Molecular Characterization of Lys49 and Asp49 Phospholipases A2 from Snake Venom and Their Antiviral Activities against Dengue virus

Abstract

We report the detailed molecular characterization of two PLA2s, Lys49 and Asp49 isolated from Bothrops leucurus venom, and examined their effects against Dengue virus (DENV). The Bl-PLA2s, named BlK-PLA2 and BlD-PLA2, are composed of 121 and 122 amino acids determined by automated sequencing of the native proteins and peptides produced by digestion with trypsin. They contain fourteen cysteines with pIs of 9.05 and 8.18 for BlK- and BlD-PLA2s, and show a high degree of sequence similarity to homologous snake venom PLA2s, but may display different biological effects. Molecular masses of 13,689.220 (Lys49) and 13,978.386 (Asp49) were determined by mass spectrometry. DENV causes a prevalent arboviral disease in humans, and no clinically approved antiviral therapy is currently available to treat DENV infections. The maximum non-toxic concentration of the proteins to LLC-MK2 cells determined by MTT assay was 40 µg/mL for Bl-PLA2s (pool) and 20 µg/mL for each isoform. Antiviral effects of Bl-PLA2s were assessed by quantitative Real-Time PCR. Bl-PLA2s were able to reduce DENV-1, DENV-2, and DENV-3 serotypes in LLC-MK2 cells infection. Our data provide further insight into the structural properties and their antiviral activity against DENV, opening up possibilities for biotechnological applications of these Bl-PLA2s as tools of research.

Antiviral activity of animal venom peptides and related compounds

“…PLA 2 -Cdt also showed in vitro activity against HIV (Table 2) [62,64], as well as the snake venom PLA 2 s NmmCM III from Naja mossambica mossambica, taipoxin from Oxyuranus scutellatus, and nigexine from Naja nigricollis [49]. Additionally, the PLA 2 variants, Lys49 and Asp49, denominated BlK-PLA 2 and BlD-PLA 2 , from Bothrops leucurus venom (Table 2), reduced dengue viral RNA in cells treated with these compounds, and presented cytotoxic activity against DENV-infected cells in vitro [65]. BlK-PLA 2 and BlD-PLA 2 have 121 and 122 amino acid residues, respectively, including seven disulfide bonds.…”

Biochemical and functional studies of ColTx-I, a new myotoxic phospholipase A2 isolated from Crotalus oreganus lutosus (Great Basin rattlesnake) snake venom

“…The characterization of this functionally versatile group of toxins awakens medicalscientific interest due to the number of potential applications for understanding envenomation, making clinical diagnosis, developing therapeutic strategies and using these toxins as molecular and biotechnological tools in pathophysiological, taxonomic, ecological studies as well as parameters for hemostatic assays Carvalho et al, 2013;Cecilio et al, 2013;Murakami and Lambeau, 2013).…”

Dengue Patients with Early Hemorrhagic Manifestations Lose Coordinate Expression of the Anti-Inflammatory Cytokine IL-10 with the Inflammatory Cytokines IL-6 and IL-8

“…28 Both transcribed RNA were generated using MEGAscript ® High Yield Transcription Kit (Ambion, Austin, TX) and were purified using the MEGAclear ™ Kit (Ambion). 30 The RNase P internal control was used as an important strategy for accurate quantification of the RNA viral load, allowing us to monitor possible false-negative results (due to RNA degradation) and to correct for variations in the amounts of the initial sample (due to different RNA recovery), thereby allowing for PCR data normalization. 29 Standard curves were generated by 10-fold serial dilutions of transcribed RNAs ranging from 10 7 to 10 3 copies/μL and used in a one-step quantitative real-time PCR (qRT-PCR).…”

“…29 Standard curves were generated by 10-fold serial dilutions of transcribed RNAs ranging from 10 7 to 10 3 copies/μL and used in a one-step quantitative real-time PCR (qRT-PCR). 30 Quantitation was performed using the StepOnePlus System The cycling program consisted of a reverse transcription step at 50°C for 30 minutes and initial denaturation at 95°C for 2 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. The detection limit of the assay was verified from successive dilutions of the standards until detection was impossible or inaccurate.…”