Viruses 2013, 5, 605-618; doi:10.3390/v5020605
Crabohydrate-Related Inhibitors of Dengue Virus Entry
Kazuya I.P.J. Hidari *, Tomoko Abe and Takashi Suzuki
Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, and Global
COE Program for Innovation in Human Health Sciences, 52-1 Yada, Suruga-ku,
Shizuoka-shi, Shizuoka 422-8526, Japan; E-Mail: email@example.com (K.I.P.J.H.)
* Author to whom correspondence should be addressed; E-Mail: firstname.lastname@example.org
Tel.: +81-54-264-5720; Fax: +81-54-264-5723.
Received: 16 January 2013; in revised form: 1 February 2013 / Accepted: 4 February 2013 /
Published: 6 February 2013
Abstract: Dengue virus (DENV), which is transmitted by Aedes mosquitoes, causes fever
and hemorrhagic disorders in humans. The virus entry process mediated through host
receptor molecule(s) is crucial for virus propagation and the pathological progression of
dengue disease. Therefore, elucidation of the molecular mechanisms underlying virus entry
is essential for an understanding of dengue pathology and for the development of effective
new anti-dengue agents. DENV binds to its receptor molecules mediated through a viral
envelope (E) protein, followed by incorporation of the virus-receptor complex inside cells.
The fusion between incorporated virus particles and host endosome membrane under acidic
conditions is mediated through the function of DENV E protein. Carbohydrate molecules,
such as sulfated glycosaminoglycans (GAG) and glycosphingolipids, and carbohydrate-
recognition proteins, termed lectins, inhibit virus entry. This review focuses on
carbohydrate-derived entry inhibitors, and also introduces functionally related compounds
with similar inhibitory mechanisms against DENV entry.
Keywords: dengue virus; receptor; endocytosis; fusion; entry inhibitor; carbohydrate;
Viruses 2013, 5
Flaviviruses are enveloped viruses with an envelope (E) protein on the surface of the lipid bilayer
membrane. Dengue virus (DENV), which belongs to the genus Flavivirus, family Flaviviridae, is
transmitted from human to human by Aedes mosquitoes [1,2]. DENV causes febrile illness and more
serious complications, such as hemorrhagic fever disease [3,4]. There are four virus serotypes, type 1
(DENV1) to type 4 (DENV4), which have similar clinical manifestations and epidemiology in tropical
and subtropical regions of the world. At present, more than two billion people are at risk of
infection [4–7]. A previous study demonstrated DENV tissue tropism in humans and mice where
active DENV replication was occurring [8,9]. Virus antigen was detected in macrophages and dendritic
cells of the spleen and lymph nodes of both host species. These cells migrate to the lymph nodes where
DENV initially propagates and spreads to secondary replication tissues, such as bone marrow myeloid
cells and hepatocytes in the liver . To date, C-type lectins such as dendritic cell-specific ICAM3-
grabbing non-integrin (DC-SIGN) and C-type lectin domain family 5 member A (CLEC5A) [10-13],
mannose-receptor , glucose-regulating protein 78 (GRP78/Bip) , CD14 , heparan sulfate
(HS) [17–21], and glycosphingolipids, such as neolactotetraosylceramide (nLc4Cer) [22,23], have been
reported as putative receptor molecules for DENV. Most of these molecules are involved in
carbohydrate-protein interaction. The structures of DENV E proteins have been elucidated by
crystallography and NMR analyses [24–27]. These studies provided a structural basis for
understanding the molecular mechanisms of virus entry.
DENV binds to as yet undefined receptor molecules on the host cell surface, followed by
incorporation through the receptor-mediated endocytotic pathways. Fusogenic conformational changes
in the virus envelope glycoprotein (E protein) are induced by the acidic environment of the endosome,
resulting in fusion between virus particles and the host endosome membrane, and subsequent viral
disassembly [28,29]. Single-stranded virus RNA with positive polarity, approximately 11 kb in length,
which contains a single open reading frame encoding a polyprotein, is released into the cytoplasm and
acts as a template for genome replication and protein translation events [27,30] (Figure 1). Many
factors derived from host cells are thought to be involved in these entry processes of DENV infection.
Several lines of evidence regarding host factors indicate that HS or the highly sulfated forms of
glycosaminoglycans, such as chondroitin sulfate E (CSE), on the host cell surface are essential for the
entry of flaviviruses including DENV [16,18,31–35]. An understanding of the molecular interactions
mediated through virus envelope proteins in virus entry into the target cells is critical for elucidation of
the mechanisms of virus tropism, such as host, tissue, and cell preferences.
Methods for the control and prevention of DENV by safe and long-lasting vaccination have not
been established. Therefore, there is a requirement for effective antiviral agents and therapeutic
concepts for DENV infection. However, at present, no specific treatments are clinically available for
DENV infection. The virus entry process mediated through host carbohydrate molecule(s) is crucially
involved in virus propagation and the pathological progression of dengue disease. Based upon the
structures and functions of the carbohydrate molecules involved in DENV entry, several types of
inhibitors that block DENV entry into cells have been generated . This review article focuses on
the chemical and biochemical properties of carbohydrate-derived inhibitors of DENV entry, such as
GAGs, glycoproteins and glycosphingolipids, and also introduces functionally related inhibitors.
Viruses 2013, 5
2. Entry Inhibitors
Host factors and domains of virus E protein that are involved in DENV entry are likely to be useful
targets in efforts to generate inhibitors of virus infection useful in both basic research and clinical
medicine . The early stages of infection by enveloped viruses, including DENV, involve two major
processes—virus adsorption and fusion—for which target molecules may be useful for the
development of antiviral agents (Figure 1). Here, we categorize and describe two types of entry
inhibitor: 1) inhibitors of virus adsorption, and 2) inhibitors of virus-induced membrane fusion.
Figure 1. Endocytotic entry pathway of the enveloped virus life cycle.
2.1. Inhibitors of virus adsorption
Table 1 shows the chemical and antiviral properties of carbohydrate-mimetic inhibitors of DENV
adsorption. Sulfated glycosaminoglycans, such as heparin, inhibit the early step of dengue virus
infection through interaction with envelope (E) protein. Heparin binding sites for DENV consist of
basic amino acid clusters on domain III, the putative receptor-binding domain in the crystal structure
of the flavivirus E protein (Figure 2) [17,24,25,38,39]. Another GAG, chondroitin sulfate E (CSE),
significantly reduced infectivity of all dengue virus serotypes toward BHK-21 and Vero cells. Virus
binding to CSE or heparin was cross-inhibited by soluble CSE or heparin. It is suggested that common
carbohydrate determinants on CSE and heparin could be essential epitopes for interaction of DENV,
and may be responsible for DENV inhibition .
Viruses 2013, 5
Figure 2. Structure of dengue virus (DENV) EGP. The structure of EGP based on the
three-dimensional structure of PDB accession number 1OKE. The figure was prepared
Domain I Domain II
Table 1. Chemical and antiviral properties of inhibitors of virus adsorption
(dengue serotype/cell line)
(DENV2, 3/Vero, HepG2)
Viruses 2013, 5
Table 1. Cont.
PI-88 (Mw 1400-3100)
(Mw approx. 5700)
Suramin (Mw 1429)
Sulfated polysaccharides obtained from the red seaweed Gymnogongrus griffithsiae and the marine
alga Cladosiphon okamuranus, the kappa/iota/nu carrageenan derivative G3d, and fucoidan were
shown to be selective inhibitors of DENV2/3 and 2 multiplications in cells, respectively [40–42]. G3d
is an active DENV2/3 inhibitor that predominantly suppresses the initial processes of virus adsorption
and internalization. Fucoidan is comprised of carbohydrate units containing glucuronic acid and
sulfated fucose residues. This compound exclusively inhibits DENV2 infection. The infection was
inhibited when the virus was pretreated with fucoidan. Structure-activity analysis demonstrated that
glucuronic acid and the sulfated functional group from fucoidan were essential for inhibition of viral
infection. The virus particles bound exclusively to fucoidan, indicating that fucoidan interacts directly
with DENV2 E protein. Structure-based analysis suggested that Lys310 and Arg323 of DENV2 E
protein, which are conformationally proximal to the putative heparin binding residues, are critical for
the interaction with fucoidan. The variation in antiviral activity of two natural sulfated polysaccharides
glucuronic acid (Mw 288)
Viruses 2013, 5
depends on the viral serotype. Many studies have demonstrated that diverse types of sulfated
polysaccharides have anti-DENV activity [43–47].
The glycosphingolipid neolactotetraosylceramide (nLc4Cer) expressed on the cell surface of
DENV-susceptible cells, human erythroleukemia K562, and baby hamster kidney BHK-21 is
recognized by four serotypes of DENV. The non-reducing terminal disaccharide residue
Galβ1-4GlcNAc of nLc4Cer is a critical determinant for the binding of DENV2. Chemically
synthesized derivatives carrying multiple carbohydrate residues of nLc4, but not nLc4 oligosaccharide,
inhibited DENV2 infection of BHK-21 cells .
The antiviral effects of heparan sulfate (HS) mimetics, such as suramin and pentosan polysulfate
(PPS), and PI-88, have been reported against DENV. PI-88 is a mannose-containing di- to
hexasaccharide with a high degree of sulfation [48–50]. PPS, a semi-synthetic β-D-xylopyranose
polymer with a higher degree of sulfation than heparin, has been used for the prevention of
postoperative thromboembolism and treatment of interstitial cystitis [48,51]. This compound also
reduces virus infectivity by steric hindrance of virus attachment. Suramin, a symmetrical
polysulfonated naphthylamine that has been used for the treatment of human trypanosomiasis, has
been shown to be an antitumor and antiviral agent [17,52,53]. It seems that the inhibitory activity of
HS mimetics, including these compounds, is due to their association with GAG binding sites of the
putative receptor-binding domain on the DENV E protein [17,24,25,38,39]. These findings are
consistent with the interpretation that heparin and HS mimetics are inhibitors of virus adsorption.
A rationale for designing sulfated carbohydrate compounds with low molecular mass as anti-DENV
agents targeting E protein functions has been reported . Significant inhibitory activity is exerted by
3-O-Sulfated GlcA on DENV2 infection with an EC50 value of 120 M. Two negatively charged
groups, 3-O-sulfate and 6-C-carboxylic acid, appear to be essential for anti-DENV activity. Docking
simulation demonstrated the binding potential of this small compound with respect to DENV E protein,
and also showed that the distance and conformation of these negative charges on the carbohydrate may
be suitable for association with three responsible amino acid residues of E protein critically involved in
virus adsorption. Similar to other HS mimetics, this compound competitively prevents DENV
adsorption to host cells.
In most studies on inhibitors of virus-cell surface binding, experiments were carried out at 4°C
during coincubation of compounds with cells and virus particles. However, previous studies indicated
that DENV particles do not bind efficiently to the cells at this temperature [55,56]. These studies
suggest that the virus binding affinity at 4°C is lower than that at physiological temperature, and some
inhibitors of virus-cell surface binding might not be properly evaluated. Thus, some inhibitors may
block virus infection in the post-virus binding steps, such as virus-endosome membrane fusion. Further
careful characterization is required.
2.2. Inhibitors of virus-induced membrane fusion
Crystal structure analysis demonstrated that a pocket of the DENV E protein, which is located at a
hinge region between domains I and II, is occupied by the ligand, octyl-β-D-glucoside (β-OG) (Figure
2) . Compounds blocking the β-OG pocket are expected to suppress conformational changes of the
E protein that are essential for fusion between virus and host endosome membranes (Table 2).
Viruses 2013, 5
Although these compounds are not directly related with carbohydrate molecules, their inhibitory
mechanisms are possibly similar to that of β-OG. In understanding the mechanism of the action of β-
OG, it is valuable to show the functional properties of these compounds. Therefore, this review also
introduces their chemical and biochemical properties.
An in silico screen for small molecules could potentially identify candidate molecules capable of
binding to the β-OG pocket. Combinatorial computational approaches identified two tetracycline
derivatives against flaviviruses . Both compounds were tetracycline derivatives with estimated
IC50 values of 67.1 M and 55.6 M, respectively. Although this study did not utilize fusion assays,
the compounds were computationally estimated to interact with critical hydrophobic residues that
affect membrane fusion. A combination of high-throughput in silico screening with this hydrophobic
pocket and evaluation of inhibitory activity by cell-based assays identified compound 6 as a fusion
inhibitor with EC50 of 119 nM against DENV2 in A549 cells. Mechanism-of-action studies
demonstrated that the compound acts in the early step of DENV infection, causing arrest of DENV in
vesicles that colocalize with endosomes . Another compound, NITD448 that inhibits DENV fusion
reduces viral titers with an EC50 of 9.8 M. Time-of-addition experiments showed that the compound
acts via inhibition of fusion .
A doxorubicin derivate, SA-17, carrying a squaric acid amide ester moiety at the carbohydrate
group was identified as a fusion inhibitor of DENV2 with EC50 of 0.52 M. Docking simulation
experiments showed that the compound also associated with amino acid residues critical for membrane
fusion, Thr-48, Glu-49, Ala-50, Lys-51, and Gln-52, in the hydrophobic β-OG pocket of the E protein
Viruses 2013, 5
Table 2. Chemical and antiviral properties of inhibitors of virus fusion
3. Conclusion and Future Directions
Repeated infection challenge with different serotypes of DENV increases the risk of antibody-
dependent enhancement (ADE) [4,8,9]. Maximal protection to the same extent against all four
serotypes with one drug or vaccine is required to control dengue diseases, particularly dengue
hemorrhagic disorders. Inhibitors targeting host factors involved in virus entry are interesting targets
and may overcome this problem. Accumulating knowledge regarding the processes of DENV entry
into the host cell and the recent progress in the in silico techniques will contribute to the development
of a new class of DENV inhibitors, i.e., entry inhibitors.
Compound Chemical structure
(dengue serotype/cell line)
SA-17 (Mw 653)
Viruses 2013, 5
While in the in vitro assays the affinity of carbohydrate derivatives, including GAG-related
compounds, HS mimetics, and compounds non-structurally related to the DENV E protein, will
determine their antiviral activity, additional factors, such as physicochemical and pharmacological
properties, including membrane permeability and bioavailability, will be critical for the development
of effective antiviral drugs for use in vivo. Among the inhibitors of virus adsorption, heparin, other
GAGs, and sulfated polysaccharides bind to plasma proteins, resulting in significant loss of
bioavailability. The smaller size of these compounds relative to other high-molecular weight sulfated
polysaccharides tested may account for their greater in vivo efficacy based on better bioavailability. In
addition to bioavailability, the side effects of HS mimetics due to their anticoagulant activity, restrict
their use as antiviral drugs. Thus, in terms of anti-DENV application of virus adsorption inhibitors, it
will be critical to identify the minimal determinants for effective antiviral activity of sulfated
polysaccharides, including heparin, etc. The smallest molecular weight inhibitor, 3-O-sulfated GlcA,
may be a useful lead compound that is expected to show low coagulopathy and prolonged
bioavailability in addition to effective in vivo inhibition of infectivity.
The fusion process is an alternative target for the development of effective in vivo antiviral agents.
In silico virtual screening with in vitro assays generated potent anti-DENV agents as described above.
Rational screening efforts for fusion inhibitors have continued and generated more potent compounds
with better physicochemical and pharmacological properties [62–67].
A previous study indicated that peptide entry inhibitors prevent antibody-mediated enhancement of
DENV2 infection of human, FcRII bearing K562 cells in vitro . In conclusion, entry inhibitors
have great potential for use either alone or in combination for treatment of dengue diseases.
This work was supported by grants-in-aid for Scientific Research on Priority Areas (21570146 and
24570168) from the Ministry of Education, Science, Sports, and Culture of Japan. This work was also
supported by a Cooperative Research Grant (2010-A-7) of the Institute of Tropical Medicine, Nagasaki
University, and research grants of Heiwa Nakajima Foundation and Japan China Medical Association.
Conflict of Interest
The authors declare no conflicts of interest.
References and Notes
1. Kuno, G.; Chang, G.J.; Tsuchiya, K.R.; Karabatsos, N.; Cropp, C.B. Phylogeny of the genus
Flavivirus. J. Virol. 1998, 72, 73–83.
Mukhopadhyay, S.; Kuhn, R.J.; Rossmann, M.G. A structural perspective of the flavivirus life
cycle. Nat. Rev. Microbiol. 2005, 3, 13–22.
Gubler, D.J. Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic
problem in the 21st century. Trends Microbiol. 2002, 10, 100–103.
Halstead, S.B. Dengue. Lancet 2007, 370, 1644–1652.
Viruses 2013, 5
5. Kuhn, R.J.; Zhang, W.; Rossmann, M.G.; Pletnev, S.V.; Corver, J.; Lenches, E.; Jones, C.T.;
Mukhopadhyay, S.; Chipman, P.R.; Strauss, E.G.; Baker, T.S.; Strauss, J.H. Structure of dengue
virus: implications for flavivirus organization, maturation, and fusion. Cell 2002, 8, 717–725.
Mackenzie, J.S.; Gubler, D.J.; Petersen, L.R. Emerging flaviviruses: the spread and resurgence of
Japanese encephalitis, West Nile and dengue viruses. Nat. Med. 2004, 10, S98–S109.
Weaver, S. C.; Barrett, A. D. Transmission cycles, host range, evolution and emergence of
arboviral disease. Nat. Rev. Microbiol. 2004, 2, 789–801.
Balsitis, S.J.; Coloma, J.; Castro, G.; Alava, A.; Flores, D.; McKerrow, J.H.; Beatty, P.R.; Harris,
E. Tropism of dengue virus in mice and humans defined by viral nonstructural protein 3-specific
immunostaining. Am. J. Trop. Med. Hyg. 2009, 80, 416–424.
Clyde, K.; Kyle, J.L.; Harris, E. Recent advances in deciphering viral and host determinants of
dengue virus replication and pathogenesis. J. Virol. 2006, 80, 11418–11431.
10. Navarro-Sanchez, E.; Altmeyer, R.; Amara, A.; Schwartz, O.; Fieschi, F.; Virelizier, J.L.;
Arenzana-Seisdedos, F.; Desprès P. Dendritic-cell-specific ICAM3-grabbing non-integrin is
essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue
viruses. EMBO Reports 2003, 4, 723–728.
11. Tassaneetrithep, B.; Burgess, T.H.; Granelli-Piperno, A.; Trumpfheller, C.; Finke, J.; Sun, W.;
Eller, M.A.; Pattanapanyasat, K.; Sarasombath, S.; Birx, D.L.; Steinman, R.M.; Schlesinger, S.;
Marovich, M.A. DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J.
Exp. Med. 2003, 197, 823–829.
12. Chen, S.T.; Lin, Y.L.; Huang, M.T.; Wu, M.F.; Cheng, S.C.; Lei, H.Y.; Lee, C.K.; Chiou, T.W.;
Wong, C.H.; Hsieh, S.L. CLEC5A is critical for dengue-virus-induced lethal disease. Nature
2008, 453, 672–676.
13. Feinberg, H.; Mitchell, D.A.; Drickamer, K.; Weis, W.I. Structural basis for selective recognition
of oligosaccharides by DC-SIGN and DC-SIGNR. Science 2001, 294, 2163-2166.
14. Miller, J.L.; de Wet, B.J.; Martinez-Pomares, L.; Radcliffe, C.M.; Dwek, R.A.; Rudd, P.M.;
Gordon, S. The mannose receptor mediates dengue virus infection of macrophages. PLoS Pathog.
2008, 4, e17.
15. Jindadamrongwech, S.; Thepparit, C.; Smith, D.R. Identification of GRP 78 (BiP) as a liver cell
expressed receptor element for dengue virus serotype 2. Arch. Virol. 2004, 149, 915–927.
16. Chen, Y.C.; Wang, S.Y.; King, C.C. Bacterial lipopolysaccharide inhibits dengue virus infection
of primary human monocytes/macrophages by blockade of virus entry via a CD14-dependent
mechanism. J. Virol. 1999, 73, 2650–2657.
17. Chen, Y.; Maguire, T.; Hileman, R.E.; Fromm, J.R.; Esko, J.D.; Linhardt, R.J.; Marks, R.M.
Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate. Nat.
Med. 1997, 3, 866–871.
18. Germi, R.; Crance, J.M.; Garin, D.; Guimet, J.; Lorta-Jacob, H.; Ruigrok, R.W.; Zarski, J.P.;
Drouet, E. Heparan sulfate-mediated binding of infectious dengue virus type 2 and yellow fever
virus. Virology 2002, 292, 162–168.
19. Hilgard, P.; Stockert, R. Heparan sulfate proteoglycans initiate dengue virus infection of
hepatocytes. Hepatology 2000, 32, 1069–1077.
Viruses 2013, 5
20. Lin, Y.L.; Lei, H.Y.; Lin, Y.S.; Yeh, T.M.; Chen, S.H.; Liu, H.S. Heparin inhibits dengue-2 virus
infection of five human liver cell lines. Antiviral Res. 2002, 56, 93–96.
21. Hung, S.L.; Lee, P.L.; Chen, H.W.; Chen, L.K.; Kao, C.L.; King, C.C. Analysis of the steps
involved in Dengue virus entry into host cells. Virology 1999, 257, 156–167.
22. Aoki, C.; Hidari, K.I.P.J.; Itonori, S.; Yamada, A.; Takahashi, N.; Kasama, T.; Hasebe, F.; Islam,
M.A.; Hatano, K.; Matsuoka, K.; Taki, T.; Guo, C.-T.; Takahashi, T.; Sakano, Y.; Suzuki, T.;
Miyamoto, D.; Sugita, M.; Terunuma, D.; Morita, K.; Suzuki, Y. Identification and
characterization of carbohydrate molecules in mammalian cells recognized by dengue virus type
2. J. Biochem. (Tokyo) 2006, 139, 607–614.
23. Wichit, S.; Jittmittraphap, A.; Hidari, K.I.; Thaisomboonsuk, B.; Petmitr, S.; Ubol, S.; Aoki, C.;
Itonori, S.; Morita, K.; Suzuki, T.; Suzuki, Y.; Jampangern, W. Dengue virus type 2 recognizes
the carbohydrate moiety of neutral glycosphingolipids in mammalian and mosquito cells.
Microbiol. Immunol. 2011, 55, 135-140.
24. Modis, Y.; Ogata, S.; Clements, D.; Harrison, S.C. A ligand-binding pocket in the dengue virus
envelope glycoprotein. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6986–6991.
25. Modis, Y.; Ogata, S.; Clements, D.; Harrison, S.C. Variable surface epitopes in the crystal
structure of dengue virus type 3 envelope glycoprotein. J. Virol. 2005, 79, 1223–1231.
26. Zhang, W.; Chipman, P.R.; Corver, J.; Johnson, P.R.; Zhang, Y.; Mukhopadhyay, S.; Baker, T.S.;
Strauss, J.H.; Rossmann, M.G.; Kuhn, R.J. Visualization of membrane protein domains by cryo-
electron microscopy of dengue virus. Nat. Struct. Biol. 2003, 10, 907–912.
27. Zhang, Y.; Corver, J.; Chipman, P.R.; Zhang, W.; Pletnev, S.V.; Sedlalk, D.; Barker, T.S.;
Strauss, J.H.; Kuhn, R.J.; Roaamann, M.G. Structures of immature flavivirus particle. EMBO J.
2003, 22, 2604–1613.
28. McMinn, P.C. The molecular basis of virulence of the encephalitogenic flaviviruses. J. Gen. Virol.
1997, 78, 2711–2722.
29. Stiasny, K.; Kössl, C.; Lepault, J.; Rey, F.A.; Heinz, F.X. Characterization of a structural
intermediate of flavivirus membrane fusion. PLoS Pathog. 2007, 3, e20.
30. Lindenbach, B.D.; Rice, C.M. Molecular biology of flaviviruses. Adv. Virus Res. 2003, 59, 23–61.
31. Lee, E.; Lobigs, M. Substitutions at the putative receptor-binding site of an encephalitis flavivirus
alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry. J. Virol.
2000, 74, 8867–8875.
32. Lee, E.; Lobigs, M. Mechanism of virulence attenuation of glycosaminoglycan-binding variants
of Japanese encephalitis virus and Murray Valley encephalitis virus. J. Virol. 2002, 76, 4901–
33. Mandl, C.W.; Kroschewski, H.; Allison, S.L.; Kofler, R.; Holzmann, H.; Meixner, T.; Heinz, F.X.
Adaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple
heparan sulfate binding sites in the envelope protein and attenuation in vivo. J. Virol. 2001, 75,
34. Su, C.M.; Liao, C.L.; Lee, Y.L.; Lin, Y.L. Highly sulfated forms of heparin sulfate are involved
in Japanese encephalitis virus infection. Virology 2001, 286, 206–215.
Viruses 2013, 5
35. Kato, D.; Era, S.; Watanabe, I.; Arihara, M.; Sugiura, N.; Kimata, K.; Suzuki, Y.; Morita, K.;
Hidari, K.I., Suzuki, T. Antiviral activity of chondroitin sulphate E targeting dengue virus
envelope protein. Antiviral Res. 2010, 88, 236–243.
36. Alen, M.M.F.; Schols, D. Dengue Virus Entry as Target for Antiviral Therapy. J. Trop. Med.
2012, 2012, 1–13.
37. Sessions, O.M.; Barrows, N.J.; Souza-Neto, J.A.; Robinson, T.J.; Hershey, C.L.; Rodgers, M.A.;
Ramirez, J.L.; Dimopoulos, G.; Yang, P.L.; Pearson, J.L. Garcia-Blanco, M.A. Discovery of
insect and human dengue virus host factors. Nature 2009, 458, 1047–1050.
38. Rey, F.A.; Heinz, F.X.; Mandl, C.; Kunz, C.; Harrison, S.C. The envelope glycoprotein from tick-
borne encephalitis virus at 2 Å resolution. Nature 1995, 375, 291–298.
39. Thullier, P.; Demangel, C.; Bedouelle, H.; Mégret, F.; Jouan, A.; Deubel, V.; Mazié, J.C.; Lafaye,
P. Mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes:
insight into the neutralization mechanism. J. Gen. Virol. 2001, 82, 1885–1892.
40. Talarico, L.B.; Pujol, C.A.; Zibetti, R.G.; Faría, P.C.; Noseda, M.D.; Duarte, M.E.; Damonte, E.B.
The antiviral activity of sulfated polysaccharides against dengue virus is dependent on virus
serotype and host cell. Antiviral Res. 2005, 66, 103–110.
41. Hidari, K.I.P.J.; Takahashi, N.; Arihara, M.; Nagaoka, M.; Morita, K.; Suzuki, T. Structure and
anti-dengue virus activity of sulfated polysaccharide from marine algae. Biochem. Biophys. Res.
Commun. 2008, 376, 91–95.
42. Talarico, L.B.; Damonte, E.B. Interference in dengue virus adsorption and uncoating by
carrageenans. Virology, 2007, 363, 473–485.
43. Pastorino, B.; Nougairède, A.; Wurtz, N.; Gould, E.; de Lamballerie, X. Role of host cell factors
in flavivirus infection: Implications for pathogenesis and development of antiviral drugs.
Antiviral Res. 2010, 87, 281–294.
44. Noble, C.G.; Chen, Y.L.; Dong, H.; Gu, F.; Lim, S.P.; Schul, W.; Wang, Q.Y.; Shi, P.Y.
Strategies for development of Dengue virus inhibitors. Antiviral Res. 2010, 85, 450–462.
45. Ono, L.; Wollinger, W.; Rocco, I.M.; Coimbra, T.L.; Gorin, P.A.; Sierakowski, M.R. In vitro and
in vivo antiviral properties of sulfated galactomannans against yellow fever virus (BeH111 strain)
and dengue 1 virus (Hawaii strain). Antiviral Res. 2003, 60, 201–208.
46. Qiu, H.; Tang, W.; Tong, X.; Ding, K.; Zuo, J. Structure elucidation and sulfated derivatives
preparation of two alpha-D-glucans from Gastrodia elata Bl. and their anti-dengue virus
bioactivities. Carbohydr Res. 2007, 342, 2230–2236.
47. Berteau, O.; Mulloy, B. Sulfated fucans, fresh perspectives: structures, functions, and biological
properties of sulfated fucans and an overview of enzymes active toward this class of
polysaccharide. Glycobiology 2003, 13, 29R–40R.
48. Lee, E.; Pavy, M.; Young, N.; Freeman, C.; Lobigs, M. Antiviral effect of the heparan sulfate
mimetic, PI-88, against dengue and encephalitic flaviviruses. Antiviral Res. 2006, 69, 31–38.
49. Ferro, V.; Li, C.; Fewings, K.; Palermo, M.C.; Linhardt, R.J.; Toida, T. Determination of the
composition of the oligosaccharide phosphate fraction of Pichia (Hansenula) holstii NRRL Y-
2448 phosphomannan by capillary electrophoresis and HPLC. Carbohydr. Res. 2002, 337, 139–
Viruses 2013, 5
50. Yu, G.; Gunay, N.S.; Linhardt, R.J.; Toida, T.; Fareed, J.; Hoppensteadt, D.A.; Shadid, H.; Ferro,
V.; Li, C.; Fewings, K.; Palermo, M.C.; Podger, D. Preparation and anticoagulant activity of the
phosphosulfomannan PI-88. Eur. J. Med. Chem. 2002, 37, 783–791.
51. Maffrand, J.P.; Herbert, J.M.; Bernat, A.; Defreyn, G.; Delebassee, D.; Savi, P.; Pinot, J.J.;
Sampol, J. Experimental and clinical pharmacology of pentosan polysulfate. Semin. Thromb.
Hemost. 1991, 2, 186–198.
52. Voogd, T.E.; Vansterkenburg, E.L.; Wilting, J.; Janssen, L.H. Recent research on the biological
activity of suramin. Pharmacol. Rev. 1993, 45, 177–203.
53. Marks, R.M.; Liu, H.; Sundaresan, R.; Toida, T.; Suzuki, A.; Imanari, T.; Hernáiz, M.J.; Lindardt,
R. J. Probing the interaction of dengue virus envelope protein with heparin: assessment of
glycosaminoglycan-derived inhibitors. J. Med. Chem. 2001, 44, 2178–2187.
54. Hidari, K.I.P.J.; Ikeda, K.; Watanabe, I.; Abe, T.; Sando, A.; Itoh, Y.; Tokiwa, H.; Morita, K.;
Suzuki, T. 3-O-sulfated glucuronide derivative as a potential anti-dengue virus agent. Biochem.
Biophys. Res. Commun. 2012, 424, 573–578.
55. van der Schaar, H.M.; Rust, M. J.; Waarts, B.-L.; van der Ende-Metselaar; H., Kuhn, R.J.;
Wilschut, J.; Zhuang, X.; Smit, J.M. Characterization of the early events in dengue virus cell
entry by biochemical assays and single-virus tracking. J. Virol. 2007, 81, 12019–12028.
56. Zaitseva, E.; Yang, S.-T.; Melikov, K.; Pourmal, S.; Chernomordik, L.V. Dengue virus ensures
its fusion in late endosomes using compartment-specific lipids. PLoS Pathog. 2010, 6, e1001131.
57. Yang, J.M.; Chen, Y.F.; Tu, Y.Y.; Yen, K.R.; Yang, Y.L. Combinatorial computational
approaches to identify tetracycline derivatives as flavivirus inhibitors. PLoS One 2007, 2, e428.
58. Wang, Q.Y.; Patel, S.J.; Vangrevelinghe, E.; Xu, H.Y.; Rao, R.; Jaber, D.; Schul, W.; Gu, F.;
Heudi, O.; Ma, N.L.; Poh, M.K.; Phong, W.Y.; Keller, T.H.; Jacoby, E.; Vasudevan, S.G. A small
molecule dengue virus entry inhibitor. Antimicrob. Agents .Chemother. 2009, 53, 1823–1831.
59. Poh, M.K.; Yip, A.; Zhang, S.; Priestle, J.P.; Ma, N.L.; Smit, J.M.; Wilschut, J.; Shi, P.Y.; Wenk,
M.R.; Schul, W. A small molecule fusion inhibitor of dengue virus. Antiviral Res. 2009, 84, 260–
60. Modis, Y.; Ogata, S.; Clements, D.; Harrison. S.C. Structure of the dengue virus envelope protein
after membrane fusion. Nature 2004, 427, 313–319.
61. Kaptein, S.J.; De Burghgraeve, T.; Froeyen, M.; Pastorino, B.; Alen, M.M.; Mondotte, J.A.;
Herdewijn, P.; Jacobs, M.; de Lamballerie, X.; Schols, D.; Gamarnik, A.V.; Sztaricskai, F.; Neyts,
J.A. Derivate of the antibiotic doxorubicin is a selective inhibitor of dengue and yellow fever
virus replication in vitro. Antimicrob. Agents Chemother. 2010, 54, 5269–5280.
62. Schmidt, A.G.; Lee, K.; Yang, P.L.; Harrison, S.C. Small-molecule inhibitors of dengue-virus
entry. PLoS Pathog. 2012, 8, e1002627.
63. Poh, M.K.; Shui, G.; Xie, X.; Shi, P.Y.; Wenk, M.R.; Gu, F. U18666A, an intra-cellular
cholesterol transport inhibitor, inhibits dengue virus entry and replication. Antiviral Res. 2012, 93,
64. Zhou, Z.; Khaliq, M.; Suk, J.E.; Patkar, C.; Li, L.; Kuhn, R.J.; Post, C.B. Antiviral compounds
discovered by virtual screening of small–molecule libraries against dengue virus E protein. ACS
Chem. Biol. 2008, 3, 765–775.
Viruses 2013, 5
65. Liao, M.; Kielian, M. Domain III from class II fusion proteins functions as a dominant-negative
inhibitor of virus membrane fusion. J. Cell Biol. 2005, 171, 111–120.
66. Costin, J.M.; Jenwitheesuk, E.; Lok, S.-M.; Hunsperger, E.; Conrads, K.A.; Fontaine, K.A.; Rees,
C.R.; Rossmann, M.G.; Isern, S.; Samudrala, R.; Michael, S.F. Structural optimization and de
novo design of dengue virus entry inhibitory peptides. PLoS Negl. Trop. Dis. 2010, 4, e721.
67. Schmidt, A.G.; Yang, P.L.; Harrison, S.C. Peptide inhibitors of dengue-virus entry target a late-
stage fusion intermediate. PLoS Pathog. 2010, 6, e1000851.
68. Nicholson, C.O.; Costin, J.M.; Rowe, D.K.; Lin, L.; Jenwitheesuk, E.; Samudrala, R.; Isern, S.;
Michael, S.F. Viral entry inhibitors block dengue antibody-dependent enhancement in vitro.
Antiviral Res. 2011, 89, 71–74.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license