Inhibition of hepatitis C virus by the cyanobacterial protein Microcystis viridis lectin: mechanistic differences between the high-mannose specific lectins MVL, CV-N, and GNA

Abstract

Plant or microbial lectins are known to exhibit potent antiviral activities against viruses with glycosylated surface proteins, yet the mechanism(s) by which these carbohydrate-binding proteins exert their antiviral activities is not fully understood. Hepatitis C virus (HCV) is known to possess glycosylated envelope proteins (gpE1E2) and to be potently inhibited by lectins. Here, we tested in detail the antiviral properties of the newly discovered Microcystis viridis lectin (MVL) along with cyanovirin-N (CV-N) and Galanthus nivalis agglutinin (GNA) against cell culture HCV, as well as their binding properties toward viral particles, target cells, and recombinant HCV glycoproteins. Using infectivity assays, CV-N, MVL, and GNA inhibited HCV with IC50 values of 0.6 nM, 30.4 nM, and 11.1 nM, respectively. Biolayer interferometry analysis demonstrated a higher affinity of GNA to immobilized recombinant HCV glycoproteins compared to CV-N and MVL. Complementary studies, including fluorescence-activated cell sorting (FACS) analysis, confocal microscopy, and pre- and post-virus binding assays, showed a complex mechanism of inhibition for CV-N and MVL that includes both viral and cell association, while GNA functions by binding directly to the viral particle. Combinations of GNA with CV-N or MVL in HCV infection studies revealed synergistic inhibitory effects, which can be explained by different glycan recognition profiles of the mainly high-mannoside specific lectins, and supports the hypothesis that these lectins inhibit through different and complex modes of action. Our findings provide important insights into the mechanisms by which lectins inhibit HCV infection. Overall, the data suggest MVL and CV-N have the potential for toxicity due to interactions with cellular proteins while GNA may be a better therapeutic agent due to specificity for the HCV gpE1E2.

Figure 1. High resolution structures of each of the lectins used in these studies

(A) Lectins in complex with their carbohydrate ligand(s). Protein Data Bank accession codes are shown in parentheses. Protein backbones are shown as blue ribbons, and carbohydrate ligands as green sticks with oxygen atoms colored red (B). Structure of Man₉GlcNAc₂-Asn (Man-9). The minimal epitopes recognized by each lectin or the monoclonal antibody 2G12 are highlighted by shaded spheres. CV-N, 2G12: yellow, GNA: pink, MVL: blue. Glycan specificity was taken from glycan array data (Supplement Figure 1).

Figure 2. Dose-dependent effects of lectins on inhibition of HCVcc

Huh7.5 cells were infected with HCVcc expressing the structural proteins of genotypes (A) 1a/2a, (B) 1b/2a, (C) J6/JFH1, and (D) 3a/2a, following incubation of the virus with increasing amounts of the lectins CV-N, MVL, GNA, or 2G12. The percent inhibition was calculated and a dose-effect plot was created by CalcuSyn 2.0 software. Experiments were performed in duplicate and repeated in three independent experiments.

Figure 3. Soluble glycans can compete for lectin binding

Inhibition of HCVcc 1a/2a infection following incubation with lectins at their IC₅₀ concentrations, and in the presence of increasing amounts of Man-9. Error bars represent standard error of the mean. Experiments were carried out in duplicate and repeated in three independent experiments. Dotted line denotes 50% inhibition.

Figure 4. Dose-dependent inhibition of HCVcc infection at early binding and entry steps

A) Huh 7.5 cells were treated with lectins (6000 ng/ml to 46.8 ng/ml) for 1 hour prior to infection with HCVcc 1a/2a. B) Addition of lectins to cells after binding of HCVcc 1a/2a at 4°C for 1 hour C) Addition of lectins to cells after infection with HCVcc 1a/2a at 37 °C for 1 hour, and (D) for 2 hours. Dose-response plots were prepared using CalcuSyn 2.0 software. Experiments were performed in duplicate and repeated in three independent experiments.

Figure 5. FACS and confocal analysis of AlexaFluor-546-labeled lectins bound to infected and uninfected cells

A) Uninfected Huh 7.5 cells surface labeled with lectins and analyzed by flow cytometry. Unstained cells are shown in black, cells incubated with labeled lectins are shown in grey. B) Huh 7.5 cells infected with HCVcc 1a/2a and surface labeled with lectins. Confocal microscopy of (C) uninfected and (D) infected cells after surface labeling with lectins.

Figure 6. Binding analysis of immobilized HCV glycoprotein rE1E2 to antibodies and lectins by bio-layer interferometry

Each plot shows the response in nm as a function of time for three different concentrations of lectin and antibody. Blue, orange and turquoise curves correspond respectively to 5.0, 2.5 and 1.25 μM 2G12; and to 10.0, 5.0 and 2.5 μM GNA, CV-N, and MVL. The calculated best-fit curves are shown as red lines.

Figure 7. Confocal Analysis of hepatoma cells with labeled lectins

A) Huh 7.5 cells infected with HCVcc 1a/2a surface labeled with lectins and specific anti-E1E2 antibodies. B) Huh 7.5 cells infected with HCVcc 1a/2a and labeled intracellularly with lectins and specific anti-E1E2 antibodies. C) Uninfected Huh7.5 cells, stained with DAPI and anti-E1E2 as controls.

Figure 8. Dose-response effect of combined MVL, CV-N, and GNA lectins on HCVcc infection

Dose-dependent inhibition by (A) CV-N, GNA and CVN & GNA; (B) MVL, GNA and MVL & GNA; and (C) CV-N, MVL and CV-N & MVL. HCVcc assays were carried out with lectin solutions starting at concentrations 8 times their respective IC₅₀ values, with 2-fold serial dilutions ending at 1/32 of their respective IC₅₀ values The observed shift (black arrow) shows the combined effect of the lectins. (D) Combination index (CI) for each lectin mixture. The CI values are plotted as the mean of value +/− SD. CI values less than 0.85 indicate synergism, 0.85-1.1 additive effects, and greater than 1.1 antagonism. Data are representative of three independent experiments each carried out in duplicate. The dose–effect plot and combination indices were calculated with CalcuSyn 2.0 (www.biosoft.com).

Figure 9. Dose-response effect of combined lectins and neutralizing monoclonal antibody on HCVcc infection

Dose-dependent inhibition by (A) CV-N, mAb41 and CVN & mAb41; (B) MVL, mAb41 and MVL & mAb41; and (C) GNA, mAb41 and GNA & mAb41. Assays were performed as described for Figure 8. (D) Combination index (CI) for each lectin/mAb41 mixture. The CI was plotted as the mean of two independent experiments each carried out in duplicate. Error bars represent SD. CI values less than 0.85 indicate synergism, 0.85-1.1 additive, and greater than 1.1 antagonism. The dose–effect plot and combination indices were calculated with CalcuSyn 2.0 (www.biosoft.com).

Figure 10. Sialylated glycans can compete for GNA binding

Inhibition of HCVcc 1a/2a infection following incubation of GNA at IC₅₀ concentrations with increasing amounts of carbohydrates is shown. α2-6 sialyl lactosamine competes for GNA binding. Bars represent standard error of the mean. Experiments were carried out in duplicate and repeated in three independent experiments. Horizontal dotted line represents 50% inhibition.