Title: The role of DC-SIGN and the mode of action of entry inhibitors in dengue virus infection
Other Titles: De rol van DC-SIGN en het werkingsmechanisme van opname-inhibitoren in dengue virus infectie
Authors: Alen, Marijke
Issue Date: 12-Jun-2012
Abstract: Dengue virus can be considered as the most important emerging mosquito-borne virus in the world. This RNA virus is only transmitted by a vector, the Aedes aegypti and the Aedes albopictus mosquito. Due to a lack of vector control, massive urbanization and deforestation, global warming and an increase in the international transport, the virus and its vector is spread all over the world. Nowadays, 3.6 billion people are at risk of getting infected with dengue virus (DENV) of which 50 to 100 million people become infected every year. There is an urgent need for a vaccine or antiviral therapy, unfortunately, until today, no vaccine or antiviral compound is available. During the viral replication cycle following a mosquito bite, every step can be a target for antiviral therapy. Our research is focused on the inhibition of the first step, namely the initial viral attachment to the host cell. In the skin, dendritic cells (DC) are present and they capture the virus via DC-SIGN. Next, the virus escapes lysosomal degradation and can be transported to the secondary lymphoid organs to activate the immune system. In the first Chapter, we investigated the role of DC-SIGN in DENV infections and found that DC-SIGN renders unsusceptible cells susceptible for DENV infection. We further optimized cellular assays and read-out of infections by flow cytometry using specific DENV antibodies. Previously, DC-SIGN has been shown to capture HIV and subsequently transmit the virus to CD4+ T-cells. The interaction between HIV and DC-SIGN could be interrupted with carbohydrate-binding agents (CBAs) specifically recognizing the N-glycans present on the viral surface. The aim of our study was to evaluate the antiviral profile of several CBAs, mainly plant lectins, in DENV infection. We demonstrated a dose-dependent antiviral activity of three purified plant lectins Hippeastrum hybrid agglutinin (HHA), Galanthus nivalis agglutinin (GNA) and Urtica dioica agglutinin (UDA), against DENV infection in Raji/DC-SIGN+ cells.In the second Chapter, we focused on the use of monocyte-derived DC (MDDC), to mimic as much as possible a natural infection process. We optimized the isolation and differentiation of MDDC from fresh donor blood by analyzing the expression of several cellular markers by flow cytometry. After differentiation, MDDC highly express DC-SIGN and the cells were found susceptible for DENV infection. The use of MDDC to study the antiviral potency of the CBAs against DENV has much more clinical relevance then using laboratory-adapted tumor cell lines. We were able to demonstrate a consistent, serotype- and cell type-independent antiviral activity of the three plant lectins, HHA, GNA and UDA against DENV. In addition, we investigated in more detail the mechanism of action in binding assays in Raji/DC-SIGN+ cells. These data indicated that the CBAs did not bind to cellular membrane proteins but specifically interact with the viral envelope proteins. Moreover, following DENV infection in MDDC, the maturation process initiated by DENV was characterized by a downregulation of DC-SIGN and an upregulation of the costimulatory molecules CD80 and CD86. The CBAs were also able to inhibit these DENV induced maturation processes. In the third Chapter, we characterized the molecular target of the CBAs on DENV. We were able to generate a resistant DENV to HHA in C6/36 mosquito cells. After genotype and phenotype characterization we identified two specific mutations in the viral E-protein: N67D and T155I. These mutations resulted in a unique DENV lacking both N-glycosylation sites which was still able to replicate efficiently in insect and mammalian cells. Remarkably, the HHAres DENV was not able anymore to infect DC-SIGN+ cells, such as Raji/DC-SIGN+ cells or MDDC. These data indicate that DENV needs N-glycans on the viral envelope to infect the first target cells in the human body. We further suggested that targeting the N-glycans is a promising target for antiviral therapy. Finally, in Chapter 4, we investigated the antiviral activity of a new class of lantibiotics, the labyrinthopeptins. Previously, it was shown that the labyrinthopeptins exhibit antiviral activity against HSV and HIV and here we evaluated the antiviral potency against DENV. We demonstrated a broad spectrum dose-dependent and cell type-independent antiviral activity of the labyrinthopeptins against DENV. A time of drug-addition assay revealed that, comparable to the CBAs, the labyrinthopeptins do not interact with cellular proteins, but specifically interact with virus envelope proteins and subsequently prevent viral entry. In addition, we set up a fusion assay in the mosquito cell line C6/36 as DENV-infected C6/36 cells can form syncytia in the presence of low pH medium. We proved that the labyrinthopeptins, comparable to HHA, could prevent viral fusion or syncytia formation. This also indicates that the labyrinthopeptins interact with the viral E-protein. The fact that the labyrinthopeptins could still inhibit HHAres DENV, lacking both N-glycans, indicates that these compounds do not target the N-glycans, but specifically interact with the fusion peptide of the E-protein. Moreover, we analyzed the effect of the CBAs and the labyrinthopeptins on the induction of several cytokines and chemokines in DENV infected MDDC. We found that RANTES, MIP-1α, MIP-1ß and TNF-α, which are important in the DENV pathogenesis, are elevated in DENV infected MDDC cultures and this induction can efficiently be inhibited with the CBAs and the labyrinthopeptins. These data indicate that the entry inhibitors, such as CBAs and labyrinthopeptins, are both valuable as novel anti-DENV agents to elucidate the viral entry process of DENV into the host cell and to help future development of better and more specific and potent DENV entry inhibitors.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Laboratory of Virology and Chemotherapy (Rega Institute)

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