The Low-Density Lipoprotein Receptor-Related Protein-1 Is Essential for Dengue Virus Infection

Abstract

Dengue virus (DENV) causes the most prevalent and rapidly spreading arboviral disease of humans. It enters human cells by receptor-mediated endocytosis. Numerous cell-surface proteins were proposed as DENV entry factors. Among these, the phosphatidylserine receptor TIM-1 is the only one known to mediate virus internalization. However, several cellular models lacking TIM-1 are permissive to DENV infection, suggesting that other receptors exist. Here, we show that the low-density lipoprotein receptor-related protein-1 (LRP1) binds DENV virions by interacting with the DIII of the viral envelope glycoprotein. DENV infection is effectively inhibited by the purified receptor at 5 × 10^-8 mol/L, and the interaction of the envelope protein with LRP1 is also blocked by a natural ligand of LRP1. The depletion of LRP1 causes 100-fold lower production of infectious virus than controls. Our results indicate that LRP1 is another DENV receptor, thus becoming an attractive target to evaluate for the development of effective antiviral drugs against DENV.

Keywords: cellular receptor; domain III; virus–receptor interaction.

Figure 1

LRP1 is essential for DENV2 infection. (A) Western blotting analysis of the silencing of LRP1 expression on cells transfected with scramble shRNA (SC, red circles) or with LRP1-shRNA (green squares). LRP1 was detected using the Mab 5A6 that recognizes the 85 kDa β chain of the receptor [44]. Detection of βactin was performed on the same membrane. (B) Effect of the transduction of LRP1-shRNA on DENV2 infection. Transduced cells were infected at day 7 with DENV2 (moi of 0.1) or vesicular stomatitis virus (VSV, moi 0.35). Virus yield was measured on 24 h post-infection supernatants. GraphPad Prism V5.3. was used for the unpaired Student’s t-test, where *** p < 0.001.

Figure 2

Purified sLRP1 inhibits DENV2 infection. (A) Chromatographic profile of the affinity chromatography using receptor-activated α2M as immobilized ligand. PT: pass-through. Numbers in parenthesis identify the lane in SDS-PAGE (B) and Western blotting analysis (C). In (C), membrane was probed with an anti-LRP1 Mab (MCA1965) that recognizes the alpha chain of the receptor. Microsomal fraction (FM) of Huh7 cells was used as control of migration of α chain of LRP1. (D) Binding analysis of purified sLRP1 to recombinant RAP protein using SPR. RAP protein was immobilized on the surface of the chip, and purified sLRP1 (green line) was injected and diluted in HBS-Ca at 1 µg/mL. Red line corresponds to the application of the initial sample of the affinity chromatography at the same protein concentration. Resp. Diff denotes the response obtained by automatically subtracting the signal of the reference channel from that of the specific channel. (E) Inhibition of DENV2 infection in Huh7 cells after incubation with chromatography fractions. Results from one representative experiment out of three independent experiments. Error bars represent the range of three sample replicates.

Figure 3

Interaction of DENV2 with ligand-binding clusters of LRP1. Schematic representation of (A) LRP1 and (B) the recombinant proteins sLRP1-CII, sLRP1-CIII and sLRP1-CIV. (C) Evaluation of the interaction of recombinant proteins sLRP1-CII, sLRP-CIII and sLRP1-CIV with purified DENV2. (D) Interaction of sLRP1-CIV with recombinant preparations of the ectodomain of the E protein of DENV serotypes 1, 3 and 4 (DENVE1, DENVE3 and DENVE4) by SPR. sLRP1-CIV was immobilized in the surface of the chip and recombinant DENV E proteins were injected and diluted in HBS-Ca at 10 µg/mL. Resp Diff: signal obtained by online subtraction of background control channel. (E) Binding competition by ELISA. Plates were coated with DENVE1 protein, and the binding of sLRP1-CIV at 5 µg/mL was evaluated after the incubation for 30 min at 37 °C with EGF or the LRP1 ligand protein RAP, both proteins used at 5 µg/mL. BC: background control. Results are representative of two independent analyses. Error bars represent the range of three replicates. GraphPad Prism V5.3. was used for the unpaired Student’s t-test, where *** p < 0.001.

Figure 4

Interaction of recombinant DENV E DIII proteins with LRP1. (A) Multiple sequence alignment of DIIIE1–4 proteins (residue numbers according to DENV1, 2 and 4). The alignment was performed using the ClustalW application [49]. The arrows denote β-strands. Residues are colored according to the physico-chemical properties and conservation according to ESPript program [50]. (B) Interaction of DIIIE1–4 proteins with sLRP1-CII, sLRP1-CIII or sLRP1-CIV by ELISA. Results are mean and SEM of three independent experiments. An unpaired Student’s t-test was used to compare the response against sLRP protein against BSA control, where * p < 0.05, and *** p < 0.001. (C) Binding analysis of sLRP1 to immobilized DIIIE1 by SPR. sLRP1 at 1 µg/mL was loaded over the DIIIE2 and DIIIE1 surfaces. For this later analysis, sLRP1 injection was performed without or after pre-incubation with RAP at (DIIIE1/RAP), or receptor-activated α2M (DIIIE1/α2M*). Resp. Diff.: non-specific signal was subtracted online using a reference channel without any immobilized protein. (D) Blocking of sLRP1-DENV2 binding. A concentration of sLRP1-CIV at 5 µg/mL was incubated with a concentration of 10 µg/mL of DIIIE1–4 for 1 h before the addition to DENV2-coated plates. Bound sLRP1-CIV was detected using an anti-human Fc Ig-POD conjugate. (E) Binding to LRP1 present in the microsomal fraction of Huh7 cells. Pull down assay using recombinant DIIIE1–4 proteins as bait and detection by Mass spectrometry using Selected Reaction Monitoring. MF: microsomal fraction. BC: background control. Data show transition signals of the surrogate peptide ₃₆₅IVFPHGITLDLVSR₃₇₈ (Swissprot Accession number Q07954). Samples were analyzed in duplicate. Similar results were obtained for other five proteotypic peptides (Supplementary Table S1). (F) Cartoon representation of the 3D structure of DIII. In yellow, the β-strands and lysine residues are represented, shown in wire-frame representation. Residue numbers correspond to DENV1, 2 and 4.