Dengue Virus NS1 Uses Scavenger Receptor B1 as a Cell Receptor in Cultured Cells

Abstract

The dengue virus NS1 is a multifunctional protein that forms part of replication complexes. NS1 is also secreted, as a hexamer, to the extracellular milieu. Circulating NS1 has been associated with dengue pathogenesis by several mechanisms. Cell binding and internalization of soluble NS1 result in endothelial hyperpermeability and in the downregulation of the innate immune response. In this work, we report that the HDL scavenger receptor B1 (SRB1) in human hepatic cells and a scavenger receptor B1-like in mosquito C6/36 cells act as cell surface binding receptors for dengue virus NS1. The presence of the SRB1 on the plasma membrane of C6/36 cells, as well as in Huh7 cells, was demonstrated by confocal microscopy. The internalization of NS1 can be efficiently blocked by anti-SRB1 antibodies, and previous incubation of the cells with HDL significantly reduces NS1 internalization. Significant reduction in NS1 internalization was observed in C6/36 cells transfected with siRNAs specific for SRB1. In addition, the transient expression of SRB1 in Vero cells, which lacks the receptor, allows NS1 internalization in these cells. Direct interaction between soluble NS1 and the SRB1 in Huh7 and C6/36 cells was demonstrated in situ by proximity ligation assays and in vitro by surface plasmon resonance. Finally, results are presented indicating that the SRB1 also acts as a cell receptor for Zika virus NS1. These results demonstrate that dengue virus NS1, a bona fide lipoprotein, usurps the HDL receptor for cell entry and offers explanations for the altered serum lipoprotein homeostasis observed in dengue patients. IMPORTANCE Dengue is the most common viral disease transmitted to humans by mosquitoes. The dengue virus NS1 is a multifunctional glycoprotein necessary for viral replication. NS1 is also secreted as a hexameric lipoprotein and circulates in high concentrations in the sera of patients. Circulating NS1 has been associated with dengue pathogenesis by several mechanisms, including favoring of virus replication in hepatocytes and dendritic cells and disruption of the endothelial glycocalyx leading to hyperpermeability. Those last actions require NS1 internalization. Here, we identify the scavenger cell receptor B1, as the cell-binding receptor for dengue and Zika virus NS1, in cultured liver and in mosquito cells. The results indicate that flavivirus NS1, a bona fide lipoprotein, usurps the human HDL receptor and may offer explanations for the alterations in serum lipoprotein homeostasis observed in dengue patients.

Keywords: NS1; Zika virus; dengue; dengue virus; scavenger receptor B1.

FIG 1

Detection of the scavenger receptor B1 (SRB1) in liver, mosquito, and kidney derived cell lines. (A) Confluent cell monolayers were fixed and stained for SRB1 (red) and nuclei counterstained with DAPI (blue). Bars = 10 μm. (B) The presence of the SRB1 on the cell plasma membrane was corroborated by total internal reflection fluorescence (TIRF) microscopy, containing cells for SRB1 (red) and wheat germ agglutinin (green) to label the cell membrane.

FIG 2

Internalization of DENV and ZIKV rNS1 in Huh7 (A), C6/36 (B), and Vero cells (C). Cells were preincubated with recombinant NS1 (3.5 μg/well), and at the indicated times cells were washed, fixed, permeabilized, and stained for intracellular NS1 (red). Cell nuclei were counterstained with DAPI. Panels A.1 and B.1 correspond to cells incubated with heat denatured NS1. Bars = 10 μm. (D, E). Quantification of the NS1 signal. Results are the mean ± SD of at least 100 different cells per condition. (F) DENV and (G) ZIKV titers after C6/36 cells preincubation or not with recombinant NS1 (light gray bars) or heat denatured NS1 (dark gray bars). Monolayers of C6/36 cells were preincubated for 1.5 h with the indicated concentrations of either DENV or ZIKV rNS1. After washing, the cells were infected with the corresponding virus at MOI = 3 for 1 h, the infection allowed to proceed, and the supernatants, collected at 24 hpi, titrated by plaque assay in Vero cells. PFU, plaque forming units. Results are the mean ± SD of at least 2 independent experiments. *, P = 0.05.

FIG 3

Inhibition of DENV NS1 internalization by antibodies against SRB1. Huh7 cells (A) and C6/36 cells (C) were incubated with anti-SRB1 antibodies at the indicated concentrations for 1 h; after extensive washing, cells were incubated with DENV NS1 (3.5 μg/well) for 1.5 h. Finally, cells were fixed, permeabilized, and labeled for NS1. Cells were analyzed by confocal microscopy. (B, D). The levels of NS1 inside the cells were quantified by analyzing at least 10 different fields and expressed as the mean ± SD of fluoresce arbitrary units (FAU). Three independent experiments were carried out. Differences in FAU were compared for significance using an ANOVA test. *, P = 0.05. Bars = 10 μm.

FIG 4

Competition assays between human HDL and recombinant DENV and ZIKV NS1. (A) Huh7 and (C) C6/36 cells were incubated with HDL at the indicated concentrations for 1.5 h; after extensive washing, cells were incubated with DENV and ZIKV NS1 (3.5 μg/well) for 1.5 h. Finally, cells were fixed, permeabilized, and labeled for NS1. Cells were analyzed by confocal microscopy. (B, D) The levels of NS1 inside the cells were quantified by analyzing at least 10 different fields and expressed as the mean ± SD of fluoresce arbitrary units (FAU). Three independent experiments were carried out. Differences in FAU were compared for significance using an ANOVA test. *, P = 0.05. Bars = 10 μm.

FIG 5

Silencing SRB1 expression in C6/36 cell. Confluent C6/36 monolayers were transfected with siRNAs targeting the SRB1, or an irrelevant siRNA, as a control. Silencing efficiency was assayed by immunofluorescence (A, B) and Western blot (E). (C) Transfected cells were incubated with DENV NS1 and 1.5 h, washed, and processed for immunofluorescence. (D) The levels of NS1 inside the cells were quantified by analyzing at least 10 different fields and expressed as the mean ± SD of fluoresce arbitrary units (FAU). Three independent experiments were carried out. Differences in FAU were compared for significance using an ANOVA test. *, P < 0.001. Bars = 10 μm.

FIG 6

Expression of the SRB1 receptor in Vero cells. (A) Immunofluorescence of transfected, nonpermeabilized cells stained for the SRB1 at 48 hpt. (B) Cells lysates from transfected and nontransfected cells were analyzed for the expression of SRB1 by Western blot. *, MWM corresponding to 60 kDa. (C) Transfected cells incubated with DENV NS1 for 1.5 h, fixed, permeabilized, and labeled with anti-NS1 (green) and anti-SRB1 (red) antibodies. (D) Nontransfected cells incubated with NS1 and both primary antibodies. Cell were analyzed by confocal microscopy. Nuclei were counterstained with DAPI. At least 3 independent experiments were carried out. Bars = 20 μm.

FIG 7

Proximity ligation assays for dengue and Zika virus NS1 and the SRB1 in human and mosquito cells. Cells incubated with recombinant DENV NS1 (A, C) or ZIKV NS1 (E, G) at a concentration of 3.5 μg/well for 45 min, then fixed and processed following the manufacturer’s instructions. Positive interactions between NS1 and SRB1 are visualized as red dots. Cell nuclei were counterstained with DAPI. B1, D1, F1, and H1 cells were incubated with DENV and ZIKV NS1, but primary anti-NS1 antibody omitted. B2, D2, F2 and H2 cells not incubated with NS1 and incubated with both primary antibodies. (I) Quantification of PLA signal. Results are the mean ± SD of at least 100 cells observed per condition. Experiments were carried out three times, and typical results are shown. Bars = 20 μm.

FIG 8

Surface plasmon resonance sensorgrams obtained for the binding interaction of DENV NS1 with the human SRB1. Increasing concentrations (color lines) of recombinant NS1 were tested for interaction with SRB1 immobilized on a Biacore sensor chip. Binding curves were expressed in resonance units (RU) as a function of time (seconds). Solid black lines through the curve show model fit for the calculation of interaction affinities.