Marijke Alen

Antiviral activity of carbohydrate-binding agents and the role of DC-SIGN in dengue virus infection

Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) is an important binding receptor for dengue virus (DENV) that recognizes N-glycosylation sites on the viral E-glycoprotein. DENV cannot bind nor infect the human B-cell line Raji/0. However, DENV productively infects Raji/DC-SIGN(+) cells that constitutively express DC-SIGN on their surface. IL-4-treated monocytes, expressing high levels of DC-SIGN, are also susceptible for DENV infection. Several carbohydrate-binding agents (CBAs), such as the plant lectins HHA, GNA (mannose-specific) and UDA (N-acetylglucosamine-specific), inhibited dose-dependently the binding of DENV and subsequently viral replication in Raji/DC-SIGN(+) cells (EC(50): 0.1-2.2 microM). These CBAs were clearly more active against DENV in IL-4-treated monocytes (EC(50): 4-56 nM). However, the CBAs were devoid of antiviral activity in DENV-susceptible Vero-B (DC-SIGN(-)) cells, demonstrating cell type-dependent differences in viral entry mechanisms.

A derivate of the antibiotic doxorubicin is a selective inhibitor of dengue and yellow fever virus replication in vitro.

ABSTRACT A doxorubicin derivate, SA-17, that carries a squaric acid amide ester moiety at the carbohydrate (α-l-daunosaminyl) group was identified as a selective inhibitor of in vitro dengue virus (DENV) serotype 2 replication (50% effective concentration [EC50] = 0.34 ± 0.20 μg/ml [0.52 ± 0.31 μM]). SA-17 is markedly less cytostatic than the parent compound, resulting in a selectivity index value of ∼100. SA-17 also inhibits yellow fever virus 17D (YFV-17D) replication (EC50 = 3.1 ± 1.0 μg/ml [4.8 ± 1.5 μM]), although less efficiently than DENV replication, but proved inactive against a variety of enveloped and nonenveloped viruses. SA-17 inhibits in vitro flavivirus replication in a dose-dependent manner, as was assessed by virus yield reduction assays and quantification of viral RNA by means of real-time quantitative reverse transcriptase PCR (RT-qPCR) (∼2 to 3 log reduction). The anti-DENV activity was confirmed using a Renilla luciferase-expressing dengue reporter virus. Time-of-drug-addition studies revealed that SA-17 acts at the very early stages of the viral replication cycle (i.e., virus attachment and/or virus entry). This observation was corroborated by the observation that SA-17, unlike the nucleoside analogue ribavirin, does not inhibit the replication of DENV subgenomic replicons. Preincubation of high-titer stocks of DENV or YFV-17D with ≥5 μg/ml SA-17 resulted in 100% inhibition of viral infectivity (≥3 log reduction). SA-17, however, did not prove virucidal.

Broad antiviral activity of carbohydrate-binding agents against the four serotypes of dengue virus in monocyte-derived dendritic cells.

Background Dendritic cells (DC), present in the skin, are the first target cells of dengue virus (DENV). Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) is present on DC and recognizes N-glycosylation sites on the E-glycoprotein of DENV. Thus, the DC-SIGN/E-glycoprotein interaction can be considered as an important target for inhibitors of viral replication. We evaluated various carbohydrate-binding agents (CBAs) against all four described serotypes of DENV replication in Raji/DC-SIGN+ cells and in monocyte-derived DC (MDDC). Methodology/Principal Findings A dose-dependent anti-DENV activity of the CBAs Hippeastrum hybrid (HHA), Galanthus nivalis (GNA) and Urtica dioica (UDA), but not actinohivin (AH) was observed against all four DENV serotypes as analyzed by flow cytometry making use of anti-DENV antibodies. Remarkably, the potency of the CBAs against DENV in MDDC cultures was significantly higher (up to 100-fold) than in Raji/DC-SIGN+ cells. Pradimicin-S (PRM-S), a small-size non-peptidic CBA, exerted antiviral activity in MDDC but not in Raji/DC-SIGN+ cells. The CBAs act at an early step of DENV infection as they bind to the viral envelope of DENV and subsequently prevent virus attachment. Only weak antiviral activity of the CBAs was detected when administered after the virus attachment step. The CBAs were also able to completely prevent the cellular activation and differentiation process of MDDC induced upon DENV infection. Conclusions/Significance The CBAs exerted broad spectrum antiviral activity against the four DENV serotypes, laboratory-adapted viruses and low passage clinical isolates, evaluated in Raji/DC-SIGN+ cells and in primary MDDC.

Sulfated Escherichia coli K5 polysaccharide derivatives inhibit dengue virus infection of human microvascular endothelial cells by interacting with the viral envelope protein E domain III.

Dengue virus (DENV) is an emerging mosquito-borne pathogen that causes cytokine-mediated alterations in the barrier function of the microvascular endothelium, leading to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). We observed that DENV (serotype 2) productively infects primary (HMVEC-d) and immortalized (HMEC-1) human dermal microvascular endothelial cells, despite the absence of well-described DENV receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) or the mannose receptor on the cell surface. However, heparan sulfate proteoglycans (HSPGs) were highly expressed on these cells and pre-treatment of HMEC-1 cells with heparinase II or with glycosaminoglycans reduced DENV infectivity up to 90%, suggesting that DENV uses HSPGs as attachment receptor on microvascular endothelial cells. Sulfated Escherichia coli K5 derivatives, which are structurally similar to heparin/heparan sulfate but lack anticoagulant activity, were able to block DENV infection of HMEC-1 and HMVEC-d cells in the nanomolar range. The highly sulfated K5-OS(H) and K5-N,OS(H) inhibited virus attachment and subsequent entry into microvascular endothelial cells by interacting with the viral envelope (E) protein, as shown by surface plasmon resonance (SPR) analysis using the receptor-binding domain III of the E protein.

Crucial role of the N-glycans on the viral E-envelope glycoprotein in DC-SIGN-mediated dengue virus infection.

We generated in the mosquito cell line C6/36 a dengue virus (DENV) resistant to Hippeastrum hybrid agglutinin (HHA), a carbohydrate-binding agent (CBA). The genotype and phenotype were characterized of the HHA resistant (HHA(res)) DENV compared to the wild-type (WT) DENV. Sequencing the structural proteins of HHA(res) resulted in two mutations, N67D and T155I, indicating a deletion of both N-glycosylation sites on the viral envelope E-glycoprotein. The HHA(res) DENV could replicate in mammalian and mosquito cells that are lacking dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) expression. In contrast, DC-SIGN expressing human cells namely monocyte-derived dendritic cells as well as DC-SIGN-transfected cells were no longer susceptible to HHA(res) DENV. This demonstrates a crucial role of the N-glycans in the E-glycoprotein in the infection of dendritic cells, which constitute primary target cells of DENV during viral pathogenesis in the human body.

Broad Antiviral Activity of Carbohydrate-Binding Agents Against Dengue Virus Infection

© 2012 Schols and Alen, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Broad Antiviral Activity of Carbohydrate-Binding Agents Against Dengue Virus Infection