different source to evaluate if
this property is related to the particular structural
characteristics of this type of carrageenans. The iota-
carrageenan provided by Sigma-Aldrich is isolated from
Eucheuma spinosa. M3a is an homogeneous polysulfate
isolated from Meristiella gelidium constituted predomi-
nantly by iota-carrageenan (88–90 %) and with minor
amounts of nu- (6–8 %) and kappa- (4 %) disaccharide
repeating units (de S.F-Tischer et al., 2006). The carragee-
nan M3a reduced DENV-2 yields in a dose-dependent
manner (Fig. 1a, b), with EC
50
values of 4.6±0.6 and
0.93±0.05 mgml
21
in C6/36 HT and Vero cells,
respectively. These results allow us to conclude that the
ability to interfere with the multiplication of DENV-2 in
mosquito cells seems to be a consistent property of the
iota-carrageenans, independently of their origin.
The effect of iota-carrageenan against DENV-2 infection in
C6/36 HT cells was also demonstrated by monitoring viral
antigen expression in infected cells by immunofluorescence
assay. C6/36 HT cells expressing viral antigen were not
detected in cultures infected with DENV-2 in the presence
of iota-carrageenan, whereas untreated cultures or cultures
treated with heparin exhibited a similar amount of DENV-2
antigen-positive cells (Fig. 1c).
Mode of antiviral action of iota-carrageenan
against DENV-2 in mosquito and Vero cells
To characterize the antiviral activity of iota-carrageenans in
mosquito cells, the time-course of the inhibitory effect was
analysed by a time-of-addition experiment. For compar-
ative purposes, Vero cells were assayed simultaneously in
the same type of assay. As previously reported for other
sulfated polysaccharides (Talarico et al., 2005, 2007), the
antiviral effectiveness in Vero cells of both iota carragee-
nans was restricted to the first hour after infection. The
highest inhibitory effect was observed when the compound
was added to cells together with virus (time 0) or
immediately after adsorption at 1 h post-infection (p.i.)
(Fig. 2a, b). Surprisingly, the response of C6/36 HT cells
was very different: a similar level of inhibition, greater than
90 %, was observed if the commercial iota-carrageenan was
added either together with the virus or at any time after
adsorption, up to 8 h after infection (Fig. 2a). For M3a,
virus yields were also diminished more than 50–70 % even
if the carrageenan was added between 5 and 8 h p.i.
Results presented in Fig. 2(a, b) suggested the possibility of
an effect of the iota-carrageenan either producing the
inactivation of virus particles released from infected cells (if
compound remains cell-associated and has virucidal
activity, it could interfere in viral spread from infected
cells during infectious centre assay incubation) or inducing
a cell refractory state to DENV-2 infection when added to
the cell. To test these presumptions, the pre-treatment of
virions or cells was performed separately before infection.
DENV-2 infectivity was not affected by pre-incubation of
virions with compound (Fig. 2c), demonstrating a lack of
inactivating properties against DENV-2 of iota-carrageenan.
When C6/36 HT cells were pre-treated with iota-
carrageenan during 2 h at 33 uC and compound was
eliminated before infection with DENV-2, virus yields at
48 h p.i. were reduced by about 1.5 log (Fig. 2c). In fact,
the EC
50
against DENV-2 by cell pre-treatment in C6/36
HT cells was 6.2±0.3 mgml
21
, similar to the above-
mentioned value of EC
50
obtained when treatment was
exerted during all the period of virus infection (data from
Fig. 1b). The carrageenan M3a exhibited a similar
behaviour, whereas heparin and lambda-carrageenan did
not exert any inhibitory effect on DENV-2 infection of C6/
36 HT cells by pre-treatment (data not shown). The pre-
incubation of Vero cells with either the commercial iota-
carrageenan or M3a did not induce any refractory state to
the subsequent DENV-2 infection (Fig. 2c), indicating
that the inhibitory activity of iota-carrageenans against
DENV-2 by cell pre-treatment before infection is depen-
dent on the type of host cell.
To further assess the differential target for DENV-2
inhibition by iota-carrageenan in Vero and C6/36 HT
Inhibition of dengue virus
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cells, the effect of this compound on virus adsorption in
both cells was analysed by measuring the number of cell-
bound viral RNA molecules after 1 h of DENV-2 infection
at 4 uC in the presence of carrageenan. A significant
reduction in the amount of DENV-2 RNA attached to Vero
cells was detected by real-time RT-PCR, whereas virus
binding to C6/36 HT cells was not affected by the
carrageenan (Fig. 3).
Results shown in Fig. 3 appeared to confirm the proposed
role of HS for DENV-2 binding in Vero cells (Chen et al.,
1997; Ma
´rtinez-Barra
´gan & del Angel 2001; Germi et al.,
2002) and suggested the possibility of a different primary
receptor for DENV-2 in C6/36 HT cells. To support this
conclusion, virus adsorption was determined compara-
tively in both cells under two experimental approaches to
affect HS in cell membrane. First, cells were treated with
heparinase I to remove GAG-related molecules. After this
treatment, DENV-2 adsorption to Vero cells was highly
inhibited, whereas the attachment to C6/36 HT cells was
not significantly affected (Fig. 4a). The enzyme specificity
for GAG degradation was tested by immunostaining of cell
surface with an anti-HS antibody. The abundant presence
of HS on Vero cell surface was highly reduced after
treatment with heparinase I, whereas HS was almost
undetectable in untreated or treated C6/36 HT cells (Fig.
4b). The level of GAG sulfation was also reduced by
growing cells in sulfate-free medium added of sodium
chlorate (Baeuerle & Huttner, 1986). Again, under this
condition DENV-2 adsorption to Vero cells was inhibited,
but adsorption to mosquito cells was not affected (Fig. 4a).
As control of the desulfation treatment, cells treated with
chlorate were supplemented with sodium sulfate, which
reversed the desulfation process and restored DENV-2
binding to Vero cells (Fig. 4a). Then, the failure of iota-
carrageenan to affect DENV-2 adsorption to C6/36 HT
Fig. 1. Antiviral activity of sulfated polysaccharides against DENV-2. (a, b) Vero (a) and C6/36 HT (b) cells were infected with
DENV-2 (m.o.i. of 0.1) in the absence or presence of each compound. Virus yields were determined at 48 h p.i. Each value is
the mean of duplicate assays±SD. (c) C6/36 HT cells were mock infected or infected with DENV-2 in the absence or presence
of 50 mgml
”1
iota-carrageenan or heparin. At 48 h p.i., immunofluorescence staining was carried out using anti-DENV E
antibody. Magnification, ¾100.
L. B. Talarico and others
1334 Journal of General Virology 92
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cells appears to be related to the low presence of adequate
HS in surface of these cells and, consequently, virus is
bound to another type of cell membrane component.
Finally, to corroborate the dissimilarities observed in the
mechanism of antiviral activity in Vero and C6/36 HT cells,
we intended to generate resistance to iota-carrageenan by
serial passage of DENV-2 in a type of host cell, either
mammalian or mosquito, in the presence of compound,
and then to study comparatively the response to the
variants in both cell types. We chose to select the variants
by passage in Vero cells because they were more susceptible
to the antiviral action of the iota-carrageenan than the
mosquito cells (Fig. 1). After three passages of DENV-2 in
the presence of iota-carrageenan, the virus designated
DENV-2-iota 3 exhibited a high resistance to iota-
carrageenan, whereas control virus passaged similarly in
the absence of compound, named DENV-2-CV 3, main-
tained the antiviral susceptibility (Fig. 5a). By contrast,
when the antiviral assay was carried out in C6/36 HT cells
both variants exhibited the same profile in the dose-
response curves (Fig. 5b). From data shown in Fig. 5(a, b),
the values of EC
50
for DENV-2-iota-3 were calculated as
.50 and 1.3 mgml
21
in Vero and C6/36 HT cells,
respectively, confirming the presence of a differential target
for this polysaccharide in mosquito and mammalian cells.
Fig. 2. Influence of time of treatment with iota-carrageenans on anti-DENV-2 activity. (a, b) Vero and C6/36 HT cells were
infected with DENV-2 and maintenance medium (MM) containing 20 or 50 mgml
”1
of iota-carrageenan (a) or M3a (b),
respectively, was added simultaneously with virus (time 0) or at the indicated times after infection. At 10 h p.i. medium was
discarded and an infectious centre assay was performed in Vero cells. (c) Pre-treatment of cells: cells were pre-incubated with
compound during 2 h; then, cells were washed and infected with DENV-2. Virus yields were determined at 48 h p.i. Pre-
treatment of virus: DENV-2 suspensions were incubated with compound for 45 min and the remaining infectivity was
determined by plaque assay. Each value is the mean of duplicate assays±SD.
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This conclusion was further assessed by evaluating the
kinetics of virus adsorption of these viruses in both cells.
The adsorption of the resistant variant DENV-2-iota 3 to
Vero cells was highly reduced in comparison to control
virus, indicating that the mechanism leading to resistance
to iota-carrageenan in Vero cells is an alteration in virion
ability to interact with HS in cell surface (Fig. 5c). By
contrast, DENV-2-iota-3 adsorption to C6/36 HT cells was
not impaired (Fig. 5d).
Effect of iota-carrageenan on cell proliferation
and protein synthesis
Data from Figs 1(b) and 2(c) suggested that the antiviral
activity of iota-carrageenan against DENV-2 in mosquito
cells may be mainly due to a long lasting effect exerted by
any cellular factor triggered as response of the cell to the
presence of the compound. The effects of treatment with
these compounds on the host cell were analysed by
monitoring proliferation of actively growing cells in the
presence of carrageenans at concentrations close to the range
used in antiviral determinations. When cytotoxicity of
carrageenan was determined by the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method in
uninfected confluent cultures of Vero and C6/36 HT cells
after 48 h of treatment, as in the antiviral assay, no significant
reduction in cell viability was detected (Fig. 6a). It must be
noted that all DENV infections in the experimental protocols
presented in this study were carried out in confluent cell
monolayers. When iota-carrageenan was added at 2.5 h after
seeding the cells, the increase in Vero cell number after 48 h
of exposure to the compound was not significantly altered in
comparison with compound treatment on confluent cells
(Fig. 6a). By contrast, the proliferation ability of C6/36 HT
cells was affected by iota-carrageenan with a reduction in the
viability percentage of growing cells treated with compound
respect to untreated cells of around 30–38% in the range of
assayed concentrations (Fig. 6a).
The activity of iota-carrageenan on cell metabolism was
further studied by analysing cell protein synthesis after
compound treatment. As seen in Fig. 6(b), no inhibition of
protein synthesis in iota-carrageenan-treated Vero cells was
Fig. 3. Effect of iota-carrageenan on virus adsorption. DENV-2
was adsorbed to Vero and C6/36 HT cells for 1 h at 4 6CinMMin
the absence or presence of iota-carrageenan. Then, the amount of
adsorbed DENV-2 RNA molecules was determined by quantitative
real-time RT-PCR. Each value is the mean of duplicate
assays±SD.
Fig. 4. Effect of heparinase and sodium chlorate treatment of cells
on DENV-2 infection. (a) Vero and C6/36 HT cells were treated
with 3 U heparinase I ml
”1
for 1 h or 30 mM of sodium chlorate for
48 h at 37 or 33 6C, according to cell type. For reversal of
sulfation inhibition, one set of chlorate-treated cells was supple-
mented with 20 mM sodium sulfate. After each treatment, DENV-2
was adsorbed for 1 h at 4 6C and cell-bound infectious virus was
determined by plaque assay. Each value is the mean of duplicate
assays±SD. (b) Vero and C6/36 HT cells were treated or not with
3 U heparinase I ml
”1
as above and then anti-HS immunofluores-
cence staining was carried out. Magnification, ¾400.
L. B. Talarico and others
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observed. In C6/36 HT cells, a reduction in the general
labelling pattern of cellular polypeptides was detected, con-
firming a direct effect of this compound on mosquito cells in
accordance with the results obtained in the cell proliferation
assay. Similar results were obtained with M3a, whereas the
lambda-carrageenan did not exert any inhibition in mosquito
cell proliferation and protein synthesis (data not shown).
To further corroborate the effect of iota-carrageenan on
mosquito cells, the viability of actively growing and
confluent C6/36 HT cells after compound treatment was
also evaluated by other methods such as neutral red (NR)
and crystal violet (CV) assays. Results were comparable to
MTT data with a reduction in the number of proliferative
cells proportional to the compound dose (Fig. 6c).
In conclusion, a subtle but reiterative alteration was
induced in mosquito cells after treatment with iota-
carrageenan, which may be involved in the inhibition
observed in DENV-2 multiplication in C6/36 HT cells.
DISCUSSION
Mosquitoes and mammals are the natural hosts of DENV
which is successfully transmitted through alternate cycles
Fig. 5. DENV-2 variants in Vero and C6/36 cells. (a, b) Vero (a) and C6/36 HT (b) cells were infected with DENV-2-iota 3 or
DENV-2-CV 3 (m.o.i. of 0.1) in the absence or presence of iota-carrageenan. Extracellular virus yields were determined at 48 h
p.i. (c, d) Vero (c) and C6/36 HT (d) cells were incubated at 4 6C with DENV-2-iota 3 or DENV-2-CV 3 (m.o.i. of 1), and cell
bound infectious virus was determined. Each value is the mean of duplicate assays±SD.
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L. B. Talarico and others
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of virus replication in each host forcing the virus to
maintain its ability to infect both types of cells. The analysis
of the antiviral activity against DENV-2 of carrageenans
reported here has shown a differential susceptibility of C6/
36 HT and Vero cells, taken as models of mosquito and
mammalian cells, depending on the structural class of
polysaccharides: all polysaccharides blocked DENV-2
infection in Vero cells, but only iota-carrageenans were
virus inhibitors in mosquito cells.
The lack of antiviral activity against DENV-2 of heparin,
lambda- and kappa-carrageenan in mosquito cells was in
accordance with previous reports of polysulfates, which
were effective against DENV infection in diverse mam-
malian cells, but were totally inactive in mosquito cell lines
C6/36 HT and AP61 (Talarico et al., 2005; Thaisomboonsuk
et al., 2005). By contrast, the two evaluated iota-carrageenans
showed an exceptional ability to interfere with DENV-2
multiplication in both cells, but with distinctive differ-
ences in their inhibitory action. First, mosquito cells were
less susceptible in comparison to mammalian cells.
Second, the mechanism of inhibition was strikingly
different: in Vero cells the activity of iota-carrageenan
was exerted only at the initiation of the cycle, affecting
virion binding, whereas in mosquito cells comparable
levels of inhibition were obtained if compound was added
to cells together with the virus, after 8 h of infection or by
cell pre-treatment. The response to iota-carrageenan in
mosquito cells is totally different to that usually described
for polysulfates not only against DENV but also against
other enveloped viruses (Damonte et al., 2004).
From these results it can be concluded that the inhibition
of DENV-2 multiplication in Vero cells appeared to be
exerted by interference with the initial interaction of
glycoprotein E with HS but the inhibition in C6/36 HT
cells is independent of HS. The expression level of GAG in
Vero cells is high as we could detect here and was reported
by other investigators (Avirutnan et al., 2007), whereas HS
in C6/36 HT membrane appeared sparsely distributed with
a very weak staining. Not only the level of GAG expression
in a cell may influence the virus–cell interaction, but also
its structural characteristics since certain ligands require
specific HS structures for binding. For instance, a highly
sulfated form of HS is the reported target for DENV-2 and
other flaviviruses to initiate infection in diverse mam-
malian cells (Barth et al., 2006; Chen et al., 1997). HS has
been extensively studied in a wide variety of animals,
including mammals, birds and flies (Bishop et al., 2007),
but very little information is known about it in insects of
medical importance. Sinnis et al. (2007) reported the first
isolation of HS in Anopheles stephensi, the vector for
Plasmodium parasites causing malaria, finding a lower
degree of sulfation with respect to human liver HS.
Although no information on Aedes HS composition is
presently available, if the sulfated state of HS in Aedes is
similar to Anopheles and considering the low HS expression
observed in C6/36 HT cell surface reported here, the
participation of HS for DENV entry appears to be minimal
in comparison to mammalian cells, providing an explana-
tion for our results. Accordingly, structural analyses
indicated that HS-binding sites of the domain III of
DENV-2 E glycoprotein are essential for binding to BHK-
21 cells but are not involved in attachment to mosquito
cells (Hung et al., 2004). Also, recent investigations have
reported the identification of diverse proteins with virus-
binding ability as receptors for DENV-2 in C6/36 cells
(Chee & AbuBakar, 2004; Kuadkitkan et al., 2010;
Paingankar et al., 2010; Salas-Benito et al., 2007) without
apparent involvement of HS, whereas in Vero and other
epithelial mammalian cells HS was proposed as primary
attachment receptor to concentrate virions on the cell
surface and trigger the interaction with a second receptor
of protein nature for virus internalization (Martı
´nez-
Barraga
´n & del Angel, 2001).
Like heparin, lambda- and iota-carrageenans are more
heavily sulfated than most tissue-derived HS (Esko &
Selleck, 2002), a characteristic that usually supported the
great antiviral potency of these compounds in mammalian
cells. Carrageenans consist of linear chains of alternating
(1-3)-b-D-galactopyranoses and (1-4)-a-D-galactopiranoses
(or 3,6-anhydrogalactopyranoses) (Damonte et al., 2004).
They differ in the amount and position of sulfate groups:
iota-carrageenan contains two sulfates per disaccharide
repeating unit at axial positions, lambda- has close to three
equatorial sulfates and kappa-only one. Other difference
is related to the monosaccharide composition: lamba-
carrageenans are constituted by galactose units whereas
iota- and kappa- contain equal amounts of galactose and
3,6 anhydrogalactose, with higher hydrophobic character.
Therefore, iota-carrageenans show a combination of ionic
and hydrophobic zones in the same macromolecule, not
found in lambda- and kappa-carrageenans. This particular
structure may allow specific interactions that probably
account for the differential effects among carrageenans
observed in mosquito cells.
Polysaccharide samples may contain contaminating sub-
stances from the natural source that remain after the
Fig. 6. Effect of iota-carrageenan on cell proliferation and protein synthesis. (a) To measure the effects on actively growing or
confluent Vero and C6/36 HT cells, iota-carrageenan was added 2.5 or 24 h after cell seeding in 96-well plates, respectively.
After 48 h of incubation, the ratio of viable cells in drug-treated and mock-treated cultures was determined by MTT assay and
expressed as percentage cell viability. (b) Vero and C6/36 HT cells were incubated for 12 h with or without 20 mg iota-
carrageenan ml
”1
at 37 or 33 6C, respectively. Then, cells were labelled with 100 mCi EXPRE
35
S-
35
Sml
”1
for 4 h and
polypeptides were electrophoresed. (c) Actively growing or confluent cells were treated with iota-carrageenan as in (a), and cell
viability was determined by NR or CV method. Each value is the mean of duplicate assays±SD.
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extraction and purification processes. To assess that the
effect detected in C6/36 HT cells was effectively caused by
the polysaccharide, the commercial iota-carrageenan was
extensively dialysed against water in a membrane with a
cut-off size of 250 kDa. The antiviral activity against
DENV-2 of this dialysed sample was similar to that
obtained with original carrageenan (data not shown),
indicating that the inhibition is not due to an additional
effect produced by any small molecule associated to the
polysaccharide. Furthermore, a similar behaviour to that
here observed for DENV-2 in C6/36 HT cells has been
reported in vertebrate cells for human papillomavirus
(HPV), a DNA virus also requiring cellular HS for
infection (Buck et al., 2006). In addition to blocking the
initial interaction of capsid HPV protein with cells, iota-
carrageenan also exerts a post-attachment inhibitory effect
on HPV infectivity up to 12 h after infection that does not
involve HS.
In conclusion, results presented here reveal the ability of
iota-carrageenan to attack different targets of DENV-2
infection depending on the type of host cell: virus
adsorption in Vero cells or apparently a still unidentified
cellular pathway in C6/36 cells. This interesting observation
of cell-dependent mode of action is a signal to be
considered when any antiviral agent is evaluated in a
particular cell system. In addition, new evidence is
provided here about the failure for HS involvement in
the initial interaction of DENV-2 in mosquito cells
contrasting with mammalian cell infection. Further research
is required to fully understand the HS-independent
mechanism of inhibition of iota-carrageenan in mosquito
cells. A recent genome RNA interference screen in insect
cells identified 116 candidate host factors required for
infection, several of them predicted to be membrane-
associated in consistency with the remodelling of cellular
membranes observed in DENV-infected cells (Sessions
et al., 2009). Although the possibility of a post-entry viral
target in the antiviral response to iota-carrageenan observed
in C6/36 HT cells cannot be totally discarded, our results
point out to an interference with any of the cellular products
found associated to DENV-2 infection.
METHODS
Compounds and antibodies. Commercial iota-, lambda- and
kappa-carrageenans, and heparin were purchased from Sigma-
Aldrich. M3a is a homogeneous carrageenan fraction obtained from
M. gelidium as described previously (de S.F-Tischer et al., 2006).
Stock solutions of compounds were prepared in distilled water at
2mgml
21
.
The mouse IgG mAb against DENV E glycoprotein was from Abcam,
the anti-HS IgM mAb was from Seikagaku Corporation, goat anti-
mouse IgG conjugated to FITC was from Sigma-Aldrich and FITC-
conjugated goat polyclonal anti-mouse IgM was from Chemicon
International Inc.
Cells and virus. Vero (African green monkey kidney) cells were
grown in Eagle’s minimum essential medium (MEM) (Gibco)
supplemented with 5 % FBS. For maintenance medium (MM), the
serum concentration was reduced to 1.5 %. The C6/36 HT mosquito
cell line from A. albopictus, adapted to grow at 33 uC, was cultured in
L-15 Medium (Leibovitz) supplemented with 0.3 % tryptose phos-
phate broth, 0.02 % glutamine, 1 % MEM non-essential amino acids
solution and 5 % FBS.
The stocks of DENV-2 strain NGC were prepared in C6/36 HT cells
and titrated by plaque formation in Vero cells.
Antiviral assay. Antiviral activity was evaluated by a virus yield
inhibition assay. C6/36 HT and Vero cells grown in 24-well
microplates (3.0610
5
cells per well) were infected with DENV-2 at
an m.o.i. of 0.1 in the presence of different concentrations of the
compounds, two wells per concentration. After 48 h of incubation at
33 (for C6/36 HT cells) or 37 uC (for Vero cells), cell supernatants
were collected and the virus yields were determined by plaque
formation in Vero cells. The EC
50
values were calculated as the
compound concentration able to reduce virus plaques by 50 %. All
determinations were performed twice and each in duplicate.
Indirect immunofluorescence assay. Infection of 1610
5
C6/36
HT cells grown in coverslips with DENV-2 (m.o.i. of 0.1) was
performed in the presence or absence of iota-carrageenan or heparin
(50 mgml
21
). At 48 h post-infection, cell monolayers were washed
with cold PBS and fixed in methanol for 15 min at 220 uC for
cytoplasmic immunofluorescence. Indirect staining was carried out by
using anti-DENV mouse mAb and FITC-labelled goat anti-mouse
IgG. After a final washing with PBS, cells were stained with Evans Blue
and mounted in a glycerol solution containing 1,4-diazabicyclo[2, 2,
2]octane (Dabco) and visualized on a fluorescence microscope
Olympus BX51.
Time of addition experiment. C6/36 HT and Vero cells (3.0610
5
cells per well) were infected with DENV-2 (m.o.i. of 1) in either MM
containing 50 or 20 mgml
21
of M3a or iota-carrageenan, respectively,
(time 0) or MM without compound. After 1 h adsorption at 4 uC,
unadsorbed virus was removed, cultures were washed with PBS, and
MM with compound was added immediately (time 0 and 1 h p.i.) or
at 2, 3, 5 and 8 h p.i. An infected culture without compound
treatment was performed simultaneously as virus control. At 10 h
after infection, cell supernatants were withdrawn, the monolayers
were washed with PBS and trypsinized with 0.25 % trypsin-EDTA to
disperse cells. Protease treatment was stopped by adding 2 mM PMSF
in PBS containing 3 % BSA (PBS-3 % BSA), then cells were
resuspended in MM, and the final cell suspensions were serially
10-fold diluted and plated onto confluent monolayers of Vero cells.
After 1.5 h incubation at 37 uC, monolayers were overlaid with MM
containing 1 % methylcellulose. Infectious centres were counted after
6 days of incubation.
Effect of pre-treatment of cells or virus with compound prior to
infection. Pre-treatment of cells: cells (3.0610
5
cells per well) were
pre-incubated with MM containing different compound concentra-
tions during 2 h at 33 or 37 uC. Then, cells were thoroughly washed
with PBS, and infected with DENV-2 (m.o.i. of 0.1) in the absence of
compound. Virus yields were determined at 48 h p.i.
Pre-treatment of virus: a DENV-2 suspension (1610
6
p.f.u. ml
21
)
was incubated in MM containing or not different compound
concentrations at 37 uC during 45 min, and then the remaining
infectivity was titrated by p.f.u.
Cell viability assays. Cell viability was measured by three different
assays in resting and actively growing cells. For resting cells, confluent
cultures in 96-well plates (5610
4
cells per well) were exposed for 48 h
to serial twofold compound dilutions, three wells for each
concentration, and then viability was tested. To monitor proliferation
L. B. Talarico and others
1340 Journal of General Virology 92
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On: Fri, 11 Sep 2015 01:31:12
of actively growing cells, 1610
4
cells were seeded in 96-well plates
and allowed to adhere during a 2.5 h incubation period at 33 or 37 uC
for C6/36 HT and Vero cells, respectively. Thereafter, compound was
added and incubation followed during 48 h before viability
determination.
In the MTT method, 10 ml of MM containing MTT (final
concentration 0.5 mg ml
21
) was added to each well. After 2 h of
incubation at 33 (C6/36 HT cells) or 37 uC (Vero cells), the
supernatant was removed, ethanol was added to each well and
absorbance was measured at 595 nm.
In NR assay, cells were incubated for 2 h with NR (100 mgml
21
)
dissolved in MEM without serum in darkness. Then the supernatant
was removed, cells were washed with PBS, elution medium (EtOH/
AcCOOH, 50/1 %) was added and absorbance at 540 nm was
recorded.
For CV assay, cells were fixed with 10 % formaldehyde during 15 min.
Then, cells were washed with water and stained with CV (0.05 % CV
in 10 % EtOH) during 30 min. After washing, colourant was eluted
with EtOH/AcCOOH, 50/0.1 % and absorbance was recorded at
590 nm.
Cell protein synthesis. C6/36 HT and Vero cells (3.0610
5
cells per
well) were incubated in the presence or absence of compound during
11 h at 33 or 37 uC, respectively. Then, cell monolayers were washed
with PBS and incubated in methionine-cysteine-free medium in the
presence or absence of compound for 1 h, and then labelled by the
addition of 100 mCi EXPRE
35
S-
35
S (NEN Dupont) ml
21
for 4 h. After
labelling, cells were washed with PBS and lysed in sample
electrophoresis buffer [5 % SDS, 2 % 2-mercaptoethanol, 10 %
glycerol (v/v) and 0.005 % bromophenol blue in 0.0625 M Tris/
HCl, pH 6.8]. Cell lysates were sonicated for 1 min, boiled during
2 min and loaded for electrophoresis on 15 % SDS-polyacrylami de
slab gels (lysates corresponding to1.0610
5
cells per well). Protein
bands were visualized by fluorography.
Virus-binding assay. C6/36 HT and Vero cells grown in six-well
microplates (1.2610
6
cells per well) were infected with DENV-2
(m.o.i. of 1) in the absence or presence of 50 or 20 mgml
21
of iota-
carrageenan, respectively. After 1 h incubation at 4 uC, cells were
washed extensively with cold PBS to remove unadsorbed virus and
total RNA was extracted from cells with TRIzol (Invitrogen)
according to the manufacturer’s instructions. The amount of cell
bound viral RNA was quantified by real-time RT-PCR as described
previously (Talarico & Damonte, 2007).
Heparinase treatment. C6/36 HT and Vero cells in six-well
microplates (1.2610
6
cells per well) were incubated for 1 h at 33
or 37 uC, respectively, in the absence or presence of 3 U heparinase I
(Sigma-Aldrich) ml
21
. Then, cells were washed with PBS and infected
with DENV-2 (m.o.i. of 1). After 1 h adsorption at 4 uC, cells were
washed with cold PBS and disrupted by freezing and thawing. The
amount of infectious bound virus was then measured by plaque
formation.
To assess the effect of heparinase I on cell surface-exposed HS
residues, C6/36 HT and Vero cells grown in coverslips were treated
with the enzyme as above and then fixed with 4 % paraformaldehyde
followed by permeabilization with 0.2 % Triton X-100. After fixation,
cells were washed with PBS containing 0.5 % BSA (PBS-0.5 % BSA),
and immunolabelled with monoclonal anti-HS and FITC-conjugated
goat polyclonal anti-mouse IgM. After a final washing with PBS-0.5 %
BSA, cells were mounted in a glycerol solution containing 2.5 %
Dabco.
Inhibition of cellular sulfation. C6/36 HT and Vero cells (1.2610
6
cells per well) were grown in standard MEM or sulfate-free MEM
added with either 30 mM of the sulfation inhibitor sodium chlorate
or 30 mM sodium chlorate plus 20 mM sodium sulfate for reversal of
sulfate inhibition. After 48 h incubation at 33 or 37 uC, according to
cell type, all cultures were washed with MEM without serum, infected
with DENV-2 and adsorbed virus was then measured as for
heparinase treatment.
Isolation of drug-resistant variant viruses. Vero cells grown in
six-well microplates (1.2610
6
cells per well) were infected with
DENV-2 (m.o.i. of 0.1) in the presence of iota-carrageenan during
virus adsorption and throughout the period of incubation.
Supernatants were collected at 4–7 days p.i. and titrated by plaque
formation. The supernatant with the highest virus titre was selected to
perform the next passage and serial passages were continued in this
manner. The compound concentration was 1 mgml
21
for the initial
two passages and 2 mgml
21
for passage 3. The EC
50
of each passage
against iota-carrageenan was determined in Vero cells to monitor the
appearance of resistant variant viruses. Simultaneously, control
passages of DENV-2 in Vero cells without carrageenan were also
performed.
ACKNOWLEDGEMENTS
The authors acknowledge Dr Juan Carlos Calvo (Depto. Quı
´mica
Biolo
´gica, FCEN, UBA-IBYME, Buenos Aires, Argentina) for
providing the anti-heparan sulfate IgM mAb and FITC-conjugated
goat polyclonal anti-mouse IgM, Juan Mondotte and Dr Andrea
Gamarnik for technical assistance in real-time RT-PCR technique.
This work was funded by grants from Agencia Nacional para la
Promocio
´n Cientı
´fica y Tecnolo
´gica (ANPCyT), Consejo Nacional de
Investigaciones Cientı
´ficas y Te
´cnicas (CONICET), Universidad
de Buenos Aires, Argentina, PRONEX-Carboidratos (Fundac¸a
˜o
Arauca
´ria/CNPq) and MCT/CNPq/MS/CT-Sau
´de (554671/2006-9),
Brazil. L. B. T. and E. B. D. are members of Research Career from
CONICET. M. E. D. and M. D. N. are Research Members of the
National Research Council of Brazil (CNPq). D. R. D acknowledges a
doctoral scholarship from CNPq.
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