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The Secreted Form of Dengue Virus Nonstructural Protein NS1 Is Endocytosed by Hepatocytes and Accumulates in Late Endosomes: Implications for Viral Infectivity

Sophie Alcon-LePoder,^1, Marie-Thérèse Drouet,¹ Pascal Roux,² Marie-Pascale Frenkiel,¹ Michel Arborio,³ Anne-Marie Durand-Schneider,⁴ Michèle Maurice,⁴ Isabelle Le Blanc,⁵ Jean Gruenberg,⁵ and Marie Flamand^1*

U.P. Interactions Moléculaires Flavivirus-Hôtes, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France,¹ Centre d'Imagerie Dynamique, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France,² Service d'Anatomo-pathologie, Hôpital d'Instruction des Armées du Val de Grce, 74 Boulevard de Port Royal, 75005 Paris, France,³ INSERM U 538, Faculté de Médecine de Saint Antoine, 27 rue de Chaligny, 75012 Paris, France,⁴ Department of Biochemistry, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva 4, Switzerland⁵

Received 11 November 2004/ Accepted 27 May 2005

The flavivirus nonstructural protein NS1 is expressed as three discrete species in infected mammalian cells: an intracellular, membrane-associated form essential for viral replication, a cell surface-associated form that may be involved in signal transduction, and a secreted form (sNS1), the biological properties of which remain elusive. To determine the distribution of the dengue virus (DEN) sNS1 protein in vivo, we have analyzed by immunohistological means the tissue tropism of purified DEN sNS1 injected intravenously into adult mice. The sNS1 protein was found predominantly associated with the liver, where hepatocytes appeared to represent a major target cell. We further showed that sNS1 could be efficiently endocytosed by human Huh7 and HepG2 hepatocytes in vitro. After its internalization, the protein was detected intracellularly for at least 48 h without being substantially degraded. Colocalization studies of sNS1 with markers of the endolysosomal compartments revealed that the protein was specifically targeted to lysobisphosphatidic acid-rich structures reminiscent of late endosomes, as confirmed by electron microscopy. Intracellular accumulation of sNS1 in Huh7 cells enhanced the fluid phase uptake of rhodamine-labeled dextran. Furthermore, preincubation of Huh7 cells with sNS1 increased dengue virus production after infection with the homologous strain of DEN-1 virus. Our results demonstrate that the accumulation of DEN sNS1 in the late endosomal compartment of hepatocytes potentializes subsequent dengue virus infection in vitro, raising the possibility that sNS1 may contribute to viral propagation in vivo.

Supplemental material for this article may be found at http://jvi.asm.org.

Present address: UMR 1161, Virologie, Ecole Nationale Vétérinaire d'Alfort, 7 Avenue du Général de Gaulle, 94704 Maisons-Alfort, France.

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