The TIM and TAM families of phosphatidylserine receptors mediate dengue virus entry

Abstract

Dengue viruses (DVs) are responsible for the most medically relevant arboviral diseases. However, the molecular interactions mediating DV entry are poorly understood. We determined that TIM and TAM proteins, two receptor families that mediate the phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, serve as DV entry factors. Cells poorly susceptible to DV are robustly infected after ectopic expression of TIM or TAM receptors. Conversely, DV infection of susceptible cells is inhibited by anti-TIM or anti-TAM antibodies or knockdown of TIM and TAM expression. TIM receptors facilitate DV entry by directly interacting with virion-associated PtdSer. TAM-mediated infection relies on indirect DV recognition, in which the TAM ligand Gas6 acts as a bridging molecule by binding to PtdSer within the virion. This dual mode of virus recognition by TIM and TAM receptors reveals how DVs usurp the apoptotic cell clearance pathway for infectious entry.

Figure 1. TIM Receptors Enhance DV Infection

(A) Surface levels of TIM molecules on 293T parental (red line) and transduced cells (blue line) were evaluated by flow cytometry using polyclonal anti-TIM antibodies. Gray shading represents cell staining with a control IgG. (B–E) Parental and 293T cells expressing TIM receptors (B) were challenged with DV2-JAM. Infection levels were assessed by flow cytometry using the anti-NS1 mAb. Parental, TIM-expressing 293T (C) were infected with DV2-JAM. Supernatants were collected 48 hr later. Virus titers were determined by plaque assay and expressed as plaque forming unit per ml. TIM receptors are used by the four DV serotypes (D). TIM receptors are shared by other flaviviruses (E). Data are represented as mean ± SEM of at least three independent experiments. See also Figure S1.

Figure 2. TIM-1 and TIM-4 Molecules Bind to DV and Enhance Virus Endocytosis

(A) Western blot analysis of DV2-JAM preincubated with control Fc, NKG2D-Fc, TIM-1-Fc, or TIM-4-Fc bound to protein A-agarose beads. Pulled-down virus was detected using the 4G2 anti-DV E protein mAb. (B) Control Fc, NKG2D-Fc, or TIM-1-Fc were coated on 96-well plates and incubated with DV2-JAM for 1 hr at 4°C. Bound virus was detected using the biotinylated 4G2 mAb and HRP-conjugated streptavidin See also Figure S2. (C) Cells transfected with an empty vector or plasmids encoding TIM-1 or TIM-4 were incubated 1 hr at 4°C with DV2-JAM. Bound virus was detected by flow cytometry using the 4G2 mAb. (D) Cells were incubated with DV2-JAM for 2 hr at 37°C and treated with proteinase K. Total RNA was extracted from infected cells, and DV2 viral RNA level was determined by real-time quantitative PCR with human GAPDH as endogenous control. Results are expressed as the fold difference using expression in 293T infected cells as calibrator value. (E) Parental and CHO-745 cells expressing TIM-1, TIM-4, or DC-SIGN were incubated with DV2-JAM for 1 hr at 4°C and shifted at 37°C for 45 min. Cells were labeled with DAPI (blue) and the anti-DV E protein 4G2 (green) to detect virus uptake in unpermeabilized and permeabilized cells. (F) 293 T cells were transfected with plasmid encoding for TIM-1 WT, TIM-1 lacking its entire cytoplasmic tail (TIM-1 Δcyt), or an empty vector (pcDNA3). Cells were stained for TIM-1 expression (left panel) and challenged with DV2-JAM (right panel). Data are represented as mean ± SEM of at least three independent experiments.

Figure 3. TIM-1 and TIM-4 Molecules Recognize PtdSer Associated with DV Particles

(A) Sucrose gradient purified DV2 particles were coated on well plates and incubated with the anti-PtdSer 1H6 mAb. See also Figure S3. (B) DV2-JAM preincubated with Annexin V (ANX5; 25 μg/ml) was used to infect the indicated cells. Infection was normalized to infection without ANX5. (C) TIM molecules mutated in the PtdSer binding domain do not mediate DV infection. Left panel: Cell surface expression of WT and TIM-1 or TIM-4 mutants. Right panel: Transfected cells were infected with DV2-JAM. Data are represented as mean ± SEM of at least three independent experiments.

Figure 4. TYRO3 and AXL Enhance DV Infection

(A) Surface levels of TAM molecules on 293T parental (red) and cells stably expressing human TYRO3 and AXL (blue). Gray: isotype control Ab. (B and C) Cells were challenged by DV2-JAM and infection was assessed 48 hr later. Immunofluorescence (B) with an anti DV prM glycoprotein (2H2) (red) and DAPI (blue). Flow cytometry analysis (C) with an anti-NS1 mAb. See also Figure S4. (D) Gas6 enhances DV binding to TYRO3- and AXL-expressing cells. Cells were incubated for 90 min at 4°C with DV2-JAM in serum-free medium containing Gas6 (1 μg/ml) or PBS (mock). Mean fluorescent intensity was measured by flow cytometry and normalized to that in noninfected cells. (E) 293T cells expressing WT AXL or a molecule mutated in the Gas6-binding site (E66R/T84R) were incubated with DV2-JAM in serum-free medium containing Gas6 (1 μg/ml) or PBS (mock). After 3 hr, medium was replaced by medium supplemented with 10% FBS. The inset displays wild-type and mutant AXL surface expression. (F) Plastic-coated DV2-JAM (10⁷ FIU) was incubated with Gas6 or Gas6ΔGla (2 μg/ml) for 1 hr. Bound Gas6 was detected by ELISA using a goat polyclonal anti-Gas6 (10 μg/ml) Ab. (G) Plastic-coated DV2-JAM particles (10⁷ FIU) were incubated with the indicated Fc-chimeras (2 μg/ml) in the presence or absence of Gas6 or Gas6ΔGla (2 μg/ml). Bound Fc-chimeras were detected using an HRP-conjugated anti-human IgG. (H) Cells were incubated with Gas6ΔGla (1 μg/ml) for 30 min before and during a 3 hr-incubation with DV2-JAM. Data are represented as mean ± SEM of at least three independent experiments. **p < 0.001, ***p < 0.0001. See also Figure S5.

Figure 5. TIM-1 and AXL Expression and DV Infection in Cell Lines

(A) Surface level of TIM-1 and AXL on cell lines was monitored by flow cytometry. Gray shading represents cell staining with a control Ab. (B) The indicated cells were challenged with DV2-JAM. (C) A549 cells were incubated with DV2-JAM for 1 hr at 4°C in the presence of goat polyclonal anti-TIM-1, anti-AXL, or normal goat IgG as control. Mean fluorescent intensity was measured by flow cytometry and normalized to binding in presence of control IgG. Data are shown as one representative flow cytometry analysis (left panel) and are represented as mean ± SEM of at least three independent experiments (right panel).

Figure 6. Endogenous TIM-1 and AXL Molecules Mediate DV Infection

(A and B) Huh7.5.1 (A) and A549 (B) cells were infected with the indicated DV strains or HSV-1 in the presence of polyclonal goat anti-TIM-1, anti-AXL, or normal goat IgG as control. The levels of infected cells were normalized to infection in presence of control IgG. (C) Representative immunofluorescence analysis of A549 infected with DV2-JAM in the presence of the indicated Ab. Green anti-prM 2H2, Blue DAPI. Scale bar: 100 μm. (D) A549 cells were transfected by the indicated siRNA, and TIM-1 and AXL expression was assessed by flow cytometry after 2 days, at the time of infection. Cells were infected with DV2-JAM or HSV-1. Infection was normalized to infection in nontargeting (siNT) siRNA-transfected cells. (E) A549 cells were infected with DV2-JAM or HSV-1 preincubated with different concentrations of ANX5. (F) and (G) Human primary kidney epithelial cells (F) or astrocytes (G) were infected with DV in the presence of control IgG or an anti-AXL Ab. Insets display AXL cellular expression at the time of infection. Data are represented as mean ± SEM of at least three independent experiments. **p < 0.001, ***p < 0.0001. See also Figure S6.

Figure 7. Model of PtdSer Acquisition by DV Particles and the TIM and TAM Receptor-Mediated Enhancement of DV Infection

(A) Proposed model of direct and indirect (bridging) recognition of DV by the TIM and TAM receptors. (B) Schematic representation of DV particle acquiring phosphatidylserine (PdtSer) upon budding into the ER lumen.