Phospholipase A₂ Isolated from the Venom of <i>Crotalus durissus terrificus</i> Inactivates <i>Dengue virus</i> and Other Enveloped Viruses by Disrupting the Viral Envelope

<div><p>The <i>Flaviviridae</i> family includes several virus pathogens associated with human diseases worldwide. Within this family, <i>Dengue virus</i> is the most serious threat to public health, especially in tropical and sub-tropical regions of the world. Currently, there are no vaccines or specific antiviral drugs against <i>Dengue virus</i> or against most of the viruses of this family. Therefore, the development of vaccines and the discovery of therapeutic compounds against the medically most important flaviviruses remain a global public health priority. We previously showed that phospholipase A₂ isolated from the venom of <i>Crotalus durissus terrificus</i> was able to inhibit <i>Dengue virus</i> and <i>Yellow fever virus</i> infection in Vero cells. Here, we present evidence that phospholipase A₂ has a direct effect on <i>Dengue virus</i> particles, inducing a partial exposure of genomic RNA, which strongly suggests inhibition via the cleavage of glycerophospholipids at the virus lipid bilayer envelope. This cleavage might induce a disruption of the lipid bilayer that causes a destabilization of the E proteins on the virus surface, resulting in inactivation. We show by computational analysis that phospholipase A₂ might gain access to the <i>Dengue virus</i> lipid bilayer through the pores found on each of the twenty 3-fold vertices of the E protein shell on the virus surface. In addition, phospholipase A₂ is able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for developing broad-spectrum antiviral drugs.</p></div

Analysis of the exposure of DENV-2 genomic RNA.

<p>DENV-2 was first treated with PLA₂-CB, crotoxin (8 ng/µL each) or PBS at 37°C and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences among groups (*p<0.05).</p

Analysis of the exposure of DENV-2 RNA.

<p>DENV-2 was first treated with proteinase K, Triton X-100 and PBS and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (**p<0.01).</p

Virucidal assay.

<p>DENV-2 was treated with different concentrations of PLA₂-CB (a) and crotoxin (b) and then used to infect Vero cells for 72 h. The antiviral effect of the toxins was evaluated by determining the virus titer in the cell culture supernatant by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (*<i>p</i><0.05, **p<0.01).</p

Pre-treatment assay.

<p>Vero cells were treated with different concentrations of PLA₂-CB (a) and crotoxin (b) and then infected with DENV-2 for 72 h. The antiviral effect of the toxins was evaluated by determining the virus titer in the cell culture supernatant by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated cells (*p<0.05, **p<0.01).</p

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

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

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

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