Heparan sulfate proteoglycans initiate dengue virus infection of hepatocytes

Dengue viruses (DEN) cause a broad spectrum of clinical manifestations incl uding potentially life-threatening conditions such as hemorrhagic shock syn drome and less frequently acute hepatitis with liver failure and encephalop athy. In addition, dengue viruses provide a potential model to understand t he initiation of hepatocyte infection by the structurally closely related h epatitis C virus (HCV), because this virus at present cannot be grown in ce ll culture. Although the initial steps of viral infection are a critical de terminant of tissue tropism and therefore pathogenesis, little is known abo ut the molecular basis of binding and endocytic trafficking of DEN or of an y other flavivirus. Our studies revealed that binding of radiolabeled DEN t o the human hepatoma cell line HuH-7 was strictly pH dependent and substant ially inhibitable by the glycosaminoglycan heparin. ligand-blot analysis, p erformed as a viral overlay assay, showed two heparan sulfate (HS) containi ng cell-surface binding proteins resolving at 33 and 37 kd, Based on the se nsitivity of unprotected virus and the viral binding site on the cell surfa ce to trypsin, viral internalization was quantified as an increase in tryps in protected virus over time. Virus trafficking to the site of degradation was inhibited by pH dissociation of the clathrin coat and dependent on IP3- mediated homotypic endosomal fusion, These findings confirm the hypothesis that binding and internalization of DEN by hepatocytes are mediated primari ly by IIS containing proteoglycans and suggest that flaviviruses traffic th e major clathrin-dependent endocytic pathway during infection.