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JOURNAL OF VIROLOGY, Apr. 2005, p. 4557–4567 0022-538X/05/$08.00 0 doi:10.1128/JVI.79.8.4557–4567.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 79, No. 8

Heat Shock Protein 90 and Heat Shock Protein 70 Are Components of Dengue Virus Receptor Complex in Human Cells
Jorge Reyes-del Valle, Salvador Chavez-Salinas, Fernando Medina, and Rosa M. del Angel* ´Departamento de Patologı Experimental, Centro de Investigacion y de Estudios Avanzados del IPN, Mexico City, Mexico ´a ´
Received 8 July 2004/Accepted 3 December 2004

Dengue virus requires the presence of an unidentified cellular receptor on the surface of the host cell. By using a recently published affinity chromatography approach, an 84-kDa molecule, identified as heat shock protein 90(HSP90) by matrix-assisted laser desorption ionization–time of flight mass spectrometry, was isolated from neuroblastoma and U937 cells. Based on the ability of HSP90 (84 kDa) to interact with HSP70 (74 kDa) on the surface of monocytes during lipopolysaccharide (LPS) signaling and evidence that LPS inhibits dengue virus infection, the presence of HSP70 was demonstrated in affinity chromatography eluatesand by pull-down experiments. Infection inhibition assays support the conclusion that HSP90 and HSP70 participate in dengue virus entry as a receptor complex in human cell lines as well as in monocytes/macrophages. Additionally, our results indicate that both HSPs are associated with membrane microdomains (lipid rafts) in response to dengue virus infection. Moreover, methyl- -cyclodextrin, araft-disrupting drug, inhibits dengue virus infection, supporting the idea that cholesterol-rich membrane fractions are important in dengue virus entry.

Dengue (DEN) virus, the most important arthropod-borne human pathogen, represents a serious public health threat. DEN virus is transmitted to humans by the bite of the domestic mosquito, Aedes aegypti, and circulates in nature as four distinctserological types (DEN-1 to -4). DEN virus has been recognized in over 100 countries, and 2.5 billion people live in areas where DEN virus is endemic (16). The clinical manifestations of DEN virus infection range in severity from a simple self-limited febrile illness known as dengue fever to a hemorrhagic fever (DHF) and potentially fatal hemorrhagic shock syndrome. Each year, more than 50 millioncases of dengue fever and several hundred thousand cases of DHF occur. During the past 8 years the incidence of dengue has grown in areas of endemicity, particularly in the American region. A specific treatment or vaccine is not yet available. DEN virus is an enveloped virus that belongs to the Flaviviridae family. Mature virions are icosahedral, 50 nm in diameter, and contain a single-strand andpositive-polarity RNA as genome of about 10.7 kb (21). The DEN virus genome encodes three structural proteins (envelope glycoprotein, E; membrane, M; and capsid, C) and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). E protein is the major structural protein exposed on the surface of the particle, and it arrays in homodimers, parallel to viral surface. Recently, the structureof DEN virus E protein has been determined by X-ray crystallography (24). Each monomer consists of three domains: the structurally central amino-terminal domain I that organizes the structure; the dimerization domain II that contains the hydrophobic fusion peptide essential for virus-cell fusion; and finally the carboxy-terminal immunoglob* Corresponding author. Mailing address: Departamento dePatologı Experimental, Centro de Investigacion y de Estudios Avanzados ´a ´ del IPN, Av. I.P.N. 2508, Col. San Pedro Zacatenco, Mexico City D.F., C.P. 07360, Mexico. Phone: (5255) 5061-3800, ext. 5647 or 3345. Fax: (5255) 5747-7107. E-mail: rmangel@mail.cinvestav.mx. 4557

ulin (Ig)-like domain III, which has been proposed to function as the binding site for cellular receptors (11, 22). The first...