The secreted form of dengue virus nonstructural protein NS1 is endocytosed by hepatocytes and accumulates in late endosomes: implications for viral infectivity

Abstract

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.

FIG. 1.

Detection of the DEN sNS1 protein in mouse liver. Mice were injected intravenously with purified DEN-1 sNS1 (25 μg) or PBS and sacrificed at 1, 3, 6, or 24 h postinjection. Histological sections of paraffin-embedded livers were immunostained with purified NS1-specific polyclonal rabbit antibodies and a fluorescein isothiocyanate-labeled secondary antibody. Cell nuclei were counterstained with propidium iodide and samples were analyzed by confocal microscopy (63× magnification). sNS1 localized in punctate cytoplasmic structures (green). Bar, 10 μm.

FIG. 2.

DEN sNS1 protein is efficiently internalized in human hepatocytes. Subconfluent Huh7 cells were incubated for 6 h with DEN-1 sNS1 (10 μg/ml) (B to D) or PBS (A). Cells were fixed directly (B) or after 24 h or 48 h of chase (C, D). sNS1 was detected by immunofluorescence using specific rabbit polyclonal antibodies and a cyanin-3-labeled secondary antibody. Bars, 10 μm.

FIG. 3.

Stability of the internalized sNS1 protein in human hepatocytes. Huh7 cells were incubated for 6 h with DEN-1 sNS1 (10 μg/ml) or PBS, and lysed directly (6 h) or after 24 h or 48 h of chase (6 h/24 h and 6 h/48 h, respectively). Proteins were separated by SDS-PAGE and analyzed by immunoblotting. The internalized sNS1 protein was probed using a mix of specific monoclonal antibodies and an alkaline phosphatase-conjugated secondary antibody. Samples were unheated, except for a control in which purified sNS1 was heated for 3 min at 95°C to convert the dimeric subunit into monomers.

FIG. 4.

sNS1 protein accumulates in LBPA-rich structures. Huh7 cells were incubated for 6 h with DEN-1 sNS1 (10 μg/ml) or PBS, followed by 24 h or 48 h of chase (6 h/24 h and 6 h/48 h, respectively). Intracellular localization of sNS1 was analyzed by immunofluorescence and confocal microscopy. The sNS1 protein was detected using NS1-specific rabbit polyclonal antibodies and a cyanin-3-conjugated secondary antibody (red). Late endosomes were labeled using a lysobisphosphatidic acid (LBPA)-specific mouse monoclonal antibody and a fluorescein isothiocyanate-conjugated secondary antibody (green). Colocalization of sNS1 and LBPA is revealed on the merged images (yellow). Bars, 5 μm.

FIG. 5.

Ultrastructural morphology of sNS1-positive structures in human hepatocytes. Huh7 cells were incubated with HRP-labeled DEN-1 sNS1 (10 μg/ml) for 6 h followed by a 24-h chase (A) or for 48 h without chase (B to F). sNS1-positive structures stained with DAB are revealed by electron microscopy as dense cytoplasmic vacuoles 0.5 to 1 μm in diameter, some of which contained distinct internal vesicular bodies. Connection of an NS1-positive vacuole to a nonlabeled tubule was occasionally observed (arrow). Bars, 500 nm.

FIG. 6.

sNS1 protein increases the endocytic activity of human hepatocytes. (A) Huh7 cells were incubated for 48 h with PBS (a and c) or DEN-1 sNS1 (10 μg/ml) (b and d) and further incubated for 6 h with PBS (a and b) or tetramethylrhodamine-labeled dextran (Rhod-dextran, 2 mg/ml) (c and d). Cells were fixed and stained with anti-NS1 rabbit polyclonal antibodies (a and b) or untreated (c and d) and observed by confocal microscopy. (B) Rhodamine-labeled dextran signals were quantified, and control values from PBS-treated cell samples were arbitrarily set to 1. Means and standard deviation from two independent experiments are shown.

FIG. 7.

sNS1 protein enhances dengue virus production by human hepatocytes. Huh7 cells were incubated for 24 h with DEN-1 sNS1 (10 μg/ml) or PBS, and infected for 2 h with the homologous DEN-1 FGA/89 virus at a multiplicity of infection of 2, 10, or 50. (A) Cell supernatants were harvested at 24 h postinfection and DEN-1 virus titers were determined by focus-forming assay in AP61 cells. (B) Ratios of DEN-1 viral titers from infected Huh7 cells treated with sNS1 versus PBS. Means and standard deviations of duplicate experiments are shown.