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