Dengue virus NS1 protein conveys pro-inflammatory signals by docking onto high-density lipoproteins

Abstract

The dengue virus nonstructural protein 1 (NS1) is a secreted virulence factor that modulates complement, activates immune cells and alters endothelial barriers. The molecular basis of these events remains incompletely understood. Here we describe a functional high affinity complex formed between NS1 and human high-density lipoproteins (HDL). Collapse of the soluble NS1 hexamer upon binding to the lipoprotein particle leads to the anchoring of amphipathic NS1 dimeric subunits into the HDL outer layer. The stable complex can be visualized by electron microscopy as a spherical HDL with rod-shaped NS1 dimers protruding from the surface. We further show that the assembly of NS1-HDL complexes triggers the production of pro-inflammatory cytokines in human primary macrophages while NS1 or HDL alone do not. Finally, we detect NS1 in complex with HDL and low-density lipoprotein (LDL) particles in the plasma of hospitalized dengue patients and observe NS1-apolipoprotein E-positive complexes accumulating overtime. The functional reprogramming of endogenous lipoprotein particles by NS1 as a means to exacerbate systemic inflammation during viral infection provides a new paradigm in dengue pathogenesis.

Keywords: Arbovirus; accessory protein; hemorrhagic fever; lipoprotein particle; molecular pathogenesis; virulence factor.

Figure 1. DENV NS1 binds human high‐density and low‐density lipoproteins

1. Size exclusion chromatography profile of NS1 pull‐down experiments showing a clear shift after incubation in complete or heat‐inactivated human serum (solid and dotted black lines, respectively) from the same healthy donor compared to the NS1 protein alone (blue line). NS1 protein interaction partners were identified by SDS‐PAGE and N‐terminal sequencing as the Apolipoprotein B scaffold of the low‐density lipoproteins (LDL) in the first SEC elution peak, and the ApoA‐I protein scaffold of the high‐density lipoproteins (HDL) in the second elution peak.

2. Biolayer interferometry (BLI) profiles corresponding to the binding of NS1 at various concentrations respective to human HDL (left panel) and human LDL (central panel) particles. The concentration‐dependence of the steady‐state signal corresponding to the binding of NS1 to HDL (black dots) and LDL (white dots) is shown on the right‐hand side panel. The measurements were replicated at least three times using novel biosensors and samples. Data points and error bars correspond to the mean ± SD.

3. Typical sedimentation coefficient distribution of NS1 or human HDL alone or pre‐incubated together (mixture of NS1 and HDL at a 1:1 or 5:1 mass ratio) monitored using an interferometric detector. Peaks were integrated for all the detectors (interference and absorbance at 280 nm). The calculated stoichiometries are indicated for each peak. Solutions were equilibrated at 20°C for 2 h before sedimentation velocity analysis.

Figure EV1. The secreted form of DENV‐2 NS1 binds bovine HDL and LDL

1. A–C

   The NS1 protein was expressed in drosophila S2 cells cultured either in InsectXpress protein‐free medium (Lonza) supplemented with 5% complete fetal bovine serum (FBS) or without FBS. NS1 was purified on a Streptactin affinity column before analysis in a size exclusion column (superdex 200 16/60). (A) Typical chromatograms obtained with purified secreted DENV‐2 NS1 (blue line) and DENV‐2 NS1 bovine complexes (red line) are shown. (B) Denaturing SDS‐PAGE analysis of 1 fraction every 4 ml from 38 to 75 ml elution volume. Molecular weights of protein standards (Std) are expressed in kDa. Proteins were detected by UV (Stain‐free, Biorad). The three major protein bands identified by mass‐spectrometry are bovine ApoB (> 250 kDa), DENV‐2 NS1 (~50 kDa), and bovine ApoA‐I (~25 kDa). (C) Negative‐stain electron micrographs of purified hexameric NS1 alone (left panel) or purified as a complex with ApoA‐I (NS1‐HDL, central panel) or with ApoB (NS1‐LDL, right panel). Bar: 100 nm. Data are derived from at least two independent experiments.

Figure EV2. Co‐immunoprecipitation of native NS1 and apolipoproteins from DENV‐infected cell supernatants supplemented with bovine or human serum

1. A, B

   Vero cells infected with DENV (strain 16,681) at an MOI of 1 or uninfected were cultured for 3 days in media supplemented with 10% FBS or 10% human serum. Supernatants were clarified by centrifugation and immunoprecipitated with (A) anti‐NS1 MAb 17A12 or anti‐ApoA‐I PAbs or (B) with anti‐NS1 MAb 17A12 or anti‐ApoE PAbs. The resulting products were separated by SDS‐PAGE on stain‐free gels, submitted to UV light and visualized in a gel imager (G‐Box, Syngene) (A, B), or transferred onto a PVDF membrane treated with biotinylated anti‐ApoE PAb and streptavidin‐HRP (B). Data shown are representative of two technical replicates.

Figure EV3. Dose‐dependent binding of NS1 to HDL particles monitored by biolayer interferometry

1. A–C

   Biolayer interferometry (BLI) profiles corresponding to the binding of NS1 at various concentrations respective to human HDL particles. Data from NS1 binding to (A) HDL loaded biosensors, (B) antibody activated biosensors and (C) the corresponding subtraction. Data was colored according to the NS1 concentration as 800 nM in Magenta, 400 nM in violet, 200 nM in blue, 100 nM in sky blue, 50 nM in green, 25 nM in apple green, 12.5 nM in orange and 6.25 nM in red.

Figure 2. Analysis of NS1‐HDL complexes by electron microscopy reveals the presence of NS1 dimers on the surface of HDL particles

1. A, B

   Electron microscopy observations from left to right: a representative image, followed by the three most representative classes of (A) purified HDL particles and (B) NS1‐HDL complexes. White bar: 50 nm, Black bar: 20 nm.

2. C

   Fitting of the NS1 dimer 3D structure into the most abundant class of NS1‐HDL complexes, pointing to a collapse of the NS1 hexamer into its hydrophobic dimeric blocks that can then anchor into the HDL lipid phase.

3. D

   Differential scanning calorimetry (DSC) of NS1 alone (blue line) or of an NS1‐HDL mixture at a 2.5:1 molar ratio (orange). Of note, the HDL particles alone did not generate any signal in the temperature range tested.

4. E

   Binding inhibition of hexameric NS1 to HDL with anti‐ApoA‐I polyclonal antibodies (anti‐ApoA‐I Ab) measured by BLI.

Figure EV4. Electron microscopy analysis of NS1‐HDL complexes

1. A, B

   Classes of NS1 and NS1‐HDL purified species from negative‐stain electron microscopy (EM) images. NS1 and NS1‐HDL were purified as described in Material and Methods. The corresponding protein fractions were recovered from size exclusion chromatography and analyzed by negative‐stain EM. Automated acquisitions were performed using EPU software and images were acquired using a Falcon II direct detector. Images were CTF‐corrected (phase flip) and sorted using the XMIP software 84. Sizes of squares are reported at the bottom right of each panel.

2. C

   Representative electron microscopy image of negatively‐stained NS1‐HDL complexes bound to Fab 17A12. NS1‐HDL complexes were formed at a 1:1 molar ratio, purified by SEC and further incubated with Fab 17A12 at a molar ratio of 3 Fab:1 NS1‐HDL complex. Samples were spotted on glow discharged grids and contrasted with 2% uranyl actetate. Images were acquired on a Tecnai F20 microscope operated at 200 kV using EPU software (Thermo‐Fisher, USA) on a Falcon II camera, under low dose conditions. Bar: 20 nm. Image representative of at least four different fields.

Figure 3. The NS1‐HDL lipoprotein complex triggers pro‐inflammatory signals in human primary macrophages

1. A–D

   Human primary macrophages were incubated for 24 h with the different potential effectors (NS1, HDL, mix NS1‐HDL) or with control suspensions (PBS buffer, LPS, mix LPS‐HDL). LPS stimulation was used as a positive control in the presence or absence of HDL and provided values consistent between experiments. Phosphate buffer used in the SEC purification step was used as a negative control. Cell culture supernatants were clarified and tested with a Luminex assay to quantify the amount of (A) TNF‐α, (B) Il‐6, (C) Il‐1ß and (D) Il‐10 released in the extracellular medium. Data reported on the graphs correspond to biological replicates of macrophages isolated from four blood donors (n = 9 for TNF‐α, Il‐6 and Il‐1ß, n = 5 for Il‐10). Data represent mean ± SEM. A Mann‐Whitney test was used to assess the statistical significance of differences observed between mean cytokine levels in different cell culture supernatants. Not significant: ns, *P < 0.05, *** P < 0.001.

Figure EV5. Standard calibration curves

1. A–E

   Detection of the DENV NS1 protein and NS1‐ApoA‐I, NS1‐ApoB or NS1‐ApoE complexes by sandwich ELISA. (A) Detection of the purified DENV NS1 has been described previously¹¹. (B) NS1‐ApoA‐I, NS1‐ApoB or NS1‐ApoE complexes were formed in normal plasma spiked with purified NS1 at a known concentration. Capture of the NS1‐ApoA‐I, NS1‐ApoB or NS1‐ApoE complexes were carried out using an anti‐NS1 monoclonal antibody (MAb17A12). The detection of immobilized complexes was performed with an anti‐ApoA‐I, anti‐ApoB or anti‐ApoE polyclonal antibody followed by a species‐specific secondary antibody. The concentration of the NS1‐ApoA‐I, NS1‐ApoB or NS1‐ApoE complexes is reported on the basis of 100% NS1 bound to (C) HDL, (D) LDL or (E) ApoE‐positive lipoprotein particles, respectively. Detection limits of the NS1, NS1‐ApoA‐I, NS1‐ApoB or NS1‐ApoE assays were set as twice the mean value of signals obtained with normal human plasma in the absence of NS1, which corresponded to 0.5, 17, 5 and 15 ng of an equivalent NS1 concentration per milliliter, respectively.

2. F

   Determination of the HDL molecular weight by mass photometry. Purified HDL were diluted at 5 µg/ml and deposited on the coverslip. Measurements were performed according to the procedure described in Wu and Piszczek (2021). The results show an average mass of 162 kDa for the overall distribution.

Figure 4. Different biological and virological parameters measured in human plasma

1. A–H

   DENV‐infected hospitalized patients from the Kampong Cham Referral Hospital, Cambodia, presented either dengue with warning signs or severe dengue. Two blood samples were recovered for each patient on the day of hospital admission and during a follow‐up visit that occurred before discharge from the hospital (on average 4 days apart). (A) Number of patient samples tested over the hospitalization period for their levels of (B) NS1, (C) NS1‐ApoA‐I, (D) NS1‐ApoE and (E) NS1‐ApoB complexes in addition to (F) total cholesterol, (G) HDL‐cholesterol and (H) triglycerides. NS1‐ApoA‐I and NS1‐ApoB complexes are representative of NS1‐HDL and NS1‐LDL complex species, respectively, while the NS1‐ApoE‐positive complexes remain to be fully characterized. Errors bars indicate SEM.

2. I

   Purified NS1 from different flaviviruses (yellow fever, YF; ZIKA; West Nile, WN; Japanese encephalitis, JE) were spiked in normal human plasma and NS1‐ApoA‐I complexes were further detected by ELISA. Data represent the mean values of two technical replicates.