Lectin-dependent enhancement of Ebola virus infection via soluble and transmembrane C-type lectin receptors

Abstract

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes.

Figure 1. MBL enhances HIV-EBOV GP infection of HEK293F cells in low complement conditions.

We used luciferase-encoding HIV-EBOV-GP virion-like particles to infect 5×10³ adherent HEK293F cells. Viruses (350 pg p24/100 µl) were preincubated with physiological concentrations of rhMBL (0.1–10 µg/ml) in (A) DMEM or (B) MBL-deficient (<0.6 ng/ml) 5% human serum for 1 hour at 37°C. We analyzed infection rates after 40 hours. Luciferase absorbance values were adjusted for cell viability by normalizing to alamarBlue fluorescence units (resazurin reduction assay) and results are expressed as Adjusted Luciferase Values. Significant differences are shown. * and ** p<0.05 (HIV-VSV-G vs both other virions at 0 and 0.1 µg/ml supplemental rhMBL, respectively), † and ‡ p<0.05 (HIV- envelope negative vs both other virions at 1 and 10 µg/ml supplemental rhMBL, respectively). Also shown in (B) is the reduced capacity of rhMBL to enhance infection by HIV-EBOV-ΔGP NTDL6 (mutated GP lacks 217 amino acids in the heavily glycosylated mucin-rich region) compared with that for HIV-EBOV GP, which contains intact mucin-rich regions (1.3- vs 17.2−fold enhancement, respectively, p<0.001). Experiments were performed twice in quadruplicate. (C) We preincubated HIV-EBOV GP with serial dilutions of serum from three individuals with undetectable (<0.6 ng/ml; LYPB/LYPB), intermediate (2,181 ng/ml; LYPA/HYPD) or high (5,424 ng/ml; HYPA/LYQA) MBL concentrations. Shown are luciferase values. * p<0.05 (high level MBL vs low or intermediate level MBL at 1% serum dilution), **, † and ‡ p<0.05 (all pairwise comparisons at 2%, 5% and 10% serum dilutions, respectively), # p<0.05 (all pairwise comparisons at 30% serum dilution). Experiments were performed twice in quadruplicate. (D) We preincubated HIV-EBOV-GP virion-like particles with 5% serum from 35 ethnically diverse individuals. Shown are associations between level of infection and native MBL activity (mannan-binding or C4 cleavage) for each individual (r² = 0.83, serum C4 cleavage activity; r² = 0.85, serum MBL-mannan binding). Experiments were performed in quadruplicate. (E) We preincubated HIV-EBOV-GP virion-like particles with 5% heat-inactivated serum (56°C for 30 minutes) from three individuals with varying MBL levels before infection of cells. (F) We preincubated HIV-EBOV GP virion-like particles with media or sera (diluted to 1–50%) that were complement component 2 (C2)-replete or depleted, and that lacked or contained approximately equivalent MBL concentrations (535–650 ng/ml). In addition, C2 depleted serum was reconstituted with recombinant human C2 (6.5 µg/ml) for comparison. Experiments were performed twice in quadruplicate.

Figure 2. MBL interacts with HIV-EBOV GP via MBL carbohydrate recognition domains.

We preincubated 5% serum containing native human MBL (3,621 ng/ml) with (A) 0, 1 and 10 mM of hexose monosaccharides or EDTA diluted in media, or (B) 0–100 µg/ml of mannan or polydisperse polyethylene glycol (PEG)(D) at room temperature for 30 minutes. Then we incubated the serum with HIV-EBOV GP (1200 pg p24/100 µl) at 37°C for 1 hour before infecting adherent HEK293F cells. Luciferase values were adjusted for cell viability using alamarBlue (resazurin reduction assay). We observed relatively more toxicity associated with 10 mM EDTA but this did not invalidate our results because of our adjustment for cell viability. (C) We repeated the previous experiments with 3F8, an anti-human MBL monoclonal antibody or an IgG1 isotype control (preincubation at 37°C for 30 minutes). Significant differences are shown. (D) We preincubated HIV-EBOV GP virion-like particles with cyanovirin (0–600 nM) at 37°C for 1 hour before incubating the particles with 5% serum in the presence or absence of rhMBL. Luciferase values were adjusted for cell viability. Experiments were performed twice in quadruplicate.

Figure 3. MBL targets N-linked glycans on viral and cellular surfaces.

The cleavage sites of two endoglycosidases are shown (A,B). N-glycosidase F (PNGase F) is an amidase that cleaves the linkages between the innermost GlcNAc and asparagine residues within high-mannose, hybrid and complex oligosaccharides of N-linked glycoproteins, thereby producing carbohydrate-free peptides without any potential ligands for MBL. Endoglycosidase H (endo H) cleaves linkages within the diacetylchitobiose stem of high-mannose of N-linked glycoproteins, thereby generating a truncated sugar molecule with one N-acetylglucosamine residue (a potential target for MBL) remaining on the asparagine. Man, mannose; GlcNAc, N-acetylglucosamine; asn, asparagine; × and y, various oligosaccharides; n = 2–150 residues. We preincubated HIV-EBOV-GP virion-like particles (1200 pg p24/100 µl) at 37°C for 1 hour with (C) PNGase F or endo H (0–10,000 U/ml), or (D) the same concentrations of heat inactivated enzymes. Then we incubated the viruses with 5% MBL-deficient serum in the presence or absence of rhMBL at 37°C for 1 hour before infecting HEK293F cells. Significant differences are shown. (E) We preincubated HEK293F cells at 37°C for 1 hour with chemicals (tunicamycin, swainsonine or deoxynojirimycin) that inhibit various stages of N-linked glycosylation. Then we infected cells with HIV-EBOV-GP virion-like particles (1200 pg p24/100 µl) that had been preincubated with 5% MBL-deficient serum and supplemented with various concentrations of rhMBL. Significant differences are shown. * and **, p<0.001 (all pairwise comparisons at 1 and 10 µg/ml rhMBL, respectively). (F) We cultivated HEK293F and HEK293S (deficient in N-acetylglucosaminyltransferase I) cells in 5% MBL-deficient serum which was supplemented with various concentrations of rhMBL. We infected cells with HIV-EBOV-GP virion-like particles (1200 pg p24/100 µl) in the absence or presence of 1 µg/ml tunicamycin. Statistical differences among inhibitors at various rhMBL concentrations are shown. *, ** and †, p<0.005 (all pairwise comparisons at 0.1, 1 and 10 µg/ml rhMBL, respectively). Luciferase values were adjusted for cell viability using alamarBlue (resazurin reduction assay). All experiments were performed twice in quadruplicate.

Figure 4. MBL mediates HIV-EBOV GP infection via the canonical macropinocytosis pathway for EBOV but with less dependence on actin.

We preincubated HEK293F cells with (A) EIPA (5-(N-Ethyl-N-isopropyl)amiloride, a potent and specific inhibitor of Na⁺/H⁺ exchanger activity), (B) methyl-β-cyclodextrin (extracts or sequesters cholesterol from the plasma membrane), (C) latrunculin B (blocks actin polymerization), (D) cytochalasin D (inhibits actin microfilament function), (E) nocodazole (disrupts microtubules), or (F) jasplakinolide (disrupts microtubules) in 5% MBL-deficient serum in the absence or presence of rhMBL at 37°C for 1 hour. We then infected cells with HIV-EBOV-GP virion-like particles (1200 pg p24/100 µl). Percentages of infected cells are relative to DMSO controls. Luciferase values were adjusted for cell viability. Experiments were performed twice in quadruplicate. Significant differences are shown. (G) Absorbance values of an ELISA assay are shown indicating the difference in amount of rhMBL within the physiological range that binds to immobilized mannan or FITC-dextran (1 µg/100 µl). (H) We preincubated FITC-dextran with various concentrations of rhMBL at 37°C for 30 minutes and then added the products to PMA-stimulated (10 ng/ml), IL-4-supplemented (100 ng/ml) THP-1 cells at 37°C for 1 hour. We measured FITC-dextran uptake by flow cytometry and reported the results as mean fluorescence intensity (geometric mean fluorescence × percentage of cells). Experiments were performed twice in triplicate.

Figure 5. MBL enhances HIV-EBOV GP infection of THP-1 cells and human monocyte-derived macrophages.

(A) We stimulated 5×10⁴ THP-1 cells with PMA (10 ng/ml) and supplemented the cells with IL-4 (100 ng/ml) for 72 hours. We preincubated HIV-EBOV GP or HIV-env negative virion-like particles (1200 pg p24/100 µl) with or without rhMBL before infecting differentiated adherent THP-1 cells cultivated in 5% MBL-deficient serum. (B) We cultivated 2.5×10⁵ PBMC derived from human single-donor buffy coat samples in RPMI-1640 with 10% FBS and stimulated the cells with M-CSF (50 ng/ml) to induce differentiation of monocyte-derived macrophages. We infected cells with HIV-EBOV GP (WT), HIV-EBOV-ΔGP NTDL6 (NTDL6, mutated GP lacks 217 amino acids in the heavily glycosylated mucin-rich region) or HIV-env negative (env neg) in the presence or absence of rhMBL. The box plot represents outliers (dots), 10^th and 90^th percentiles (whiskers), 25^th and 75^th percentiles (box) and median values (line). Significant differences in infection rates are shown. Luciferase values were adjusted for cell viability using alamarBlue (resazurin reduction assay) for all the above experiments, which were performed twice in quadruplicate.

Figure 6. RNA interference screen of candidate cellular receptors for EBOV and MBL.

(A–D) We targeted 24 candidate lectin, scavenger and other putative receptors using pLKO.1 lentiviral vectors that expressed 4 or 5 unique short hairpin RNA (shRNA) constructs per gene. We transduced HEK293F cells in quadruplicate using 4.6×10⁸ viral particles (shRNA-expressing vectors or empty control vectors) with hexadimethrine bromide (6 µg/ml) at 37°C for 18 hours. We selected transduced cells with 5 µg/ml puromycin over 48 hours and determined cell viability with alamarBlue reagent (resazurin reduction assay). We then infected cells in quadruplicate with HIV-EBOV GP virion-like particles (1000 pg p24/100 µl) with or without rhMBL. After 48 hours we measured rates of single-round infection (luciferase assay) and adjusted results for cell viability. Percentage change in infection was normalized to the empty pLK0.1 control vector (CTRL). Shown are positive hits (A, C) which were defined as ≥66% reduction in infection by at least two shRNA constructs for any particular gene. Reductions in protein expression produced by shRNAs (western blots; B, D) relative to that produced by the empty pLK0.1 control vector are shown. Relative densitometry was performed with ImageJ (NIH) by adjusting for variations in the actin loading controls (adjusted relative densities for CLEC6A: lane 1, 0.60; lane 2, 0.56; lane 3, 0.51; lane 4, 0.35; control, 1.0. C1QBP: lane 1, 0.32; lane 2, 0.52; lane 3, 0.47; control 1.0).

Figure 7. MBL enhances infections by wild type-like EBOV and other glycosylated virions.

(A) We preincubated wild type-like EBOV-eGFP (1976 Mayinga variant) with media alone or 5% MBL-deficient serum with or without rhMBL at 37°C for 1 hour and then infected 4×10⁴ HEK293T cells (multiplicity of infection, 0.1) at 37°C for 1 hour. We measured cellular fluorescence after 72 hours of incubation in fresh media. Comparisons are with baseline values, * p = 0.028; ** p = 0.037. (B) We preincubated native Hendra and Nipah viruses (10,000 TCID₅₀/ml) with 10% heat-inactivated MBL-deficient serum with or without rhMBL and then infected Vero E6 cells at 37°C for 1 hour. After 24 hours, infection was detected by chemiluminescence-based viral protein immunoassays. Comparisons are with baseline values, *p = 0.001; ** p = 0.029. (C) We preincubated 250 µl West Nile virion-like particle-GFP with media alone or 2% MBL-deficient human serum, with or without rhMBL at 37°C for 1 hour and then transduced 1×10⁴ HEK293T cells. Cells were detached using TrypLE and washed three times with PBS at 4°C. Rates of transduction were assayed by flow cytometry. Comparisons are with baseline values, * p = 0.002; ** p = 0.001. WT refers to wild type; mutant refers to glycosylation mutant of WNV E protein; hMBL refers to human MBL.

Figure 8. Proposed model of MBL-mediated macropinocytosis of EBOV.

MBL carbohydrate recognition domains (CRD) bind to highly glycosylated mucin-rich regions of EBOV GP and the MBL-virion complex is presented to the cell surface. Then MBL binds to cognate cellular receptors, such as C1QBP or calreticulin via MBL collagenous stalks. In this manner, MBL concentrates virus at the cell surface and may facilitate cross-linking of EBOV to its cognate receptor or to other attachment factors such as dectin-2, which may facilitate subsequent membrane fusion and internalization. The relatively large size of EBOV particles (800 to 1,400 nm) is amenable to bulk fluid-phase uptake pathways such as macropinocytosis which is the canonical pathway for EBOV entry. Our data indicate that MBL also mediates internalization of virus via macropinocytosis but suggests that MBL-mediated uptake preferentially utilizes microtubules compared with the canonical EBOV pathway which is dependent on both microtubules and actin.