Recombinant human L-ficolin directly neutralizes hepatitis C virus entry

L-ficolin is a soluble pattern recognition molecule expressed by the liver that contributes to innate immune defense against microorganisms. It is well described that binding of L-ficolin to specific pathogen-associated molecular patterns activates the lectin complement pathway, resulting in opsonization and lysis of pathogens. In this study, we demonstrated that in addition to this indirect effect, L-ficolin has a direct neutralizing effect against hepatitis C virus (HCV) entry. Specific, dose-dependent binding of recombinant L-ficolin to HCV glycoproteins E1 and E2 was observed. This interaction was inhibited by soluble L-ficolin ligands. Interaction of L-ficolin with E1 and E2 potently inhibited entry of retroviral pseudoparticles bearing these glycoproteins. L-ficolin also inhibited entry of cell-cultured HCV in a calcium-dependent manner. Neutralizing concentrations of L-ficolin were found to be circulating in the serum of HCV-infected individuals. This is the first description of direct neutralization of HCV entry by a ficolin and highlights a novel role for L-ficolin as a virus entry inhibitor

Multiple effects of toxins isolated from Crotalus durissus terrificus on the hepatitis C virus life cycle

Hepatitis C virus (HCV) is one of the main causes of liver disease and transplantation worldwide. Current therapy is expensive, presents additional side effects and viral resistance has been described. Therefore, studies for developing more efficient antivirals against HCV are needed. Compounds isolated from animal venoms have shown antiviral activity against some viruses such as Dengue virus, Yellow fever virus and Measles virus. In this study, we evaluated the effect of the complex crotoxin (CX) and its subunits crotapotin (CP) and phospholipase A2 (PLA2-CB) isolated from the venom of Crotalus durissus terrificus on HCV life cycle. Huh 7.5 cells were infected with HCVcc JFH-1 strain in the presence or absence of these toxins and virus was titrated by focus formation units assay or by qPCR. Toxins were added to the cells at different time points depending on the stage of virus life cycle to be evaluated. The results showed that treatment with PLA2-CB inhibited HCV entry and replication but no effect on HCV release was observed. CX reduced virus entry and release but not replication. By treating cells with CP, an antiviral effect was observed on HCV release, the only stage inhibited by this compound. Our data demonstrated the multiple antiviral effects of toxins from animal venoms on HCV life cycle

Multiple effects of toxins isolated from Crotalus durissus terrificus on the hepatitis C virus life cycle.

Hepatitis C virus (HCV) is one of the main causes of liver disease and transplantation worldwide. Current therapy is expensive, presents additional side effects and viral resistance has been described. Therefore, studies for developing more efficient antivirals against HCV are needed. Compounds isolated from animal venoms have shown antiviral activity against some viruses such as Dengue virus, Yellow fever virus and Measles virus. In this study, we evaluated the effect of the complex crotoxin (CX) and its subunits crotapotin (CP) and phospholipase A2 (PLA2-CB) isolated from the venom of Crotalus durissus terrificus on HCV life cycle. Huh 7.5 cells were infected with HCVcc JFH-1 strain in the presence or absence of these toxins and virus was titrated by focus formation units assay or by qPCR. Toxins were added to the cells at different time points depending on the stage of virus life cycle to be evaluated. The results showed that treatment with PLA2-CB inhibited HCV entry and replication but no effect on HCV release was observed. CX reduced virus entry and release but not replication. By treating cells with CP, an antiviral effect was observed on HCV release, the only stage inhibited by this compound. Our data demonstrated the multiple antiviral effects of toxins from animal venoms on HCV life cycle

Multiple effects of toxins isolated from <i>Crotalus durissus terrificus</i> on the hepatitis C virus life cycle

<div><p><i>Hepatitis C virus</i> (HCV) is one of the main causes of liver disease and transplantation worldwide. Current therapy is expensive, presents additional side effects and viral resistance has been described. Therefore, studies for developing more efficient antivirals against HCV are needed. Compounds isolated from animal venoms have shown antiviral activity against some viruses such as <i>Dengue virus</i>, <i>Yellow fever virus</i> and <i>Measles virus</i>. In this study, we evaluated the effect of the complex crotoxin (CX) and its subunits crotapotin (CP) and phospholipase A₂ (PLA₂-CB) isolated from the venom of <i>Crotalus durissus terrificus</i> on HCV life cycle. Huh 7.5 cells were infected with HCVcc JFH-1 strain in the presence or absence of these toxins and virus was titrated by focus formation units assay or by qPCR. Toxins were added to the cells at different time points depending on the stage of virus life cycle to be evaluated. The results showed that treatment with PLA₂-CB inhibited HCV entry and replication but no effect on HCV release was observed. CX reduced virus entry and release but not replication. By treating cells with CP, an antiviral effect was observed on HCV release, the only stage inhibited by this compound. Our data demonstrated the multiple antiviral effects of toxins from animal venoms on HCV life cycle.</p></div

Inhibitory activity of the toxins on HCV replication.

<p>Huh-7.5 cell line stably expressing SGR-luc-JFH-1 were treated with PLA₂-CB (A), CX (B), CP (C) at specific concentrations for 48 h. The effective concentration of inhibition (EC₅₀), the cytotoxic concentration of 50% (CC₅₀), and the selectivity index (SI = CC₅₀/EC₅₀) were calculated (D). Expression of HCV NS5A protein was measured 48 h post-treatment using western blotting assays (E). Mean values of three independent experiments each measured in triplicate including the standard deviation are shown. P < 0.001 was considered significant (*).</p

Effect of toxins on HCV replication.

<p>Huh7.5 cells were infected with JFH-1 HCVcc for 4 h, Then cells were washed extensively to remove virus and treated with toxins. Replication levels were assessed by performing the focus formation units assay 48 h post-infection. PBS was used as negative control and CsA as positive control for replication inhibition. Mean values of three independent experiments each measured in triplicate including the standard deviation are shown. P < 0.001 was considered significant (A). In an attempt to determine de antiviral mechanism of action of these toxins, synthesized dsRNA was incubated with each toxin for 45 min and submitted to electrophoresis in 1% agarose gel. PBS was used as negative control and Doxorubicin (DOX) was used as intercalating positive control (B).</p

Effect of toxins on lipids droplets.

<p>Huh-7.5 cell line stably expressing SGR-luc-JFH-1 were treated with 10 μg/mL of CX, CP or PLA₂-CB at 37° for 48 hours. Cell were fixed and nuclei, Lipid droplets (LDs) and viral protein NS5A were labelled with DAPi (blue), BODYPI 493/503 (green) and antibodies against NS5A (red), respectively. PBS was used as untreated control. Scale bars, 200 nm.</p

Effect of the toxins on HCV infectivity.

<p>Infectious supernatant and toxins were added in different times to the cells and intracellular virus was titrated 48 h post-infection by analyzing focus-forming units per milliliters (Ffu/mL). For entry assay, Huh-7.5 cells were infected with JFH-1 HCVcc and toxins were immediately added. After 4 h, the supernatant was replaced by fresh medium after repeated washes with PBS to remove completely the inoculum (A). For virucidal assay, JFH-1 HCVcc particles were incubated with toxins for 1 h prior to the infection. After that, the inoculum was used to infect naïve Huh-7.5 cells for 4 h. Cells were extensively washed and medium was added (B). In the pre-treatment assay, cells were previously treated with toxins for 1 h, washed to completely remove toxins and infected with JFH-1 virus for 4 h. Cells were then washed to virus removal and replaced with fresh media for up to 48 h post-infection (C). PBS was used as negative control and EGCG as control of entry blockage. Mean values of three independent experiments each measured in triplicate including the standard deviation are shown. P < 0.001 was considered significant.</p

Effect of toxins on CD81 cell receptors.

<p>Huh-7.5 cells were incubated with CD81/TAPA1 antibody and 10 μg/ml of CX, PLA₂-CB or PBS. Then cells were washed and incubated with secondary antibody Alexa Fluor 594. Cells were fixed with 4% paraformaldehyde and labelled for nuclei with DAPI and analyzed on Fluorescence microscopy ZEN lite 2012.</p

Crystal structure of the complex crotoxin from <i>Crotalus durissus terrificus</i> venom.

<p>The basic subunit (PLA₂-CB) is displayed in blue (A). The overall structure of crotoxin complex (B). The three chains of acid subunit (crotapotin) is shown in red [α], light pink [β] and pink [γ] and (C) (PDB ID: 3R0L).</p