Indian Journal of Clinical Biochemistry, 2005, 20 (2) 92-103

MOLECULAR MECHANISM OF PATHOGENESIS OF DENGUE VIRUS : ENTRY AND FUSION WITH TARGET CELL
Seema and S.K.Jain* Department of Biotechnology, Hamdard University, New Delhi 110 062, India
ABSTRACT Dengue fever is one of the major health problems in India. Interaction with specific receptor(s) at the cell surface is one of the first events in the pathogenesis of Dengue virus. However, relatively little is known about these receptors. Cellular receptors in human monocytes and mouse neural cells are main target for the viral infection. The envelope protein of the virus (E-protein) plays important role in attachment of virus to target cells and their interaction with cellular receptors. The modulation of receptor gene(s) and/or protein(s) can be used as a method for interfering with virus entry and can thus become a new method for disease prevention. The receptors can be purified by affinity chromatography using E-protein as ligand. It has been reported that addition of highly sulfated heparan sulfate prevents E-protein binding to target cells suggesting that heparan sulfate is utilized by dengue envelope protein to bind to target cells. KEY WORDS Cell surface receptors, Dengue fever, Glycosaminoglycans, Heparan sulfate, Viral attachment . INTRODUCTION The entry of virus into target cell is facilitated by a number of activities taking place at the host cell. The host cell provides surface receptors, shows endocytic activity, triggers signals for penetration, intracellular transport and so on, and the viruses take advantage of these processes to establish the infection. The early phase of viral infection has remained a relatively neglected field inspite of their obvious importance for understanding the viral replication, cell tropism and pathogenesis. Identification of cellular receptors for virus is often hampered by a weak affinity between individual viral proteins and receptor molecules and the receptors are often present in low numbers on the cell surface. However, it is possible to incorporate sufficient radioactive label into virus particles to follow their fate even at low multiplicity. Modern immunochemical methods have proved powerful tool in identifying receptors. A number of inhibitors that block the productive infection pathway of large groups of viruses have been identified and characterized. In vivo and in vitro systems have been Author for Correspondence : Dr. S.K. Jain Head Department of Biotechnology Hamdard University, Hamdard Nagar New Delhi 110062, India E-mail : skjain@jamiahamdard.ac.in Indian Journal of Clinical Biochemistry, 2005 developed for studying the viral integration by membrane fusion (reviewed by Marsh and Heleniust, 1989). The step-by-step itinerary of the entry pathway of certain viruses, such as the Semliki Forest Virus (SFV) is now known (Helenius et al, 1988; Kielian and Helenius, 1986). In this review we have focused on some of the cellular receptors involved in binding of dengue virus serotypes to illustrate general mode for a dengue virus infection. DENGUE VIRUS The dengue virus belongs to genus Flavivirus (family Flaviviridae) that includes about 70 distinct viruses, all of which are serologically related and in majority of cases, maintained in nature by transmission from hematophagous arthropod vectors (mosquito or ticks) to vertebrate hosts (reviewed by Monath and Heinz, 1996). More than 50% of the flaviviruses have been associated with human diseases, and of these the most important in terms of disease incidence are dengue (DEN) virus (types 1 to 4), yellow fever (YF) virus, Japanese encephalitis (JE) virus, and tick borne encephalitis (TBE) virus. DEN virus is present in tropical and subtropical areas around the world, as these provide the ecological conditions required for maintaining the natural cycles of the virus. There are four dengue virus serotypes: DEN-1, DEN-2, DEN-3 and DEN-4. All flaviviruses have common group epitopes on the envelope protein that results in extensive cross-reaction in serological tests, making unequivocal serologic diagnosis of flaviviruses difficult. This is especially true of the four dengue virus 92

Indian Journal of Clinical Biochemistry, 2005, 20 (2) 92-103 serotypes. Infection with one dengue serotype provides lifelong immunity to that virus, but there is no cross protective immunity to the other serotypes. Thus persons living in a dengue-endemic area can get infected with all four serotypes during their lifetime. The structure of many flaviviruses (including DEN virus) has been determined by using a combination of cryo-electron microscopy and fitting the known structure of glycoprotein-E into electron density map to a resolution of ~11. The virus core within a lipid bilayer has a less ordered structure than the external icosahedral scaffold of 90 glycoprotein-E dimers. The three E monomers per icosahedral asymmetric unit do not have quasi-equivalent symmetric environment. In addition, structures of immature dengue and yellow fever virus particles were determined to 16 and 25 resolution, respectively (Fuller et al., 1995). The closely similar structures show 60 icosahedrallyorganized, trimeric spikes on the particle surface. Each spike consists of three prM:E heterodimers, where E is an envelope glycoprotein and prM is the precursor to the membrane protein M. The pre-peptide components of the prM proteins in each spike cover the fusion peptides at the distal ends of the E glycoproteins (Wang et al., 1999). Each heterodimer is associated with an E and a prM transmembrane density. These transmembrane densities represent either an EE or prM-prM antiparallel coil, due to which each protein spans the membrane twice. The carboxy terminus of each protein remains on the exterior of the viral membrane, consistent with the predicted membrane-spanning domains of the unprocessed polyprotein (Hung et al., 1999). The structure suggests that flaviviruses employ a fusion mechanism in which the distal beta barrels of domain II of the glycoproteinE are inserted into the cellular membrane. TRANSMISSION AND INFECTION WITH DENGUE VIRUS The primitive enzootic transmission cycle of dengue virus involves canopy-dwelling Aedes species mosquitoes and lower primates in the rain forests of Asia and Africa (Gubler et al., 1988). A number of Aedes (Stegomyia) mosquito species may act as vector in these situations, including Ae. aegypti, Ae. albopictus, Ae. polynesiensis, and some members of Ae. scutellaris group. From a public health standpoint, the urban endemic/epidemic cycle is the most important transmission cycle. The viruses are maintained in an Ae. aegypti-human-Ae. aegypti cycle, with periodic epidemic occurring at 3 to 5 year intervals. Humans are infected with dengue virus by the bite of an infective Aedes mosquito (Gubler et al., 1988) that prefers to lay its eggs in water filled household. Adult Ae. aegypti mosquitoes are unobtrusive, prefer to rest indoors and feed on humans during daylight hours. The female mosquitoes Indian Journal of Clinical Biochemistry, 2005 often disrupt the feeding process at slightest movement and return to the same or a different person to continue feeding moments later. The Ae. aegypti females may thus feed on several persons during a single blood meal and transmit dengue virus to multiple persons within a short period of time (Gubler, 1979, 1981, 1984). This behavior makes Ae. aegypti an efficient epidemic vector. Dengue fever (DF) is an acute febrile illness with chills, headache, retro-ocular pain, body ache and arthralgia in more than 90% of cases accompanied by nausea or vomiting and a maculopapular rash resembling measles that last 2-7 days in about 60% of cases. This is followed by sharp pain in the muscles and joints with sensations as if bones are breaking, that gave the name break bone fever to DF. Another name, saddle back fever has also been used because of regular temperature fluctuations in the patient. The symptoms last for about a week and death is uncommon. However, the co-infection with one of the other dengue viruses results in dengue hemorrhage fever (DHF), resulting in appearances of rashes on the face and extremities, and severe vomiting and shock ensues that may lead to death of the patient. WHO reported DF/DHF to be the second (after malaria) most important tropical infectious disease, with about 2.5 billion people living in high risk areas for dengue transmission. Each year about 50 to 100 million cases of DF recur that lead to 500,000 hospitalizations and 25,000 deaths (WHO, 1999). The spread of viral infection depends on the transfer of the viral genome and accessory proteins from an infected cell to uninfected cells that requires a sequence of discrete events. Depending on the virus, these include virus assembly in the infected cell, release of infective virions from the infected cell into the extracellular space, attachment of the virus to receptor structures on the host cell surface, their internalization by endocytosis, penetration through a host cell membrane, uncoating of the viral genome, and its intra-cytoplasmic relocation to nucleus and/or other sites within the cell (Reviewed by Marsh and Helenius; 1989). DENGUE VIRUS STRAIN VARIATION Little is presently known about the role of virusspecified factors in the transmission and pathogenesis of dengue. Considerable microevolution of dengue virus strains found in studies employing monoclonal antibody analysis (Vazeille et al., 1999), RNA finger printing (Lorenz et al., 1984) or sequencing of selected regions of the genome. As variants with similar genetic structure have been found within a specific geographic location, the studies elucidating the movements of dengue viruses and source of epidemics suggest that genetic variation in virus strains may determine 93

Indian Journal of Clinical Biochemistry, 2005, 20 (2) 92-103 virulence and explain the changing patterns of disease. All the four endemic dengue viruses have evolved independently from sylvatic progenitors by adapting to peridomestic mosquito vectors and human reservoir hosts. The rise of urban civilizations associated with peridomestication of Ae. aegypti and Ae. albopictus mosquitoes provided the opportunity for adaptation and have resulted in the emergence of dengue in urban areas of tropics. This hypothesis predicts that endemic dengue virus strains are more efficient at infecting urban mosquitoes such as Ae. aegypti and Ae. albopictus with sylvatic versus urban strains. This hypothesis was tested by retrospectively examining evidence for adaptation of epidemic and endemic versus sylvatic strains of DEN-2 to Ae. albopictus and Ae. aegypti. Second-generation offsprings of mosquitoes from different geographic regions in America and Southeast Asia were tested for their susceptibility to epidemic/endemic and sylvatic DEN-2 isolates from West Africa, Southeast Asia and Oceania. Both Aedes species were found to be highly susceptible (upto 100% infected) to endemic/epidemic DEN-2 strains after ingesting artificial blood meals but significantly less susceptible (as low as 0%) to sylvatic DEN-2 strains. These results support the hypothesis that adaptation to peridomestic mosquito vectors mediates dengue emergence from sylvatic progenitor viruses. The four independent evolutionary dengue virus emergence events (DEN-1 to DEN-4) suggest that adaptation of dengue virus to new vectors and hosts occurred repeatedly from 300 to 1,500 years ago in Asia or Oceania and resulted in emergence and changing patterns of the disease (Holmes et al., 2003). CELLULAR RECEPTORS FOR VIRUS In order to cause infection, a virus must be able to bind to cell surface. The binding occurs through interaction between the surface proteins of virion and specific viral receptors on target cell membranes. Enveloped and non-enveloped viruses have multiple identical proteins on their surface and possess the inherent capacity to bind to receptors through multivalent interactions, providing them considerable versatility in terms of binding capabilities. Virus binding has been primarily studied in tissue culture systems. A combination of factors including temperature, ionic strength, pH, composition of the medium and presence of serum play major role in initial binding of the virus to the cell (Holmberg-Holm, 1981). Techniques such as affinity isolation with whole virus or viral components, antireceptor antibodies or anti-idiotypic antibodies to the viral binding sites, chemical cross-linking, DNA transfection, and somatic cell hybrids have been used in attempts to identify specific binding components (reviewed by Sommerfelt and Marsh, 1988). However, few specific virus receptors have been unambiguously identified and still Indian Journal of Clinical Biochemistry, 2005 fewer have been demonstrated to play a functional role in virus entry in vivo. Viruses have developed a variety of strategies to recognize and bind to host cells. They can use proteins, lipids, or oligosaccharides as receptors, however, many of these receptors are the proteins involved in ligand binding, endocytosis, and cell recognition. Based on the nature, the viruses can be classified into following groups. A. High Specificity Binding/ Narrow Host Range: Viruses such as human immunodeficiency virus (HIV1 and HIV- 2) and Epstein-Barr virus (EBV) bind to components expressed only on a limited number of cell types. In case of HIV, the virions bind to CD4 molecules expressed on helper/inducer T lymphocytes, cells of monocyte-macrophage lineage, and certain cells transfected with the CD4 gene (Dalgleish et al., 1986). Although CD4 binds to envelope glycoprotein of HIV-1, a second receptor is necessary for entry and infection. The T-cell tropic strains of HIV-1 use the coreceptor CXCR4, while the macrophage tropic strains use CCR5. Both are receptors for chemokines, and their normal ligands can block HIV infection of the cell. B. High Specificity Binding/ Broad Host Range: Orthomyxoviruses and paramyxoviruses bind with considerable specificity to sialic acid, often in distinct linkage configurations. The binding site for sialic acid in influenza virus HA is highly conserved depression in the HA1 subunit at the tip of the spike protein (Wilson et al., 1991). Sialic acid is often the terminal saccharide residue on cell surface glycoproteins and glycolipids and the complex carbohydrates such as heparan sulfate proteoglycan may be involved in herpes simplex virus infection (Wu and Spear, 1995) and dengue viral infection (Wei et al., 2003). C. Broad Host Range: A number of viruses, such as the alphavirus, SFV, rhabdo virus and vesicular stomatitis virus (VSV) exhibit a broad host range in cell culture but do not appear to utilize carbohydrates as the binding component. SFV probably utilizes several cell surface proteins as receptor and can bind to MHC class1 antigens on human and murine lymphoblastoid cells (Helenius et al., 1995). However, it can also infect cells that do not express MHC molecules (Oldstone et al., 1980). D. Different Viruses Using the Same Binding Sites: Orthomyxoviruses and paramyxoviruses, which bind to sialic acid residues use same cellular receptors. Competition experiments have shown that other viruses may share these receptors also. Eighteen different retroviruses that infect cultured human cells, utilize eight distinct receptors for their binding. Though it remains unclear whether the viruses that share common receptors utilize the same epitopes or multiple epitopes are present for different viruses 94

Indian Journal of Clinical Biochemistry, 2005, 20 (2) 92-103 (Sommerfelt and Marsh, 1988). E. Viral Proteins as Virus Receptors: Membrane proteins of enveloped viruses may themselves function as virus receptors. Normally BHK-21 cells do not express a receptor for murine hepatitis virus (MHVA59), this makes cells resistant to MHV. However, cells become susceptible if preinfected with influenza. Thus virus infection can facilitate secondary infections by other viruses (Fuller et al., 1995). F. Indirect Binding: All the examples discussed so far involve direct interaction of the attachment sites present on virus with cell surface-binding components. Viruses may also bind to cell surface components in an indirect manner through interaction with an intermediate molecule. This type of interaction, for example, occurs in antibody-enhanced viral infection shown by all serotypes of DEN virus. G. Role of Cell Surface Virus Receptors: Binding facilitates viral entry by providing the initial physical association between surface of the cell and the virion. Whether virus receptors play any further role in the entry or not, varies amongst the individual viruses. Influenza virus, for example, does not require sialic acid containing receptors for penetration; the receptors are needed for binding and endocytosis. In the case of alphaviruses and rhabdoviruses the fusion activity, which underlies penetration in endosomes, is not dependent on the presence of specific surface receptors. The receptors for poliovirus may, for instance, be needed for the correct acid-induced conformational change. There is no evidence for a more direct role of receptors in penetration. ENTRY OF FLAVIVIRUSES Flavivirus entry has been studied in great detail with West Nile virus (WNV). The WNV is internalized by coated vesicles and transported to endosomes (Gollins and Porterfield, 1985). Infection is inhibited by weak bases and is therefore presumed to involve acid-induced fusion (Gollins and Porterfield, 1985,1986). This conclusion is supported by the experiments in which viruses have been induced to fuse with liposomes or the plasma membrane of target cells (Gollins and Porterfield, 1986). Penetration is pHdependent with the threshold for fusion being at about pH 6.5. The mechanism of entry into macrophages is of particular interest, as it is mediated by antibodies and presumably occurs via Fc receptors (Gollins and Porterfield, 1985; Peiris and Porterfield, 1979; Peiris et al., 1981). Gollins and Porterfield (1986) have made an important observation that has implications for therapeutic strategies against flaviviruses. It was found that among several neutralizing monoclonal antibodies to WNV, one was able to block penetration without affecting attachment or endocytic internalization, suggesting that this antibody specifically interacted Indian Journal of Clinical Biochemistry, 2005 with the acidic fusion-active form of the virus and prevented penetration to endosomes. A similar report was made for rabies virus also (Dietzschold et al., 1987). FUSION OF DENGUE VIRUS TO TARGET CELL Dengue virus is a single stranded, positive sense RNA virus with 11 kb unfragmented genome surrounded by a lipid bilayer envelope. RNA codes for three structural proteins and seven non-structural proteins. Three structural proteins are C (nucleocapsid), M (membrane associated protein) and E (envelope protein) and the seven structural proteins have been named as NS1, NS2, NS3, NS4, NS5, NS6 and NS7. The establishment of infection requires the entry of DEN virus into cells, followed by release of nucleocapsid. This is achieved by the fusion of viral membrane with a cellular membrane (reviewed by White, 1992;Hernandez et al, 1996). This may occur either at the plasma membrane or at endosomal membranes after the virus is taken inside through receptor mediated endocytosis. The conformational changes triggered by the plasma membrane due to receptorvirus binding or by the acidic pH in the endosomes lead to exposure of the fusion peptide, which interacts with the target membrane and initiates the fusion event. The presence of cholesterol in the target membrane facilitates the fusion but is not absolutely necessary for fusion (Kielian, 1995; Lu et al., 1999; Smit et al., 1999). ENTRY OF DENGUE VIRUS INTO CELLS Monocyte macrophages (m) are the principal target cells for dengue viruses. The virus E-protein mediates the attachment to cells, but the nature of host cell receptors remains elusive. The candidate receptor molecules currently being explored include proteins, Fc receptors, glycosaminoglycans (GAGS) and lipopolysaccharide binding CD14-associated molecules (Bielefeldt-Ohmann et al., 2000). The initial steps in dengue virus entry have been divided into adsorption and penetration. When BHK cells are treated with glycine, the extracellular viruses get inactivated even after their attachment to the cells but prior to penetration inside the cells. It was found that virus infection is accomplished within 2 hours of adsorption. Using dengue E protein-specific monoclonal antibodies, Hung et al. (1999) found that while three different MAbs (17-2, 46-9, and 51-3) could neutralize DEN2 through inhibition of viral attachment as well as viral penetration, only one monoclonal antibody (56-31) specifically interfered with attachment. It was, therefore concluded that the functional domains of E-protein involved in the attachment and penetration might be different. Moreover, studies with lectins indicated that carbohydrates, especially -mannose residues, 95

Indian Journal of Clinical Biochemistry, 2005, 20 (2) 92-103 present on virion glycoproteins might contribute to binding and penetration of the virus into BHK cells and mosquito C6/36 cells. Finally, virus infectivity was also inhibited by heparin that blocked both the virus attachment and the penetration, suggesting that heparan sulfate present at the cell surface plays an important role in these steps of infection (Hung et al., 1999). Defining the precise events during interaction of virus with cell surface is important to understand the pathogenesis of dengue virus. The initial processes essential for productive dengue infection have not been reported in as much detail as for other flaviviruses, such as WNV. Studies using electron microscopy have indicated that attachment is a temperature independent process that occurs at both 400C as well as at 370C, whereas viral penetration proceeds only at 370C. The penetration can occur by membrane fusion in mosquito C6/36 cells or by receptor-mediated endocytosis in monocytes (Barth, 1992; Hase et al., 1989). In presence of subneutralizing amounts of antibody, Fc receptors also mediate attachment and uptake of dengue viruses into certain target cells such as monocytes and macrophages (Golins and Porterfield, 1985; Mady et al., 1993). This entry mechanism, termed as antibodydependent enhancement (ADE), may play a role in development of DHF and the Dengue Shock Syndrome (DSS) as the consequence of sequential infections with different dengue serotypes (reviewed by Halstead, 1988). The E-protein of dengue virus, which is embedded in lipid bilayer, may mediate both the virus attachment as well as penetration into cells. E-protein is both the target as well as the modulator of host immune response. E-protein, a 60 KDa glycoprotein, contains two sites for N-linked glycosylation (Smith and Wright, 1985). Use of glycosylation site in mosquito cells appears to depend on the serotype of virus (Johnson et al., 1994). Mutations in N-linked glycosylation sites of E protein affect virus-mediated membrane fusion and neurovirulence (Guirakhoo et al., 1993; Kawano et al., 1993; Sanchez and Ruiz, 1996). However, the functional significance of the attached carbohydrates is not fully understood. Chen et al. (1996b) found that recombinant E protein specifically binds to Vero, CHO, endothelial and glial cells. In addition, this binding has also been found to be associated with heparan sulfate or GAGS. These complex charged carbohydrates are found both on the cell surface and in the extracellular matrix. Thus, highly sulfated heparan sulfate on the cell surface may serve as the initial attachment receptor for DEN-2 virus (Chen et al., 1997). However, the precise mechanism of blocking the entry steps by heparin is not clear and may involve additional receptors for dengue viruses that may be present in the cells. It has been found that DEN-2 virus can infect Indian Journal of Clinical Biochemistry, 2005 cultured monocytes through a trypsin-sensitive cellular receptor that is proteinaceous in nature (Daughaday et al., 1981). The flavivirus E-protein is a multifunctional protein which is involved in cell receptor binding and virus entry via fusion with a host cell membrane (Rice et al.,1996). Some of the functions of E protein, especially membrane fusion, are regulated by interaction with a second viral protein, prM. The association of prM with E-protein stabilizes certain pH sensitive epitopes present on E-protein, thereby preventing the conformational changes which occur at acidic pH and are essential for the activation of fusogenic activity of E protein (Allison et al., 1995). Besides its role in viral assembly, the prM protein has also been included in novel recombinant formulations in which it is generally co-expressed with E protein. The resultant E-prM complexes have been shown to be immunogenic and protective, when used in vaccine formulations against challenge with several flaviviruses, including JEV (Mason et al., 1991), yellow fever virus (Pincus et al.,1992), dengue virus (Fonseca et al., 1994) and tick borne encephalitis virus (Heinz et al). Despite the importance of the E-prM complex in flavivirus biology and vaccinology, its structure and biosynthesis are not fully understood, particularly in relation to dengue virus infection. Trypsin cleavage of E-PrM containing virus particles resulted in release of a soluble 45 KDa fragment of Eprotein, which retained the cell-binding activity. The results suggest that E-PrM interaction in dengue virus particles is largely mediated by domains at the carboxy-terminal anchoring region of E-protein, while cell-binding activity is retained in a trypsin-releasable ectodomain (Okamoto et al., 1998). Binding of virus occurs as the result of an adhesion receptor-like interaction between a viral ectodomain molecule and a co-receptor expressed on the surface of target cell. As discussed, the infection of certain cells may involve anti-dengue antibody mediated immune adherence by means of Fc domain of the antibodies bound to virus mediating binding cells such as monocytes that express Fc receptors. However, this mechanism does not explain the primary infection in patient without dengue antibody, or infection of cells lacking Fc receptors. Flaviviruses have relatively simple structure with only a single envelope protein, making it likely that the motif responsible for target cell binding is expressed by the ectodomain of the envelope virus. Vero cells have been used as target as these are derived from dengue virus infection susceptible species and are commonly used for studying in vitro infection by dengue and other viruses. Heparin had potent inhibitory activity with an inhibitory dose of 0.3 g/ml, while doses even upto 30 g/ml of heparan sulfate (HS), other GAGS, and dextran sulfate had no such effect. The heparin-binding activity of the 96

Indian Journal of Clinical Biochemistry, 2005, 20 (2) 92-103 envelope protein has been directly examined by incubating radiolabelled soluble envelope protein with immobilized heparin. It was found that the envelope proteins became bound to heparin and could be eluted with 0.5 M NaCl. When monolayer cultures of Aedes albopictus C6/36 cells (Igarashi, 1978), Vero cells (Yasamura and Kawakita, 1963) and Hep G2 cells (Aden et al., 1979) were inoculated with DEN-4 (814669 strain) and radiolabelled with [ 35 S] methionine, plaque assays showed low levels of DEN4 in the supernatant media taken from all these cells. THE ENVELOPE PROTEIN There are certain domains in envelope protein, which are important for binding to heparan sulfates of glycosaminoglycan family. These GAG-binding motifs have well defined multiple regions that are rich in basic aminoacids and are separated by sequences, which turn in a manner so that the basic regions face each other. Examination of envelope protein sequence of the dengue 2 virus revealed two such putative GAGbinding motifs, between aminoacids 284 and 310 and aminoacids 386 and 411 (C-terminal). As all flavivirus envelope proteins are highly homologous, it is possible that the dengue virus envelope protein structure can be modeled based on TBE (Chen et al., 1997). The susceptibility of a cell to DEN-4 may be determined largely at the stage of virus binding i.e. by presence of a cell surface receptor capable of binding to viral E-protein. Flavivirus entry into permissive cells apparently occurs by an endocytic mechanism following the virus binding, although there are evidences for an alternative plasma membrane penetration process also. The cell-binding event is presumed to involve the viral E glycoprotein, although this has not been directly demonstrated. Consistent with the involvement of E protein in the process of cell entry is the finding of a glycine-rich intra-molecular region (residues 98-111), conserved among all known flaviviral E- proteins, which is possibly involved in low pH catalyzed membrane fusion as well as for a pH dependent conformational change. The observation that closely related cell lines, Vero and LLC-MK2, showed much higher level of E-protein binding than did the other cell lines tested, suggests that they possess high level of the candidate receptor(s) and are good source for its isolation (Anderson et al., 1992). The Eproteins play a role in cellular tropism by being involved in binding of virus to cellular receptors and intra-endosomal acid catalysed fusion (Guirakhoo et al., 1991). Anderson et al. (1992) observed that binding of the E-protein of DEN-4 virus to cell surface receptor(s) correlated with cell susceptibility to virus infection. Further, mutations in this protein affect the virulence of the virus variants, suggesting its participation in virus binding to cellular receptor(s) (Rey et al., 1995). Indian Journal of Clinical Biochemistry, 2005 CELLULAR RECEPTOR(S) FOR DENGUE VIRUS Cellular receptors are main target for the viral infection in human monocytes and mouse neural cells. As the envelope protein causes the attachment of virus to target cell through cellular receptors, the receptors can be purified by affinity chromatography using E- protein as ligand. As E-protein can be obtained only in small quantities from virion, genetic engineering has been used for its production and the recombinant E-protein has been expressed in insect cells using baculovirus expression vector system. Addition of sulfated heparan sulfate prevents E-protein binding to target cells suggesting that heparan sulfate is utilized for viral binding to target cells (Chen et al.,1997). Highly sulfated type of heparan sulfates have been found on the surface of the target cells and their presence has been reported in a variety of human tissues. These heparan sulfate molecules are also present in the liver, which suggests that the liver might be one of the initial sites for dengue infection. Suramin, an effective drug that is used against dengue, mimics the structure of heparan sulfate and inhibits the infection by preventing the binding of dengue virus. Considerable progress has been made in identification of cell receptors for a number of viruses, however, relatively little is known regarding the nature of flavivirus receptors. The attachment is considered as the major determinant of viral host-range and tissue tropism, and diverse cell surface molecules have been identified as virus receptors. Many, if not the majority, of viruses require more than one cell surface molecules to bind and penetrate the cell (Zeng et al., 1996). Whether DEN virus uses a single ubiquitous receptor or several different receptors remains a debatable issue. DEN virus may enter macrophages by receptor-mediated endocytosis or, for Fc receptor bearing cells, by an antibody-dependent uptake mechanism. Daughaday et al. (1981) demonstrated that DEN virus infects human monocytes via a mechanism involving both proteinaceous receptor(s) and trypsin-resistant Fc receptors. The proteinacious nature of cell membrane components required for DEN-1 virus binding was confirmed when treatment with protease at a concentration that do not destroy the viability of the cells, resulted in decreased DEN-1 virus binding to both HepG2 and Vero cells by more than 50%. These results suggested the involvement of proteins in the interaction between DEN-1 virus and the cell surface. This was further confirmed when trypsin-treatment inhibited virus binding to monocytoid cells. Papain and proteinase K had a similar effect on DEN-2 and DEN-3 viruses. Pepsin treatment had no effect on virus binding at physiological pH (BielefeldtOhmann, 1998). In contrast, neuraminidase and phospholipase-C had no significant effect on virus binding (Marianneau et al., 1996). 97

Indian Journal of Clinical Biochemistry, 2005, 20 (2) 92-103 Presence of specific cell surface receptors for dengue virus became evident by experiments involving the infection of cultures of mouse brain cells containing high proportion of either neurons or astrocytes with various strains of dengue virus. The analysis by double immunofluorescence revealed that all the strains of dengue virus were tropic for neurons. Further, the astrocytes were not infected at all suggesting that virus entry requires specific cell receptors present on certain specific cells (the permissible cells) (Imbert et al., 1994). Neurotropism and neurological diseases are common features of flavivirus infection (Monath, 1989, 1990). In humans, dengue virus may eventually cause neurological diseases (Acevedo et al., 1982; Sumarmo et al., 1978; Fraser et al., 1978). In rodents and other biological hosts, all the flaviviruses are neurotropic. Even in the arthropod vectors, the ganglia are major sites for viral replication (Monath, 1989,1990). However, the populations of brain cells that are primarily infected by dengue virus have not been defined, and there is controversy over astrocytological or neural tropism (Bhamarapravati and Halstead, 1964; Craighead et al., 1966; Suri and Banerjee, 1987). Experimental studies on flavivirus infection in mice have shown a relationship between level of viremia, development of brain infection, and widespread appearance of viral antigens in nervous system, supporting the concept of hematogenous spread of the virus into central nervous system (Albrecht, 1968; Kaplan and Koveleski, 1978). A possible interaction of virus with neurotransmitter receptors has been suggested (Monath, 1989). In highly susceptible animals, such as young mice that succumb rapidly to flavivirus infection, inflamatory changes are usually mild, but immunofluorescence and electron microscopy reveal extensive viral replication and ultra-structural cytopathology (Monath, 1990). The main targets for DEN in humans are monocyte-macrophages, B-lymphocytes and bonemarrow cells. Mast cells are also the potential targets for infection in view of its Fc receptor expression (King et al., 2002). The viral envelope protein is 494 amino acid long protein with two potential glycosylation sites (Rey et al., 1995). Early studies described a cell surface protein on human monocytes that is responsible for binding of dengue virus in absence of virus-specific antibodies suggesting that it is different from the Fc receptors (FcR) for immunoglobulins (Daughaday et al., 1981). Chen et al. (1996) using a recombinant dengue virus envelop-Fc fusion protein were unable to detect binding to human monocytes other than via FcR. A series of reports describing dengue virus binding molecules on human, non-human mammalian and insect cell lines have implicated both glycoprotein (Marianneau et al., 1996; Salas-Benito and del Angel, Indian Journal of Clinical Biochemistry, 2005 1997) and glycosaminoglycans (Chen et al., 1997) in virus binding. These studies suggested that the dengue virus binding entities on the cell surface membranes might vary between different cell types. The effect of neuraminidase has given variable results; no discernible effect in some experiments and slight binding-enhancement in some other experiments. However, an inhibition has never been seen. When cells were cultured in the presence of glycosylation inhibitors such as tunicamycin (Elbein, 1984) or swainsonine (Tulsani et al., 1989), a marginal enhancing effect on DEN-2 and DEN-3 virus binding was noted. To investigate the possible involvement of glycosaminoglycans in DEN-2 virus binding to leukocytes, HL60 cells were co-incubated with DEN2 virus and heparin or heparan sulfate in the manner described by Chen et al. (1997) for the Vero cells. A significant inhibition of virus binding to the cells was seen by 10 g/ml of heparin, while an increased dose (100 g/ml) was required for 75-85% inhibition of virus binding. No further inhibition in binding was achieved by even higher doses of heparin. It has been concluded that 15-25% of DEN-2 virus binding to HL60 cells occured regardless of the availability of glycosaminoglycan receptors. It is thought that the initial binding of dengue viruses, and also that of certain other flaviviruses, to cellular receptors is dependent on the dimer-structure of the envelope protein, while the fusion requires a conformational change, mediated by low pH in the immediate vicinity of the cell membrane and resulting in formation of a trimer. To explore the effect of conformational changes to the envelope protein, dengue-2 virus was incubated at pH 5, followed by reconstitution to neutral pH. This treatment resulted in complete inhibition of infectivity of the virus and more than 60% reduction in its binding to HL60 cells, as assessed by flow cytometry (Martinez-Barragan et al., 2001). Interferon- has been shown to modulate the expression of a range of leukocyte surface molecules (usually an up-regulation). The effect of IFN- on dengue virus infection remains controversial. Fc receptor-mediated dengue virus infection of macrophage cell line U937 was reported to be stimulated, while IFN- inhibited the ab-independent and ab-dependent dengue virus infection of blood monocytes (Bielefeldt-Ohmann; 1998). The correlation between the degree of E-protein binding and the cell susceptibility of various cell lines to viral infection (Anderson et al., 1992) and the glycine-rich conserved internal element that is involved in low pH-catalyzed membrane fusion and pHdependent conformational changes confirm that Eprotein is the Viral attachment protein (VAP). Further, the binding motif for a recombinant envelope protein that inhibits infection of Vero cells by dengue virus has 98

Indian Journal of Clinical Biochemistry, 2005, 20 (2) 92-103 been mapped to be present between aminoacids 281 and 423 (Chen et al., 1996). Though its cellular counter part (the cellular receptor) needs further characterization (Salas-Benito et al., 1997). Virus overlay protein binding assays (VOPBA) with labeled virus have identified two glycoproteins of 40 KDa and 45 KDa that are located on the surface of C6/36 cells and are involved in binding of DEN 4 virus. Preincubation of C6/36 cells with specific antibodies raised against these proteins inhibited virus binding suggesting that 40 KDa and 45 KDa surface proteins are putative receptors or part of a receptor complex for dengue virus. The two proteins do not appear to be subunits of a dimer complex as in presence or absence of -mercaptoethanol both protein bands show the same molecular weight in VOPBA. Moreover, dengue virus binding does not require folding and seems to be independent of disulfide bridges (SalasBenito and del Angel; 1997). CURRENT STATUS Although some work has been carried out on DEN virus binding, the initial events of flavivirus infection have not been analysed in detail and relatively little is known about the molecular basis of the binding of flavivirus to their target cells. Many viruses, including dengue virus use heparan sulfate (the most ubiquitous member of the glycosaminoglycan family) to bind to target cells. HS could act directly as a receptor or help to concentrate these viruses on the cell surface to facilitate the interaction with specific, high affinity receptors (Germi et al., 2001). Currently, the candidate receptor molecules include proteins, Fc receptors, GAGS, and lipopolysaccharide binding CD14associated molecules (Bielefeldt-Ohmann et al., 2001). Molecules other than HS that could participate in infection of Vero cells by dengue virus have also been suggested. Labeled DEN 4 virus binds to two molecules of 74 KDa and 44 KDa with high affinity. Binding of dengue virus to 74 KDa molecule was found to be susceptible to proteases and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin, prevented dengue virus binding to both 74 KDa and 44 KDa proteins in overlay assays. However, phytohemagglutinin P did not affect the binding, suggesting that carbohydrate residues (-mannose or N-acetyl glucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immuno-fluorescence with a polyclonal antibody raised against the 74 KDa protein that was able to inhibit dengue virus infection, suggest that HS might serve as primary receptor, and may probably be responsible for concentrating virus particles on the surface of Vero cells. Other molecules such as the 74KDa protein might participate as co-receptors in viral penetration. Indian Journal of Clinical Biochemistry, 2005 The 74KDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells (Martinez-Barragan and del Angel, 2001). The endothelial cell line ECV304, derived from human umbilical cord was identified to be susceptible to DEN2 infection and was used to study the molecular mechanism of DEN-2 binding to endothelial cells (Bonner and O Sullivan, 1998; Killen and O Sullivan, 1993). When VOPBA was employed, it was found that DEN-2 binds to three membrane proteins (29, 34 and 43 KDa respectively), of ECV304 cells (Wei et al., 2003). These proteins were also identified by using radiolabelled viruses. Two of these proteins could be digested with trypsin while the 29 KDa protein is resistant to trypsin. A modified micro-plaque assay has been developed to determine the virus titres. It has been shown that DEN 2 could replicate efficiently and mature in ECV304 cells and that this cell line could be used as a model cell line for DEN-2 binding assays (Wei et al., 2003). The 3D structure of the envelope glycoprotein has highlighted the possible regions of Eprotein that can be targeted to block viral entry. This information has been used in designing potent immunogens that protect against disease. The design of small molecules capable of inhibiting entry of pathogenic microbes is appealing, as such drugs will not have to gain access to the interior of cells and are therefore expected to display increased bioavailability. It has also been pointed out that the presence of mannose residues is important for viral entry (Hung et al., 1999). Further, reports show that mosquito-grown dengue virus relies on interactions with the C-type lectin DC-SIGN to efficiently enter inside the cells (Navarro-Sanchez et al., 2003; Tassaneetrithep et al., 2003). Like all flaviviruses, dengue virus requires a replication step in the arthropod vector before its transmission in humans. In a natural infection, an infected mosquito into the skin deposits the viruses during a blood meal. Dendritic cells are normal residents of the skin and express DC-SIGN at their surface. This lectin is specific for the high-mannosetype carbohydrates that are present on virions produced in insect cells. Recently it has been seen that the two putative N-linked glycosylation sites are present in the dengue virus E-protein and both used specifically for recognition by DC-SIGN. The site corresponding to Asn-153 is conserved among many flaviviruses, whereas the Asn-67 site is unique to dengue viruses (Navarro-Sanchez et al., 2003), suggesting that this tetrameric lectin may need to interact with more than one sugar at the same time for efficient internalization of the virus. ACKNOWLEDGEMENT The financial assistance to Seema from UGC in form of a Junior Research Fellowship is gratefully acknowledged. 99

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