Virus Research 137 (2008) 225–234 Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Characterization of the interaction of domain III of the envelope protein of dengue virus with putative receptors from CHO cells Vivian Huerta ∗ , Glay Chinea, Noralvis Fleitas, Mónica Sarría, Jorge Sánchez, Patricia Toledo, Gabriel Padrón Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Habana 10600, Cuba article info Article history: Received 13 March 2008 Received in revised form 4 July 2008 Accepted 16 July 2008 Available online 21 September 2008 Keywords: Dengue Domain III Receptor Residue conservation abstract Domain III (DIII) of the envelope protein of dengue virus (DENV) contains structural determinants for the interaction with cellular receptors. In the present study a solid phase assay and recombinant fusion pro- teins containing DENV-DIII of serotypes 1 and 2 were used to study structural features of the interaction of the envelope protein with putative receptors present in the microsomal fraction of CHO cells. Recombi- nant fusion proteins showed specific interaction with proteins present in the microsomal fraction. Binding of the fusion proteins across the pH range of 5.5–8.0 resembled that of virus particles, peaking at pH 6.0. This suggests that the interaction of DIII with cell receptor(s) is strengthened at endosomal pH. The effect of reduction and carbamidomethylation of cysteine residues on the binding to the microsomal fraction and in their recognition by antibodies suggests that the region of DIII that is interacting with putative receptor(s) overlaps only partially with a dominant epitope of the antibody response. The analysis of the residue conservation profile indicates that the surface of DIII is composed typically of specific sub- complex residues with an increased representation of specific type/subtype residues found at the surface that closely correlates with the dominant neutralizing epitope. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Dengue virus (DENV) is currently one of the most important Flaviviruses in terms of incidence and people at risk (Mackenzie et al., 2004; Pugachev et al., 2005). The DENV complex consists of four closely related, but antigenically distinct serotypes: DENV1, DENV2, DENV3, and DENV4. The clinical expressions of the infection with any of the four DENV serotypes range from asymptomatic, or a mild self-limited illness, called dengue fever; to a severe and potentially life-threatening disease, dengue hemorrhagic fever (Mackenzie et al., 2004). In spite of a considerable amount of research and the development of several promising strategies, there is, as yet, no effective vaccine to protect from DENV infection (Pugachev et al., 2005; Stephenson, 2005). Neither is there a specific antiviral drug to treat the DENV-caused disease. DENV enters mammalian cells through receptor-mediated endocytosis (Barth, 1992; Krishnan et al., 2007). Cumulative results from studies on DENV receptors suggest that the virus is using a ∗ Corresponding author at: Physical Chemistry Division, Center for Genetic Engi- neering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba. Tel.: +53 7 271 6022; fax: +53 7 271 4764. E-mail address: Vivian.huerta@cigb.edu.cu (V. Huerta). multi-molecular complex where some molecules play the role of primary receptors concentrating virus particles on the cell surface and facilitating a subsequent interaction with an endocytic receptor (Halstead, 2003; Clyde et al., 2006). Experimental results have also indicated that the DENV complex could be using different proteins for cell attachment and entry, depending on the cell type (Thepparit and Smith, 2004; Jindadamrongwech et al., 2004; Reyes-Del Valle et al., 2005). Highly sulfated heparan sulfates seem to function as primary receptors in Vero, CHO, BHK-21 and several other cell lines of mam- malian origin (Chen et al., 1997; Hung et al., 1999; Germi et al., 2002; Lin et al., 2002). In dendritic cells this role corresponds to DC-SIGN, a C-type lectin which interacts very efficiently with mosquito-cell-derived glycosides of DENV (Navarro-Sanchez et al., 2003; Tassaneetrithep et al., 2003). In cells of the immune system, Fc receptors could be functioning as primary receptors concentrat- ing antibody–virus complexes on the surface while the productive infection is mediated by a different endocytic receptor (Halstead, 2003). On the virus, the envelope (E) protein is responsible for the interaction with cellular receptors (Chen et al., 1996). The E pro- tein forms a homodimer in the mature virion and each monomer is composed of three structural domains. Domain III (DIII), of the E protein of DENV, forms an Ig-like  barrel and contains one 0168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2008.07.022