- freely available
Toxins 2013, 5(10), 1780-1798; doi:10.3390/toxins5101780
Published: 15 October 2013
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.
Snake venoms have been regarded for long as excellent sources for drug discovery given their structural diversity and wide variety of biological activities [1,2,3,4]. Due to the broad range of pharmacological functions, these active components have been the subject of hundreds of scientific papers in different research fields. Snake venom PLA2s (svPLA2s) are secreted enzymes (EC 18.104.22.168) that, despite their conserved structure, these molecules have been associated with a variety of pharmacological effects . They form a family of enzymes, which require Ca2+ and catalyze the hydrolysis of glycerophospholipids at the sn-2 position of the glycerol backbone to produce free fatty acids and lysophospholipids . In addition, svPLA2s are small (approximately. 14 kDa), extremely stable due to the presence of seven disulphide bridges in the globular structure and have an active site histidine. These enzymes are classified into different groups based on their structural homology, disulphide pattern, catalytic specificity, and site of expression . Group I of svPLA2s includes, the proteins mainly found in the venom of elapid and colubrid snakes (Group IA), and pancreatic svPLA2s (Group IB), while group II is found abundantly in the venom of viperidae snakes [5,7]. The latter group is similar to the mammalian nonpancreatic, inflammatory enzymes and are the major toxic proteins of the venom. Thus, they have a relevant role in immobilization and capture of the prey by interfering with physiological processes of victims. On the other hand, group II differs from group I in having an extended C-terminal tail [6,8].
In vitro, the svPLA2s can also modulate cell adhesion and cell proliferation and have anti-angiogenic properties [9,10,11]. Furthermore, a number of studies describe the anti-microbial properties of several svPLA2s against Gram negative  and Gram positive bacteria . The readers are also referred to the recent review . In addition, some svPLA2s or their products have been shown to interfere with viral infection, mainly by inhibiting the replication of HIV-1 and HIV-2 [14,15].
Over the last forty years, dengue disease has become recognized as the world’s most important mosquito-borne viral disease. Likely due to the climate change, the mosquito and the dengue virus expands into countries, which had been considered disease-free. Moreover, the virus re-emerges in countries where the disease used to be once under medical control . It is the most rapidly spreading arboviral disease that is caused by four related and antigenically distinct viruses, which are named Dengue virus (DENV) 1–4, and belong to the Flaviviridae family. Dengue infection ranks in fifth position in the list of neglected tropical diseases in the Americas in terms of disability-adjusted life years (DAYLs) . An estimated 2.5 billion people live in approximately 100 endemic countries and are at risk of acquiring dengue viral infection . DENV is generally transmitted in a cycle involving humans and mosquito vectors, mainly Aedes aegypti. DENV causes a wide range of diseases in humans, from the acute febrile illness of dengue fever (DF) to life-threatening dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS)  that can occur in hyperendemic areas with multiple circulating serotypes. Several factors such as uncontrolled urbanization and inadequate basic urban infrastructure (e.g., unreliable water supply leading householders to store water in containers close to homes) have combined to produce epidemiological conditions in developing countries in tropics and subtropics that favor viral transmission by the mosquito vector. Consequently, the number of cases of severe dengue disease continues to grow in endemic areas of Central and South America, Southeast Asia, and other subtropical regions. Children have the highest risk of developing severe disease manifestations, further underscoring the need for effective care. The total economic burden of dengue disease in the Americas was estimated to be approx. US$ 2.1 billion per year on average . Since there is no specific drug therapy for DENV, the development of an effective vaccine remains a global public health priority .
The pit viper B. leucurus (white-tailed-jararaca) is a common venomous snake which inhabits restricted areas of the northeast of Brazil including the states of Ceará and Bahia to Espírito Santo in the southeast , where snakebite envenomations represent a relevant public health problem, B. leucurus is a leading cause of human accidents . In a previous study, we reported the isolation and partial characterization of two svPLA2s from the venom of B. leucurus. One enzyme contains lysine at position 49 (BlK-PLA2), and the other, an aspartic acid in this position (BlD-PLA2). We showed that BlK-PLA2 exhibited negligible levels of phospholipase activity as compared to that of BlD-PLA2. In addition, BlD-PLA2 did not affect platelet aggregation induced by ADP, collagen, and arachidonic acid, but strongly inhibited coagulation and was able to stimulate Erlich tumor growth, but not angiogenesis . The absence of direct correlation between catalytic activity and biological effects has led to the hypothesis that specific effects of svPLA2s are due to the presence of pharmacological sites/domains on the enzyme surface . The magnitude and severity of cases of the dengue disease, which acerbates with every new epidemic, spur the search for novel bioactive compounds with antiviral activities. Several studies dedicated to molecular mechanisms and structure-function relationships reported the toxicities of svPLA2s and their neutralization. However, little is known about the antiviral properties of these enzymes. A few reports indicate that some components of snake venoms, including svPLA2s, and the enzymes from bee venom have potential anti-viral activity . At present, there are still no antiviral drugs being tested against dengue infection in any clinical trial. Many endeavors to develop DENV specific antiviral derivatives have been undertaken, and include the development and design of a low-cost rapid diagnostic tool, as well as the identification of safe compounds, which target multiple serotypes and are effective even after the onset of severe clinical disease . In this regard, an alternative approach is to evaluate bioactive compounds such as the svPLA2s that could ultimately allow the advancement of lead structures for the development of a new antiviral agent against DENV. We therefore examined whether Bl-PLA2s have antiviral properties against DENV. The antiviral effects of Bl-PLA2s detected against DENV-1, DENV-2, and DENV-3 serotypes on cells in culture could be due to enzymatic activity or perhaps due to the presence of pharmacological domains on the enzyme surface, or may be due to both, that is specific protein-protein interaction and enzymatic activity. This is a topic that is under investigation by us. A model of the molecular mechanism of action of the neurotoxic svPLA2 ammodytoxin (AtxA) from Vipera ammodytes ammodytes, which includes specific protein-protein interaction, and enzymatic activity has been recently reported .
In this study, our primary objective was to determine the structural properties of BlK- and BlD-PLA2s and more closely investigate the potential of these molecules as antiviral agents against dengue virus 1–3 serotypes. Due to their binding properties, small size and high stability, the Bl-PLA2s may represent a convenient tool for proving interactions between cell surfaces.
2. Results and Discussion
2.1. Mass Spectrometry Analysis and Sequence Determination
Snake venoms contain PLA2s isoforms exhibiting different physiological activities including antiviral properties [15,25,28]; thus, they could serve beneficial applications in medicine and as tools for biomedical research. Two Bl-PLA2s were purified to homogeneity according to the method used in previous study . The MALDI-TOF mass spectrum analysis of these proteins showed - molecular masses of 13,978.386 for BlD-PLA2 and 13,689.220 for BlK-PLA2, respectively (Figure 1), which fall in the typical range of svPLA2s. A great number of svPLA2s share similar structural features. The complete amino acid sequences of both enzymes have been determined (Figure 2), and deposited in the UniProt (accession numbers: P86974 for D49-PLA2 and P86975 for K49-PLA2). Sequence alignment of the isoforms was made by ClustalW program . They contain 121 (BlK-PLA2) and 122 (BlD-PLA2) amino acid residues determined by automated Edman degradation of the reduced and pyridylethylated proteins and of peptides produced by digestion with trypsin (data not shown). Both polypeptides display a large amino acid sequence similarity between each other and to the members of Viperidae svPLA2s (Figure 2), including the positions of the 14 cysteine residues that may form seven disulfide bridges to stabilize the tertiary structure as proposed for homologous svPLA2s [5,30]. Irrespective of any enzymatic activity the overall structures of both isoforms are very similar except for the extended C-terminal region. The presence of two short clusters of hydrophobic/basic amino acids residues 61–71 and 105–117 (Figure 2, this work) is observed in BlK49 isoform that may be the membrane-destabilizing elements as reported for Lys49 myotoxin-II from B. asper  and others. The significantly more positive surface observed for BlK49- compared to the BlD49-PLA2 isoform is in line with the higher penetrability of most basic svPLA2s compared to acidic and neutral enzymes .
2.2. Biochemical and Immunochemical Properties
Separated proteins of the crude venom by 2D-electrophoresis (2-DE) showed two protein spots at approx. 14 kDa (at alkaline pH), with pIs of 8.18 and 9.05, corresponding to BlD- and BlK-PLA2s, respectively (Figure 3A). It is known that BlK49-PLA2s present a high isoelectric point (pI > 9) and a good stability at room temperature. Two main spots that may correspond to both isoforms were immunologically recognized by the antiserum raised against BlK isoform (Figure 3B). The SDS-PAGE (15% gel) analysis also showed that crude venom contain a band of 14 kDa (Figure 4A, lane 2) which correspond to the apparent molecular masses (14 kDa) of purified BlK- and BlD-PLA2s under reduced (Figure 4A, lanes 3–4) and non-reduced conditions (not shown). Furthermore, western blotting showed these bands at approx. 14 kDa of the transferred samples BlK- and BlD-PLA2s, which were immunologically recognized by the purified anti-BlK-PLA2 IgG (Figure 4B, lanes 3 and 4). In addition, a band at the same Mr (14 kDa) was immunologically detected in the crude venom (Figure 4B, lane 2). A number of svPLA2s interact with other proteins to form complexes or aggregates and others exist as monomers . These additional molecules help svPLA2s to express their biological properties to the greatest potency. For instance, vipoxin, a heterodimeric post-synaptic neurotoxin found in V. ammodytes meridionalis, consists of two PLA2 subunits: a basic, highly toxic PLA2 and an acidic, nontoxic and enzymatically inactive PLA2 . Furthermore, when the purified rabbit anti BlK-PLA2 IgG was tested against the Bl-PLA2s (pool) and with other snake venoms by ELISA, a strong cross-recognition was observed with Bl-PLA2s, B. leucurus as well as with B. atrox. On the other hand, Crotalus durissus terrificus, Bothrops barnetti, and B. jararaca gave only low cross-reactivity signals. Finally, very low cross-reaction was detected with L.m. muta, and there was no reaction with Micrurus lemniscatus (Elapidae) (Figure 4C).
2.3. Phylogenetic Relationships among svPLA2s
A phylogenetic tree (Figure 5) was obtained by the comparison of the sequences of representative mature proteins of group II (Viperidae) listed in Figure 2, and it also includes svPLA2s of group I (Elapidae and Hydrophiidae) by the program phylogeny . The most prominent characteristic of this tree is that the members of the different subfamilies are almost perfectly clustered and clearly separated from the other subgroup. This tree splits into two main branches, Viperidae PLA2s (pit vipers and true vipers) and Elapidae together with Hydrophiidae (Lapemis hardwickii and Aipysurus eydouxii) PLA2s, which are evidently divided. This suggests a possible evolutionary relationship between the different svPLA2s. Thus, the BlK49 PLA2 (B. leucurus) was clustered in the same branch with homologous enzymes from Bothrops and Deinagkistrodon (formerly Agkistrodon) acutus snakes, while the BlD49 isoform was clustered into the same branch with Echis carinatus (S49), Protobothrops elegans (R49), and with other Viperidae svPLA2s. In some members of the group II svPLA2s the D49 residue is replaced by serine, asparagine, or arginine and are identified as K49 [35,36]. This phylogenetic relationship appears to reflect the correlation of molecular properties and suggests that the two Bl-PLA2s represent two fundamental PLA2s species with different physiological activities. Phylogenetic differences of svPLA2s is actually the result of two independent recruitment events, elapid (group I svPLA2s) and viper venoms (group II svPLA2s) . Thus, the two independently recruited types of svPLA2s have distinct activities, which the tissue-endogenous isoforms lacks (e.g., myotoxicity, antiplatelet effect, and neurotoxicity). In accordance with this, the toxic forms contain a positively charged hotspot on the surface that is lacking in the ancestral form from the pancreas .
3. Materials and Methods
3.1. Purification of BlK-PLA2 and BlD-PLA2
Two B.leucurus snakes were captured in the north of the Minas Gerais State and kept at the serpentarium of the Ezequiel Dias Foundation (FUNED, Belo Horizonte, Brazil). Venoms from these captive specimens were collected manually by milking. The pooled samples of the freeze-dried venoms were stored at 4 °C. Bl-PLA2s enzymes were purified as described in a previous work . The homogeneity of purified svPLA2s was determined by using MALDI-TOF mass spectrometry, SDS-PAGE under reducing and non-reducing conditions and by N-terminal sequence determination. All protein concentrations throughout this study were determined using a BCA assay kit (Pierce Chemical Company, Rockford, IL, USA). Anti-BlK-PLA2 antiserum was raised in a rabbit (New Zealand 2.3 kg) using as antigen the isoform K49 as described . The IgG fraction of immune rabbit serum was purified by affinity chromatography on protein A-Sepharose. The experiments reported here were performed in accordance with the guidelines established by the Brazilian College for Animal Experimentation and approved by FUNED Animal Ethics Committee.
3.2. Protein Characterization
The molecular masses (Mr) of purified BlK- and BlD-PLA2s were determined by SDS-PAGE (15% gel) and by MALDI TOF mass spectrometry. The N-terminal and the complete amino acid sequence of each protein was determined by automated Edman degradation using a Shimadzu PPSQ-21A protein sequencer according to the manufacturer’s instructions.
3.3. MALDI-TOF Mass Spectrometry
Protein masses were determined by Matrix assisted laser desorption/ionization-time-of flight (MALDI-TOF) mass spectrometry. Spectra were recorded and analyzed using a Bruker Autoflex III Smartbeam instrument in the linear positive mode controlled by the proprietary COMPASS™ 1.2 software package. The Nd-YAG-laser power (355 nm) was manually adjusted for optimal signal appearance. A freeze-dried salt- and detergent-free sample was dissolved in few microliters of 30% ACN in 0.1% TFA. 0.5 µL was spotted on a ground steel target plate, mixed with 0.5 µL matrix solution (10 mg/mL sinapinic acid in 50% ACN, 0.1% TFA) and left to dry at room temperature. Commercially available standard protein mixtures were spotted on the same target for calibration purposes prior to sample analyses.
3.4. 2D-Electrophoresis (2-DE) and Image Analysis
The proteins (60 µg) of B. leucurus venom were separated by 2-DE. Prior to running the first dimension, the IPG strips were placed in the rehydration tray and the proteins were dissolved in the De Streak Rehydrate solution (GE Health Care, Uppsala, Sweden), 0.5% IPG buffer pH 3–10 (GE Health Care). First dimension IEF was carried out in an Ettan IPGphor 3 (GE Health Care) as described by the manufacturer. Immobiline strips 7 cm, pH 3–10 linear (GE Health Care) were employed for the first dimension separation at 20 °C using a four-phase electrophoresis program: 300 V to reach 200 Vh; 1000 V to reach 300 Vh; 5000 V to reach 4000 Vh; and 5000 V to reach 1250 Vh (total accumulated 5800 Vh with 50 µA/strip). Prior to running the second dimension the proteins in the strip were reduced and alkylated by sequential incubation in the following solutions: 75 mM Tris pH 8.8, 6 M urea, 3% glycerol, 2% SDS, 0.002% bromophenol blue (equilibration buffer-EB), 10 mg/mL DTT for 20 min; and then a solution of 25 mg/mL iodoacetamide in EB for 20 min. In addition, SDS-PAGE was done in a mini gel 7.5 cm 15% polyacrylamide gel. Proteins were visualized using Coomassie blue staining. Direct scanning and image analysis was performed using an Image Master 2D Platinum 7 (GE Health Care).
3.5. Protein Sequencing
The thiol groups of purified proteins (1.5 mg each) were S-reduced and alkylated with vinyl pyridine as described . The material was dissolved in 1 mL of 0.1 M Tris-HCl (pH 8.6), 6 M guanidine-HCl. After addition of 30 µl β-mercaptoethanol the samples were incubated first at 50 °C for 4 h under nitrogen, then with 40 µL of 4-vinyl pyridine in the dark at 37 °C for 2 h and subsequently desalted on a Vydac C4 column with a gradient of 0%–60% acetonitrile in 0.1% TFA. The S-pyridylethylated (PE) proteins were digested with trypsin (2% w/w, enzyme:substrate in 1 mL of 0.1 M ammonium bicarbonate, pH 7.9) for 3.5 h at 37 °C. The cleavage products were separated on a Vydac C18 small pore column (4.6 × 250 mm) in a linear gradient of 0%–50% acetonitrile in 0.1% aqueous TFA and sequenced using a Shimadzu PPSQ 21A protein sequencer. The primary structures of BlK- and BlD-PLA2s were compared with the sequences of other related proteins in the SWISS-PROT/TREMBL data bases using the FASTA and BLAST programs.
3.6. Immunoblot and Enzyme-Linked Immunoabsorbent Assay (ELISA)
For analysis of immunological reactivity of several different snake venoms against the anti-BlK-PLA2 IgG, western immunoblotting and ELISA were used. The BlK- and BlD-PLA2s isoforms (4 µg each) was subjected to SDS-PAGE (15% gel) under reducing conditions, then eletrophoretically transferred onto a nitrocellulose membrane according to the manufacturer’s (Bio-Rad, Richmond, CA, USA) instructions. One gel of 2-DE containing separated proteins from B. leucurus venom was also used for immunoblot assay under similar experimental conditions. ELISA plate was coated with 100 µL of 0.5 µg/well of each antigen (Bl-PLA2s or crude venoms) in 0.05 M carbonate buffer, pH 9.6. After washing with 0.05% Tween-saline, a blocking solution (2% casein in phosphate buffered saline-PBS) was added (1 h at room temperature). After two washes with the same solution, anti-BlK-PLA2 antibody previously diluted in PBS containing 0.25% casein and 0.05% Tween 20 (0.015 to 2 µg/well) was added and incubated for 1 h at 37 °C. After six washes, peroxidase-coupled anti-rabbit IgG (Sigma, St. Louis, MO, USA, diluted 1:12,000) was added and incubated for 1 h at room temperature. The wells were washed and 100 µL of peroxidase substrate OPD (0.33 mg/mL in citrate buffer, pH 5.2, in the presence of 0.012% H2O2) was added and the color reaction developed for 1 h in the dark. Absorbance was read with a micro-plate reader at 492 nm.
3.7. Cells and Viruses
Aedes albopictus cells (C6/36 Line, mosquito cells) were maintained in Leibovitz’s medium (L-15, Gibco/Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Gibco/Invitrogen), 100 U/mL penicillin G, and 100 µg/mL streptomycin (Gibco/Invitrogen) at 28 °C in a B.O.D. incubator. Rhesus Monkey Kidney Epithelial Cells (LLC-MK2 Line) were maintained with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin at 37 °C in a humidified 5% CO2 atmosphere. The viruses DENV-1 (Brasil/98), DENV-2 (SpH 125367) and DENV-3 (RibH 1) were propagated in C6/36 cells and titrated using Dulbecco plaque technique  in LLC-MK2 cells. Aliquots were stored at −80 °C until required.
3.8. Determination of the Non-Cytotoxic Concentrations
The non-cytotoxic concentrations of the compounds Bl-PLA2s (Pool, partially purified fraction containing both PLA2s) and their isoforms BlK- and BlD-PLA2s were determined by assessment of cell viability using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay (Sigma). Confluent LLC-MK2 cells monolayers (4 × 104 cells/well) in 96-well microplates were exposed to different concentrations (0.1, 1.0, 10.0, 20.0, 40.0, and 80.0 µg/mL) of Bl-PLA2s (Pool), and the purified enzymes for 48 h in a CO2 incubator. After the incubation period, cells were observed using an inverted optical microscope (Nikon, Tokyo, Japan) and washed twice with PBS buffer (136.9 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4). Then, 100 µL of MTT solution (0.5 mg/mL in DMEM) was added to each well and the plate was maintained in a CO2 incubator. After 3 h of incubation at 37 °C the plates were centrifuged at 400 × g for 10 min. The supernatant was removed and 50 µL of dimethyl sulfoxide (DMSO) was added to each well to solubilize the formazan crystals. The absorbance was measured at 540 nm using an automated Microplate Reader, Pharmacia Biotech Ultrospec 1000. The cellular viability was calculated by comparison with untreated cells, which was set to 100% viability. Each data point was the mean value of triplicates from three independent experiments. The highest concentration of each compound without any significant toxicity (p < 0.05) was considered as the maximum non-toxic concentration (MNTC).
3.10. RNA Extraction
RNA was extracted from the supernatants (140 μL) of antiviral assays using QIAamp® Viral RNA Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol. An exogenous control (10 μL of cloned RNase P, corresponding to 1 × 105 RNA copies) was added to each sample prior to RNA extraction. Finally, the RNA was eluted in 60 μL of diethyl pyrocarbonate (DEPC) treated water (Sigma) and maintained at −80 °C.
3.11. Generation of RNA Standard
Using pGEM-T Easy vector (Promega, Madison, WI, USA), two plasmids were constructed by cloning the 67 bp amplicon of 3'UTR region from DENV-1, consensus to the four serotypes of DENV , and 64 bp amplicon from Human RNAseP . The amplicons were generated by SuperScript® III Platinum One-Step qRT-PCR System (Invitrogen, Carlsbad, CA, USA) using the primers shown in Table 1, according to the manufacture’s instructions. The presence and orientation of the insert DNA were confirmed by sequencing. The plasmids were linearized by digestion with Nde I and the target sequences were amplified using MEGAscript® High Yield Transcription Kit (Ambion, Austin, TX, USA). The transcribed RNAs were treated with DNAse (Invitrogen) and purified using the MEGAclear™Kit (Ambion). The RNAs were quantified by spectrophotometry (Genequant, Amersham Pharmacia Biotec, Cambridge, England). The copy numbers of the RNAs were calculated based on the concentrations and their molecular weights and 10-fold serial dilutions of these RNAs, ranging from 107 to 103 copies/µL were used as standards in all qRT-PCRs. The detection limit of the assay was verified from successive dilutions of the standards until detection was impossible or inaccurate.
|Table 1. Probes and primer sequences for Dengue virus, DENV (3'UTR) and exogenous control (RnaseP) used in quantitative Real-Time PCR assay.|
|Target||Sequence (5'-3')||Nucleotide position|
3.12. Quantitative Real-Time PCR
Quantitative Real-Time PCR was performed using a Real-Time PCR System StepOnePlus (Applied Biosystems, Foster City, CA, USA) and SuperScript™ III Platinum® One-Step qRT-PCR System (Invitrogen). Amplifications were carried out in reaction mixtures containing 5 μL of transcribed or viral RNA, 0.5 μL of SuperScript™/Platinum® Taq Mix, 2x reaction mix buffer, 0.4 μM of primers to DENV, 0.2 μM of primers to RnaseP, 0.2 μM of TaqMan probe to DENV, 0.1 μM of TaqMan probe to RnaseP (Applied Biosystems) (see Table 1), 0.25 μL of ROX Reference Dye, 0.5 μL of RNaseOUT™ (Invitrogen), and DEPC-treated water to 25 μL final volume. The cycling program consisted of an RT step at 50 °C for 30 min, initial denaturation at 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Each reaction set was checked for contamination using negative control (all reagents included and water instead of DNA). In addition to this negative control, cells not infected were included in the assay.
3.13. Statistical Analysis
Data were expressed as means ± standard error (SE). Statistical differences were determined by the nonparametric Tukey’s Multiple Comparison Test. Pearson’s correlation (R) was used to evaluate the relationship between Cycle threshold (Ct) and quantity of RNA on standard curves of qRT-PCR. Values of p < 0.05 were considered significant.
We have described the main structural properties of two basic svPLA2s, BlK- without and BlD-PLA2s with hydrolytic activity from B. leucurus venom. The promising results observed in our antiviral assays, with decreased amounts of viral RNA quantified in cells treated with Bl-PLA2s, opening up possibilities for biotechnological applications of these molecules as research tools. Furthermore, the data suggest that, in addition to catalytic activity, the physiological role of BlPLA2s can be mediated by ligation to specific receptors on the cell membrane, which is in accord with the higher penetrability of most basic svPLA2s compared to neutral and acidic enzymes  and others. Thus, consistent with these findings, Bl-PLA2s display cytotoxic properties against DENV in vitro, suggesting that they are useful tools against DENV or as a prototype to develop anti-dengue drug.
We thank Gena Medeiros for her collaboration. This work was supported by the Brazilian Agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Grant No: 482502/2012-6 to EFS), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Grant No: 23038000825/2011-63), and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG, Grant No: CBB APQ 01791-10 to EFS), and Deustsche Forschungsgemeinschaft (DFG grant: SFB815, Project A6 to JAE).
Conflicts of Interest
The authors declare no conflicts of interest.
- Koh, D.C.I.; Armugan, A.; Jeyaseelan, K. Snake venom components and their applications in biomedicine. Cell. Mol. Life Sci. 2006, 63, 3030–3041, doi:10.1007/s00018-006-6315-0.
- Kini, R.M. Toxins in thrombosis and haemostasis, potential beyond imagination. J. Thromb. Haemost. 2011, 9, 193–208.
- Koh, C.Y.; Kini, R.M. From snake venom toxins to therapeutic-cardiovascular examples. Toxicon 2012, 59, 497–506, doi:10.1016/j.toxicon.2011.03.017.
- Lomonte, B.; Sasa, A.Y.; Gutierrez, J.M. The phospholipase A2 homologues of snake venoms, biological activities and their possible adaptative roles. Protein Pept. Lett. 2009, 16, 860–876, doi:10.2174/092986609788923356.
- Samy, R.P.; Gopalakrishnakone, P.; Kesturu, G.; Swamy, S.N.; Hemshekhar, M.; Tan, K.S.; Rowan, G.E.G.; Stiles, B.G.; Chow, V.T.K. Snake venom phospholipases A2: A novel tool against bacterial diseases. Curr. Med. Chem. 2012, 19, 6150–6162, doi:10.2174/092986712804485791.
- Doley, R.; Zhou, X.; Kini, R.M. Snake Venom Phospholipase A2 Enzymes. In Handbook of Venoms and Toxins of Reptiles; Mackessy, S.P., Ed.; CRC Press: New York, NY, USA, 2010; pp. 173–198.
- Huang, P.; Mackessy, S.P. Biochemical characterization of phospholipase A2 (trimorphin) from the venom of the Sonoran lyre snake Trimorphodon biscutatus lambda (family Colubridae). Toxicon 2004, 44, 25–43.
- Six, D.A.; Dennis, E.A. The expanding of superfamily of phospholipase A2 enzymes, classification and characterization. Biochim. Biophys. Acta 2000, 1488, 1–19.
- Bazaa, A.; Luis, J.; Srairi-Abid, N.; Kallech-Ziri, O.; Kessentini-Zouari, R. MVL-PLA2; a phospholipase A2 from Microvipera lebetine transmediterranea venom; inhibits tumor cells adhesion and migration. Matrix Biol. 2009, 28, 188–193, doi:10.1016/j.matbio.2009.03.007.
- Bazaa, A.; Pasquier, E.; Defilles, C.; Limam, I.; Kessentini-Zouari, R.; Kallech-Ziri, O.; el Battari, A.; Braguer, D.; el Ayeb, M.; Marrakchi, N.; et al. MVL-PLA2; a snake venom phospholipase A2; inhibits angiogenesis through an increase in microtubule dynamics and disorganization of focal adhesions. PLoS One 2010, 5, e10124, doi:10.1371/journal.pone.0010124.
- Khunsap, S.; Pakmanee, N.; Khow, O.; Chanhome, L.; Sitprija, V.; Suntravat, M.; Lucena, S.E.; Perez, J.C.; Sánchez, E.E. Purification of a phospholipase A2 from Daboia russelli siamensis venom with anticancer effects. J. Venom. Res. 2011, 2, 42–51.
- Samy, R.P.; Pachiappan, A.; Gopalakrishnakone, P.; Thwin, M.M.; Hian, Y.E.; Chow, V.T.K.; Bow, H.; Weng, J.T. In vitro antimicrobial activity of natural toxins and animal venoms tested against Burkholderia pseudomallei. BMC Infect. Dis. 2006, 6, 1–16, doi:10.1186/1471-2334-6-1.
- Vargas, L.J.; Londoño, M.; Quintana, J.C.; Rua, C.; Segura, C.; Lomonte, B.; Nuñez, V. An acidic phospholipase A2 with antibacterial activity from Porthidium nasutum venom. Comp. Biochem. Physiol. B 2012, 161, 341–347, doi:10.1016/j.cbpb.2011.12.010.
- Gunther, A.G.; Stegmann, T. How lysophosphatidylcholine inhibits cell-cell fusion mediated by the envelope glycoprotein of human immunodeficiency virus. Virology 1997, 235, 201–208, doi:10.1006/viro.1997.8699.
- Fenard, D.; Lambeau, G.; Valentin, E.; Lefebvre, J.C.; Lazdunski, M.; Doglio, A. Secreted phospholipases A2; a new class of HIV inhibitors that block virus entry into host cells. J. Clin. Invest. 1999, 104, 611–618, doi:10.1172/JCI6915.
- Ooi, E.E.; Gubler, D.J. Global spread of epidemic dengue, the influence of environmental change. Future Virol. 2009, 4, 571–580, doi:10.2217/fvl.09.55.
- Hotez, P.J.; Bottazzi, M.E.; Franco-Paredes, C.; Ault, S.K.; Periago, M.R. The neglected tropical diseases of Latin America and the Caribbean, a review of disease burden and distribution of a roadmap for control and elimination. PLoS Negl. Trop. Dis. 2008, 2, e300, doi:10.1371/journal.pntd.0000300.
- World Health Organization (WHO). Impact of Dengue. Available online: http//www.who.int/csr/disease/dengue/impact/em (accessed on 31 May 2013).
- Thomas, S.J.; Strickman, D.; Vuaghan, D.W. Dengue epidemiology; ecology; resurgence. Adv. Virus Res. 2003, 61, 235–289, doi:10.1016/S0065-3527(03)61006-7.
- Shepard, D.S.; Coudeville, L.; Halasa, Y.A.; Zambrano, B.; Dayan, G.H. Economic impact of dengue illness in the Americas. Am. J. Trop. Med. Hyg. 2011, 84, 200–207, doi:10.4269/ajtmh.2011.10-0503.
- Whitehead, S.S.; Blaney, J.E.; Durbin, A.P.; Murphy, B.R. Prospects for a dengue virus vaccine. Nat. Rev. Microbiol. 2007, 5, 518–528, doi:10.1038/nrmicro1690.
- Porto, M.; Teixeira, D.M. Bothrops leucurus (White-tailed-lancehead). Herpetol. Rev. 1995, 26, 156.
- Sanchez, E.F.; Eble, J.A. P-III Metalloproteinase (Leucurolysin-B) from Bothrops Leucurus Venom, Isolation and Possible Inhibition. In Drug Design of Zinc-Enzyme Inhibitors; Supuran, C.T., Winum, J.-Y., Eds.; John Wiley & Sons Inc: Hoboken, NJ, USA, 2009; pp. 789–812.
- Higuchi, D.A.; Barbosa, C.M.V.; Bincoleto, C.; Chagas, J.R.; Magalhaes, A.; Richardson, M.; Sanchez, E.F.; Pesquero, J.B.; Araujo, R.C.; Pesquero, J.L. Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom. Biochimie 2007, 89, 319–328, doi:10.1016/j.biochi.2006.10.010.
- Muller, V.D.M.; Russo, R.R.; Cintra, A.D.O.; Sartim, M.A.; Alves-Paiva, R.M.; Figueiredo, L.T.M.; Sampaio, S.V.; Aquino, V.H. Crotoxin and phospholipases A2 from Crotalus. durissus terrificus showed antiviral activity against dengue and yellow fever viruses. Toxicon 2012, 59, 507–515, doi:10.1016/j.toxicon.2011.05.021.
- Julander, J.G.; Perry, S.T.; Shresta, S. Important advances in the field of anti-dengue virus research. Antivir. Chem. Chemother. 2011, 21, 105–116, doi:10.3851/IMP1690.
- Mattiazzi, M.; Sun, Y.; Wolinski, H.; Bavdek, A.; Petan, T.; Anderluh, G.; Kohlwein, S.D.; Drubin, D.G.; Krizaj, I.; Petrovic, U. A neurotoxic phospholipase A2 impairs yeast amphiphysin activity and reduces endocytosis. PLoS One 2012, 7, e40931, doi:10.1371/journal.pone.0040931.
- Petricevich, V.L.; Mendonça, R.Z. Inhibitory potential of Crotalus durissus terrificus venom on measles virus growth. Toxicon 2003, 42, 143–153, doi:10.1016/S0041-0101(03)00124-7.
- Larkin, M.A.; Blackshilds, G.; Brown, N.P.; Chenna, R.; McGettigan, P.A.; McWilliam, H.; Valentin, F.; Wallace, I.M.; Wilm, A.; Lopez, R.; et al. Clustal and Clustal X version 2.0. Bioinformatics 2007, 23, 2947–2948, doi:10.1093/bioinformatics/btm404.
- Scott, D.L. Phospholipase A2 Structure and Catalytic Properties. Kini, R.M., Ed.; John Wiley & Sons: Chichester, UK, 1997; pp. 97–128.
- Páramo, L.; Lomonte, B.; Pizarro-Cerda, J.; Bengoechea, J.A.; Gorvel, J.P.; Moreno, E. Bactericidal activity of Lys49 and Asp49 myotoxic phaspholipases A2 from Bothrops asper snake venom. Eur. J. Biochem. 1998, 253, 452–561.
- Watanabe, L.; Fontes, M.R.; Soares, A.M.; Giglio, J.R.; Arni, R.K. Initiating structural studies of Lys49-PLA2 homologues complexed with an anionic detergent; a fatty acid and a natural lipid. Protein Pep. Lett. 2003, 10, 525–530, doi:10.2174/0929866033478726.
- Freedman, J.E.; Snyder, S.H. Vipoxin. A protein from Russell’s viper venom with high affinity for biogenic amine receptors. J. Biol. Chem. 1981, 256, 13172–13179.
- Guindon, S.; Dufayard, J.F.; Lefort, V.; Anisimova, M.; Hordijk, W.; Gascuel, O. New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst. Biol. 2010, 59, 307–321, doi:10.1093/sysbio/syq010.
- Polgar, J.; Magnenat, E.M.; Peitsch, M.C.; Wells, T.N.; Clemetson, K.J. Asp-49 is not an absolute prerequisite for the enzymic activity of low-Mr phospholipase A2, Purification; characterization and computer modelling of an enzymically active Ser-49 phospholipase A2; ecarpholin S; from the venom of Echis carinatus sochureki (saw-scaled viper). Biochem. J. 1996, 319, 965–968.
- Chijiwa, T.; Tokunaga, E.; Ikeda, R.; Tereda, K.; Ogawa, T.; Oda-Ueda, N.; Hattori, S.; Nozaki, M.; Ohno, M. Discovery of novel Arg49 phospholipase A2 isozyme from Phrotobothrops elegans venom and regional evolution of Crotalinae snake venom phospholipase A2 isozymes in the southwestern islands of Japan and Taiwan. Toxicon 2006, 48, 672–682, doi:10.1016/j.toxicon.2006.06.021.
- Fry, B.G.; Wüster, W. Assembling an arsenal, Origin and evolution of the snake venom proteome inferred from Phylogenetic analysis of toxin sequences. Mol. Biol. Evol. 2004, 21, 870–883, doi:10.1093/molbev/msh091.
- Fry, B.G.; Scheib, H.; van der Weer, L.; Young, B.; McNaughtan, J.; Ramjan, S.F.R.; Vidal, N.; Poelmann, R.E.; Norman, J.A. Evolution of an arsenal. Mol. Cell. Proteomics 2008, 7, 215–246.
- Cummings, K.L.; Tarleton, R.L. Rapid quantitation of Trypanosoma cruzi in host tissue by real-time PCR. Mol. Biochem. Parasitol. 2003, 129, 53–59, doi:10.1016/S0166-6851(03)00093-8.
- Trane, L.D.; Bedini, B.; Donatelli, I.; Campitelli, L.; Chiappini, B.; de Marco, M.A.; Delogu, M.; Buonaboglia, C.; Vaccari, G. A sensitive one-step real time PCR for detection of avian influence viruses using a MGB probe and an internal positive control. BMC Infect. Dis. 2006, 6, 87, doi:10.1186/1471-2334-6-87.
- Caldas, S.; Caldas, I.S.; Diniz, L.F.; Lima, W.G.; Oliveira, R.P.; Cecílio, A.B.; Ribeiro, I.; Talvania, A.; Bahia, M.T. Real-Time PCR strategy for parasite quantification in blood and tissue samples of experimental Trypanosoma cruzi infection. Acta Trop. 2012, 123, 170–177, doi:10.1016/j.actatropica.2012.05.002.
- Naumann, G.B.; Silva, L.F.; Silva, L.; Faria, G.; Richardson, M.; Evangelista, K.; Kohlhoff, M.; Gontijo, C.M.F.; Navdaev, A.; Rezende, F.F.; et al. Cytotoxicity and inhibition of platelet aggregation caused by an L-amino acid oxidase from Bopthrops leucurus venom. Biochim. Biophys. Acta 2011, 1810, 683–694.
- Wilson, K.J.; Yuan, P.M. Protein and Peptide Purification. In Protein Sequencing a Practical Approach; Findlay, J.B.C., Geisow, M.J., Eds.; IRL Press: Oxford, UK, 1989; pp. 3–41.
- Dulbecco, R. Microbiology. In Evolution of Microbiology and Microbes; Dulbecco, R., Ed.; Lippincott Company: Philadelphia, PA, USA, 1994; pp. 769–795.
- Gurukumar, K.R.; Priyadarshini, D.; Patil, J.A.; Bhagat, A.; Singh, A.; Shah, S.; Cecilia, D. Development of real time PCR for detection and quantitation of Dengue Viruses. Virol. J. 2009, 6, 10, doi:10.1186/1743-422X-6-10.
- World Health Organization. CDC protocol of real time RT PCR for influenza A (H1N1). Available online: http://www.who.int/csr/resources/publications/swineflu/CDCrealtimeRTPCRprotocol20090428.pdf (accessed on 27 June 2013).
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).