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Potent virucidal activity against Flaviviridae of a group IIA phospholipase A2 isolated from the venom of Bothrops asper
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Potent virucidal activity against Flaviviridae of a group IIA phospholipase A2 isolated from the venom of Bothrops asper Hebleen Brenesa, Gilbert D. Loríab, Bruno Lomontec,∗ a
Instituto Costarricense de Investigación y Enseñanza en Nutrición y Salud (INCIENSA), Tres Ríos, Cartago, Costa Rica Sección de Virología, Centro de Investigación en Enfermedades Tropicales (CIET), and Centro de Investigaciones en Hematología y Trastornos Afines (CIHATA), Universidad de Costa Rica, San José, Costa Rica c Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica b
A R T I C LE I N FO
A B S T R A C T
Keywords: Phospholipase A2 Virucidal Flaviviridae Enveloped virus Dengue Snake venom
Secreted phospholipase A2 (sPLA2) molecules are small, calcium-dependent enzymes involved in many biological processes. Viperid venoms possess gIIA sPLA2s and sPLA2-like proteins, both having homology to human gIIA sPLA2, an innate immunity enzyme. We evaluated the antiviral action of Mt–I (catalytically-active sPLA2) and Mt-II (catalytically-inactive variant) isolated from the venom of Bothrops asper, against a diverse group of viruses. Yellow Fever and Dengue (enveloped) viruses were highly susceptible to inactivation by the snake proteins, in contrast to Sabin (non-enveloped; Polio vaccine strain), and Influenza A, Herpes simplex 1 and 2, and Vesicular Stomatitis (enveloped) viruses. Titration of the antiviral effect against Dengue virus revealed Mt–I to be highly potent (IC50 0.5–2 ng/mL), whereas Mt-II was 1000-fold weaker. This large difference suggested a requirement for PLA2 activity, which was confirmed by chemical inactivation of Mt–I. A synthetic peptide representing the membrane-disrupting region of Mt-II, previously shown to have bactericidal effect, lacked antiviral action, suggesting that the weak virucidal effect observed for Mt-II is likely caused by contamination with traces of Mt–I. On the other hand, Mt–I was demonstrated to act by a direct virucidal mechanism prior to infection, and not by an independent effect on host cells, either pretreated, or exposed to Mt–I after virus infection. Interestingly, DENV2 propagated in mosquito cells was much more sensitive to the action of Mt–I, compared to human cell-propagated virus. Therefore, differences in envelope membrane composition may be crucially involved in the observed virucidal action of PLA2 enzymes.
1. Introduction Phospholipases A2 (PLA2s; EC 3.1.1.4) constitute a large superfamily of hydrolytic enzymes with specificity for the ester bond at the sn-2 position of glycerophospholipids, which release free fatty acids and lysophospholipids [1]. These enzymes are ubiquitous across the tree of life [2,3] and abundantly found in snake venoms, where they present a wide variety of bioactivities [4]. Snake venom PLA2s belong to the secreted type of enzymes (sPLA2), classified in the structural groups IB (in Elapidae snakes) and IIA (in Viperidae snakes). The former show homology to the mammalian pancreatic juice PLA2, whereas the latter are homologous to the mammalian ‘inflammatory’ PLA2, a relevant component of innate immunity [5]. All sPLA2s share a conserved catalytic mechanism based on a His/Asp dyad using Ca2+ as an essential cofactor. Although the structural scaffold of group I and group II PLA2s is essentially conserved, these small (~14 kDa) enzymes differ by the
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position of one out of their seven disulfide bonds, and by the presence of an extended C-terminal segment (5–7 amino acids) in the latter [6]. Among the group II sPLA2s of viperids, a group of natural variants exists presenting key amino acid substitutions that preclude Ca2+ binding, and hence, catalytic activity. Such proteins are commonly referred to as ‘PLA2-like’ variants or ‘Lys49 PLA2-homologues' [7,8]. Antimicrobial effects have been described for both mammalian and snake venom sPLA2s acting against bacteria [9–13], parasites [14,15], or viruses [16–22]. In some cases, these effects depend on the catalytic action of the sPLA2s, but catalytic-independent actions have also been evidenced. For example, Fenard and coworkers [21] reported that several venom sPLA2s display potent inhibition of HIV-1 replication by a mechanism not involving the catalytic activity of the enzymes. Viral diseases remain a major health burden worldwide, and more efforts are undoubtedly required to search for new molecules with antiviral actions that could lead to novel therapeutic options [22]. Reports
Corresponding author. Instituto Clodomiro Picado Facultad de Microbiología Universidad de Costa Rica, San José, 11501, Costa Rica. E-mail addresses: [email protected] (H. Brenes), [email protected] (G.D. Loría), [email protected] (B. Lomonte).
https://doi.org/10.1016/j.biologicals.2019.12.002 Received 22 October 2019; Received in revised form 20 November 2019; Accepted 8 December 2019 1045-1056/ © 2019 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Hebleen Brenes, Gilbert D. Loría and Bruno Lomonte, Biologicals, https://doi.org/10.1016/j.biologicals.2019.12.002
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gradient toward acetonitrile stepped from 0 to 25% in 5 min, 25–50% in 22 min, 50–70% in 1 min, and was sustained at 70% for 2 min, for a total run time of 30 min. The isolated proteins were collected, dried by vacuum centrifugation (Vacufuge), redissolved at 0.1 mg/mL in Minimum Essential Medium Eagle (MEM) containing 2% v/v fetal calf serum, aliquoted and stored at −80 °C until use.
on the antiviral effects of some sPLA2s of venom origin prompted us to explore the action of two proteins purified from the venom of Bothrops asper, a viperid species found in Central America. This venom contains both enzymatically-active sPLA2 (Mt–I) and catalytically-inactive sPLA2-like protein (Mt-II) isoforms [23], and could therefore provide a useful comparison to understand the role of enzymatic activity in the antiviral action of sPLA2s. Previous studies have reported virus-inhibiting effects of snake venom sPLA2s belonging to the structural group IB of enzymes [20,21]. Among group IIA counterparts, antiviral action has been described for the sPLA2 subunit of crotoxin (a heterodimeric complex found in the venom of the South American rattlesnake Crotalus durissus terrificus) [17,18,21], and for Asp49 and Lys49 sPLA2s of Bothrops leucurus [19]. In this study, we describe a potent antiviral effect against enveloped viruses of the Flaviviridae family, but not against other enveloped viruses, of Mt–I, a catalytically-active sPLA2 isoform of B. asper venom. Its virucidal action was shown to depend on enzymatic activity. In agreement with this, the non-enzymatic Mt-II variant showed a 1000fold weaker effect than Mt–I, likely caused by traces of Mt–I contamination. This is also supported by the observation that the bioactive synthetic peptide p115-129 of Mt-II was devoid of antiviral effect.
2.4. Mt–I and Mt-II antiviral activity The viruses were mixed with each of the proteins, at a final concentration of 50 μg/mL, and these mixtures were incubated for 1 h at 37 °C, followed by virus quantification. For comparison, virus was incubated with MEM 2% FBS alone and treated identically. In addition, the inhibitory concentrations IC50 and IC90 were estimated by preparing double dilutions of Mt–I and Mt-II in MEM 2% BSA and incubating each one for 1 h at 37 °C with DENV-1, followed by virus quantification as described. 2.5. Inactivation of the catalytic activity of Mt–I Mt–I was chemically modified with p-bromophenacyl bromide (pBPB) as previously described [24]. This procedure alkylates the single histidine residue at the catalytic site of the enzyme, abrogating its ability to hydrolyze phospholipids [25]. Inactivation of enzymatic activity was evaluated using the synthetic monodisperse substrate 4-nitro3-octanoylbenzoic acid (NOBA) [24].
2. Materials and methods 2.1. Cells and viruses BHK-21 and Vero cells were grown in MEM enriched with 10% FBS at 37 °C and 5% CO2; C636, MDCK, and HepG2 cells were grown with RPMI 10% FBS at 37 °C (MDCK and HepG2) and 28 °C (C636) in 5% CO2. DENV-1 (Angola strain), DENV-2 (Jamaica strain), DENV-3 (Nicaragua strain), DENV-4 (Dominica strain), YFV (17D vaccine strain), HSV-1 (ATCC VR-733), HSV-2 (ATCC VR-734), Influenza A virus (H3N2, Alice vaccine strain) and Polio virus (Sabin vaccine strain) were obtained from the virus collection of the Section of Virology, School of Microbiology, UCR. VSV-IND and VSV-NJ were kindly provided by Dr. Carlos Jiménez from the School of Veterinary Medicine, National University of Costa Rica (UNA).
2.6. Synthetic peptide 115–129 of Mt-II The amino acid sequence 115–129 (KKYRYYLKPLCKK; common numbering system of [26]), near the C-terminal region of Mt-II, was synthesized using Fmoc strategy with free N-terminus and amidated Cterminus (Peptide 2.0; Chantilly, VA, USA) and purified to > 95%. This peptide has been shown to mimick the enzymatic-independent membrane damaging action of the parent protein against eukaryiotic cells [27,28], and against bacteria [11]. 2.7. Virucidal model
2.2. Virus quantification The virucidal activity corresponds to the strategy described previously, in which the Mt–I or Mt-II were allowed to act directly on the virus particles before infecting the cell monolayer.
Virus quantification was carried out by plaque formation assay. In brief, cell monolayers were inoculated with 10−1 to 10−6 dilutions of the samples, and then adsorbed for 1 h at 37 °C and 5% CO2, agitating every 15 min. Then, cell culture media containing 1% carboxymethyl cellulose was added, and plaque formation was allowed to proceed until appropriate-sized plaques were observed. Cells were fixed with 3.7% formalin and stained with violet crystal. In the case of Influenza A virus, TPCK trypsin was added to the Dulbecco medium, which allows virus particle maturation.
2.8. Pre-infection model The uninfected monolayer was covered with 100 μL of a 50 μg/mL solution of Mt–I or Mt-II, and after 1 h incubation at 37 °C, 5% CO2, the supernatant was removed and five sterile-PBS washing steps were done. The monolayer was then infected with the virus and quantification was done as described previously.
2.3. Mt–I and Mt-II isolation 2.9. Post-infection model Crude venom of Bothrops asper was a pool obtained from more than 20 specimens kept at the Serpentarium facility of Instituto Clodomiro Picado, University of Costa Rica. The lyophilized venom (200 mg) was dissolved in 0.1 M Tris, 0.1 M KCl (pH 7.0) buffer and fractionated by cation-exchange chromatography on a CM-Sephadex C25 column (20 × 2 cm) equilibrated with the same buffer. Elution was carried out at a flow of 0.4 mL/min with a linear gradient from 0.1 M to 0.75 M KCl in 0.1 M Tris (pH 7.0). Proteins were monitored at 280 nm with a BioLogic chromatograph (Bio-Rad), and peaks corresponding to Mt–I and Mt-II, respectively, were collected and further purified by RP-HPLC as described [24], using a semi-preparative C8 column (Vydac, 10 × 250 mm, 5 μm particle) eluted at 2.5 mL/min with a gradient from water to acetonitrile, both containing 0.1% trifluoroacetic acid, in a model 1220 Agilent chromatograph monitored at 215 nm. The
Cells monolayers were adsorbed with the virus for 1 h at 37 °C and 5% CO2, to be later covered with Dulbecco medium containing 50 μg/ mL of Mt–I or Mt-II. Virus quantification proceeded as previously indicated. 3. Results and discussion 3.1. Bothrops asper sPLA2s display antiviral action against Flaviviruses An initial screening for the activity of Mt–I and Mt-II against various enveloped viruses, as well as against the Sabin vaccine strains of Polioviruses (Table 1), showed that members of Flaviviridae were highly susceptible to inhibition when exposed to either of the two 2
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Table 1 Virus strains tested in the inhibition activity experiments. Virus
Strain
Family
Enveloped
Dengue DENV-1 Dengue DENV-2 Dengue DENV-3 Dengue DENV-4 Yellow Fever YFV Herpes simplex HSV-1 Herpes simplex HSV-2 Influenza A H3N2 Sabin Poliovirus 1 Sabin Poliovirus 2 Sabin Poliovirus 3 Vesicular stomatitis VSV Vesicular stomatitis VSV
Angola Jamaica Nicaragua Dominica vaccine 17D ATCC VR733 ATCC VR734 vaccine Alice vaccine Sabin vaccine Sabin vaccine Sabin Indiana New Jersey
Flaviviridae Flaviviridae Flaviviridae Flaviviridae Flaviviridae Herpesviridae Herpesviridae Orthomyxoviridae Picornaviridae Picornaviridae Picornaviridae Rhabdoviridae Rhabdoviridae
yes yes yes yes yes yes yes yes no no no yes yes
Table 2 Antiviral potencies of Mt–I and Mt-II against DENV2, expressed as inhibitory concentrations, IC50 and IC90. Parameter
Mt–I
Mt-II
IC50 (ng/mL) IC90 (ng/mL)
1.5 1.7
2768 3058
procedure resulted in ~97% inactivation of Mt–I PLA2 activity on the NOBA substrate. When the p-BPB-treated Mt–I was comparatively titrated for virus inhibition activity, the IC50 and IC90 potency values (0.37 and 0.56 ng/mL, respectively) increased by nearly two orders of magnitude (90 and 317 ng/mL, respectively), supporting the relevance of enzymatic action in the virus inhibiting mechanism of Mt–I. Previous studies on Mt-II have shown that this PLA2-like protein has the ability to disrupt liposomes, as well as to induce lysis of various cell types in culture and local myonecrosis in vivo, in spite of lacking enzymatic activity [7]. Its C-terminal region encompassing amino acid residues 115–129 was identified as responsible for the membrane-permeabilizing effect of the protein [27] and furthermore shown to reproduce its bactericidal activity [11]. Therefore, it was of interest to explore if the synthetic peptide 115–129 of Mt-II would be capable of inhibiting Dengue viruses. As seen in Fig. 2, this peptide lacked antiviral effect even at concentrations as high as 1 mg/mL. This result is in agreement with the observed weak inhibitory effect of intact Mt-II, which we interpret as being caused by trace contamination with the enzymatically active Mt–I. Both types of proteins co-exist in the venom of B. asper, as well as in many viperid species, and their absolute chromatographic separation is often difficult to achieve [24]. Aiming to explore the mechanism of the antiviral effect of Mt–I, additional experiments were performed by pre-exposing the target cells to the protein, followed by extensive washing, prior to virus adsorption
proteins and subsequently tested for infectivity by the plaque-forming technique (Fig. 1). At a protein concentration of 50 μg/mL, the inhibition observed toward YFV and DENV1-4 strains was complete for both Mt–I and Mt-II, while effects recorded against other viruses were moderate to negligible. Following this, the potency of Mt–I and Mt-II against DENV2 was tested by titration at various protein dilutions, to estimate median (IC50) and 90% (IC90) inhibitory concentrations. Results revealed a striking difference between the effect of the two proteins, the catalytically active PLA2 (Mt–I) being about three orders of magnitude more potent than the catalytically inactive PLA2-like (Mt-II) (Table 2). The large difference in the potency of Mt–I and Mt-II suggested that the enzymatic activity of the former could be playing a role in the inhibitory effect expressed toward DENV. To evaluate this possibility, Mt–I was treated with the alkylating agent p-BPB, which modifies the single histidine residue in the catalytic center of this enzyme [24]. This
Fig. 1. Antiviral action of Bothrops asper sPLA2s (Mt–I and Mt-II) against Flaviviruses. The antiviral effect was estimated by pre-incubation of viruses with 50 μg/mL of each protein for 1 h at 37 °C, and subsequent plaque titration in cell cultures. As controls, viruses were incubated identically with medium alone (MEM 2%). (A) Titers of viruses preincubated with Mt–I or Mt-II (filled bars) or with medium (empty bars). Results are expressed as mean ± range (n = 2). (B) Expression of results as means of the titer reduction in comparison with the corresponding control. Abbreviations for viruses are as in Table 1.
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disrupt the viral envelope, in line with the previous findings of Muller et al. [18]. On the other hand, the catalytically-inactive Mt-II displayed only a weak antiviral effect (three orders of magnitude lower than Mt–I), which might either be intrinsic, or alternatively, attributable to possible trace contamination with Mt–I during chromatographic isolation. On the basis of evidence for the involvement of enzymatic activity in the action of Mt–I, added to the observation that the synthetic peptide 115–129 of Mt-II is unable to express antiviral action, we favor the second hypothesis. A previous study [19] reported a similar antiviral action against Dengue viruses for both an Asp49 PLA2 and a Lys49 PLA2-like protein, isolated from the venom of the Brazilian snake Bothrops leucurus. These two proteins have homology with Mt–I and Mt-II, respectively, evaluated in the present study. Therefore, our findings partly differ from those previously reported [19] in the sense that the virucidal activity of Mt-II (Lys49) was about three orders of magnitude lower than that of Mt–I. The question arises as to why the Bothrops asper PLA2, Mt–I, shows a marked preference for the enveloped viruses of Flaviviridae, as compared to other enveloped virus species (Fig. 1). Factors such as phospholipid composition, fluidity, and curvature of bilayers, among others, are known to influence the catalytic activity of secreted PLA2 enzymes [1]. It is reasonable to speculate that the structural organization and physico-chemical properties of the envelope of the Flaviviridae family members could favor the catalytic action of Mt–I, in contrast to envelopes of other virus families. In support of this assumption, it was observed that depending on the cell type in which a virus was propagated, its susceptibility to the viral action of Mt–I changed dramatically. DENV2 grown in human cells was much more resistant to Mt–I than DENV2 grown in mosquito cells (Fig. 4). This finding suggests that differences in envelope bilayer composition or structural organization, possibly related to the cell type or even cell compartment providing the envelope, may be crucially involved in the virucidal action of PLA2 enzymes, and warrants further studies to explore how the different replicative hosts of the virions influence the virucidal effect. Finally, the structural homology of viperid venom PLA2s such as B. asper Mt–I (present study), B. leucurus B/D PLA2 [19], or C. d. terrificus CB-PLA2 [22] with the human secreted gIIAPLA2 [3] suggest the possibility that the latter enzyme could play a relevant role in the innate immune response during Dengue infections, a hypothesis that would be interesting to investigate.
Fig. 2. Lack of antiviral action of the synthetic peptide ‘p115’ of Bothrops asper Mt-II against DENV1. The antiviral effect was determined by pre-incubation of the virus with 1000 μg/mL or 100 μg/mL of the peptide, or 50 μg/ mL of Mt–I as a positive control, for 1 h at 37 °C, and subsequent plaque titration in cell cultures (BHK-21). Bars represent mean ± range of duplicate cell cultures.
Fig. 3. Mt–I acts by a direct virucidal mechanism. DENV1 was co-incubated with Mt–I (50 μg/mL) for 1 h at 37 °C before infecting the cell (BHK-21) monolayer to quantify the virus by plaque technique. Complete abrogation of plaques demonstrates a direct virucidal mechanism. In the pre-infection model, the cell monolayer was treated for 1 h at 37 °C with Mt–I (50 μg/mL), then washed 5 times with PBS and infected with the virus. In the post-infection model, the cell monolayer was first infected with the virus, and then the Dulbecco's medium added contained Mt–I (50 μg/mL). Bars represent mean ± range of duplicate cell cultures.
(pre-infection assay), or adding the protein to cells after the adsorption of the virus (post-infection assay). Only the direct interaction between DENV1 virus and Mt–I led to the complete abrogation of the viral plaques (Fig. 3), indicating that this protein acts by a virucidal mechanism. The partial reduction in viral plaques observed in the preinfection experiment model is likely to be explained by a slight cytotoxic action of Mt–I on cells, since although this protein was tested at a sub-cytolytic concentration (50 μg/mL) it is possible that a minor cell damage may occur [29]. Altogether, our findings support the conclusion that Mt–I is capable of exerting a potent virus inhibitory activity against members of the Flaviviridae family. The mechanism involved in this effect is directly virucidal, and relies on the expression of PLA2 enzymatic activity, which would presumably hydrolyze viral envelope phospholipids and
Fig. 4. DENV2 grown in human cells is more resistant to Mt–I action than if grown in mosquito cells. DENV2 was replicated in C636 (mosquito) and HepG2 (human) cell lines, and the equivalent to 20 PFU of each virus was treated with varying Mt–I dilutions for 1 h at 37 °C before virus quantification by the plaque technique. DENV2 propagated in HepG2 was detectable at lower Mt–I dilutions, indicating that it was much more resistant to the virucidal effect of Mt–I, in comparison to DENV2 propagated in mosquito cells, which was highly susceptible. Quantification was made in BHK-21 cells. Bars represent mean ± range of duplicate cell cultures. 4
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Author contributions
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Biologicals journal homepage: www.elsevier.com/locate/biologicals
Potent virucidal activity against Flaviviridae of a group IIA phospholipase A2 isolated from the venom of Bothrops asper Hebleen Brenesa, Gilbert D. Loríab, Bruno Lomontec,∗ a
Instituto Costarricense de Investigación y Enseñanza en Nutrición y Salud (INCIENSA), Tres Ríos, Cartago, Costa Rica Sección de Virología, Centro de Investigación en Enfermedades Tropicales (CIET), and Centro de Investigaciones en Hematología y Trastornos Afines (CIHATA), Universidad de Costa Rica, San José, Costa Rica c Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica b
A R T I C LE I N FO
A B S T R A C T
Keywords: Phospholipase A2 Virucidal Flaviviridae Enveloped virus Dengue Snake venom
Secreted phospholipase A2 (sPLA2) molecules are small, calcium-dependent enzymes involved in many biological processes. Viperid venoms possess gIIA sPLA2s and sPLA2-like proteins, both having homology to human gIIA sPLA2, an innate immunity enzyme. We evaluated the antiviral action of Mt–I (catalytically-active sPLA2) and Mt-II (catalytically-inactive variant) isolated from the venom of Bothrops asper, against a diverse group of viruses. Yellow Fever and Dengue (enveloped) viruses were highly susceptible to inactivation by the snake proteins, in contrast to Sabin (non-enveloped; Polio vaccine strain), and Influenza A, Herpes simplex 1 and 2, and Vesicular Stomatitis (enveloped) viruses. Titration of the antiviral effect against Dengue virus revealed Mt–I to be highly potent (IC50 0.5–2 ng/mL), whereas Mt-II was 1000-fold weaker. This large difference suggested a requirement for PLA2 activity, which was confirmed by chemical inactivation of Mt–I. A synthetic peptide representing the membrane-disrupting region of Mt-II, previously shown to have bactericidal effect, lacked antiviral action, suggesting that the weak virucidal effect observed for Mt-II is likely caused by contamination with traces of Mt–I. On the other hand, Mt–I was demonstrated to act by a direct virucidal mechanism prior to infection, and not by an independent effect on host cells, either pretreated, or exposed to Mt–I after virus infection. Interestingly, DENV2 propagated in mosquito cells was much more sensitive to the action of Mt–I, compared to human cell-propagated virus. Therefore, differences in envelope membrane composition may be crucially involved in the observed virucidal action of PLA2 enzymes.
1. Introduction Phospholipases A2 (PLA2s; EC 3.1.1.4) constitute a large superfamily of hydrolytic enzymes with specificity for the ester bond at the sn-2 position of glycerophospholipids, which release free fatty acids and lysophospholipids [1]. These enzymes are ubiquitous across the tree of life [2,3] and abundantly found in snake venoms, where they present a wide variety of bioactivities [4]. Snake venom PLA2s belong to the secreted type of enzymes (sPLA2), classified in the structural groups IB (in Elapidae snakes) and IIA (in Viperidae snakes). The former show homology to the mammalian pancreatic juice PLA2, whereas the latter are homologous to the mammalian ‘inflammatory’ PLA2, a relevant component of innate immunity [5]. All sPLA2s share a conserved catalytic mechanism based on a His/Asp dyad using Ca2+ as an essential cofactor. Although the structural scaffold of group I and group II PLA2s is essentially conserved, these small (~14 kDa) enzymes differ by the
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position of one out of their seven disulfide bonds, and by the presence of an extended C-terminal segment (5–7 amino acids) in the latter [6]. Among the group II sPLA2s of viperids, a group of natural variants exists presenting key amino acid substitutions that preclude Ca2+ binding, and hence, catalytic activity. Such proteins are commonly referred to as ‘PLA2-like’ variants or ‘Lys49 PLA2-homologues' [7,8]. Antimicrobial effects have been described for both mammalian and snake venom sPLA2s acting against bacteria [9–13], parasites [14,15], or viruses [16–22]. In some cases, these effects depend on the catalytic action of the sPLA2s, but catalytic-independent actions have also been evidenced. For example, Fenard and coworkers [21] reported that several venom sPLA2s display potent inhibition of HIV-1 replication by a mechanism not involving the catalytic activity of the enzymes. Viral diseases remain a major health burden worldwide, and more efforts are undoubtedly required to search for new molecules with antiviral actions that could lead to novel therapeutic options [22]. Reports
Corresponding author. Instituto Clodomiro Picado Facultad de Microbiología Universidad de Costa Rica, San José, 11501, Costa Rica. E-mail addresses: [email protected] (H. Brenes), [email protected] (G.D. Loría), [email protected] (B. Lomonte).
https://doi.org/10.1016/j.biologicals.2019.12.002 Received 22 October 2019; Received in revised form 20 November 2019; Accepted 8 December 2019 1045-1056/ © 2019 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Hebleen Brenes, Gilbert D. Loría and Bruno Lomonte, Biologicals, https://doi.org/10.1016/j.biologicals.2019.12.002
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gradient toward acetonitrile stepped from 0 to 25% in 5 min, 25–50% in 22 min, 50–70% in 1 min, and was sustained at 70% for 2 min, for a total run time of 30 min. The isolated proteins were collected, dried by vacuum centrifugation (Vacufuge), redissolved at 0.1 mg/mL in Minimum Essential Medium Eagle (MEM) containing 2% v/v fetal calf serum, aliquoted and stored at −80 °C until use.
on the antiviral effects of some sPLA2s of venom origin prompted us to explore the action of two proteins purified from the venom of Bothrops asper, a viperid species found in Central America. This venom contains both enzymatically-active sPLA2 (Mt–I) and catalytically-inactive sPLA2-like protein (Mt-II) isoforms [23], and could therefore provide a useful comparison to understand the role of enzymatic activity in the antiviral action of sPLA2s. Previous studies have reported virus-inhibiting effects of snake venom sPLA2s belonging to the structural group IB of enzymes [20,21]. Among group IIA counterparts, antiviral action has been described for the sPLA2 subunit of crotoxin (a heterodimeric complex found in the venom of the South American rattlesnake Crotalus durissus terrificus) [17,18,21], and for Asp49 and Lys49 sPLA2s of Bothrops leucurus [19]. In this study, we describe a potent antiviral effect against enveloped viruses of the Flaviviridae family, but not against other enveloped viruses, of Mt–I, a catalytically-active sPLA2 isoform of B. asper venom. Its virucidal action was shown to depend on enzymatic activity. In agreement with this, the non-enzymatic Mt-II variant showed a 1000fold weaker effect than Mt–I, likely caused by traces of Mt–I contamination. This is also supported by the observation that the bioactive synthetic peptide p115-129 of Mt-II was devoid of antiviral effect.
2.4. Mt–I and Mt-II antiviral activity The viruses were mixed with each of the proteins, at a final concentration of 50 μg/mL, and these mixtures were incubated for 1 h at 37 °C, followed by virus quantification. For comparison, virus was incubated with MEM 2% FBS alone and treated identically. In addition, the inhibitory concentrations IC50 and IC90 were estimated by preparing double dilutions of Mt–I and Mt-II in MEM 2% BSA and incubating each one for 1 h at 37 °C with DENV-1, followed by virus quantification as described. 2.5. Inactivation of the catalytic activity of Mt–I Mt–I was chemically modified with p-bromophenacyl bromide (pBPB) as previously described [24]. This procedure alkylates the single histidine residue at the catalytic site of the enzyme, abrogating its ability to hydrolyze phospholipids [25]. Inactivation of enzymatic activity was evaluated using the synthetic monodisperse substrate 4-nitro3-octanoylbenzoic acid (NOBA) [24].
2. Materials and methods 2.1. Cells and viruses BHK-21 and Vero cells were grown in MEM enriched with 10% FBS at 37 °C and 5% CO2; C636, MDCK, and HepG2 cells were grown with RPMI 10% FBS at 37 °C (MDCK and HepG2) and 28 °C (C636) in 5% CO2. DENV-1 (Angola strain), DENV-2 (Jamaica strain), DENV-3 (Nicaragua strain), DENV-4 (Dominica strain), YFV (17D vaccine strain), HSV-1 (ATCC VR-733), HSV-2 (ATCC VR-734), Influenza A virus (H3N2, Alice vaccine strain) and Polio virus (Sabin vaccine strain) were obtained from the virus collection of the Section of Virology, School of Microbiology, UCR. VSV-IND and VSV-NJ were kindly provided by Dr. Carlos Jiménez from the School of Veterinary Medicine, National University of Costa Rica (UNA).
2.6. Synthetic peptide 115–129 of Mt-II The amino acid sequence 115–129 (KKYRYYLKPLCKK; common numbering system of [26]), near the C-terminal region of Mt-II, was synthesized using Fmoc strategy with free N-terminus and amidated Cterminus (Peptide 2.0; Chantilly, VA, USA) and purified to > 95%. This peptide has been shown to mimick the enzymatic-independent membrane damaging action of the parent protein against eukaryiotic cells [27,28], and against bacteria [11]. 2.7. Virucidal model
2.2. Virus quantification The virucidal activity corresponds to the strategy described previously, in which the Mt–I or Mt-II were allowed to act directly on the virus particles before infecting the cell monolayer.
Virus quantification was carried out by plaque formation assay. In brief, cell monolayers were inoculated with 10−1 to 10−6 dilutions of the samples, and then adsorbed for 1 h at 37 °C and 5% CO2, agitating every 15 min. Then, cell culture media containing 1% carboxymethyl cellulose was added, and plaque formation was allowed to proceed until appropriate-sized plaques were observed. Cells were fixed with 3.7% formalin and stained with violet crystal. In the case of Influenza A virus, TPCK trypsin was added to the Dulbecco medium, which allows virus particle maturation.
2.8. Pre-infection model The uninfected monolayer was covered with 100 μL of a 50 μg/mL solution of Mt–I or Mt-II, and after 1 h incubation at 37 °C, 5% CO2, the supernatant was removed and five sterile-PBS washing steps were done. The monolayer was then infected with the virus and quantification was done as described previously.
2.3. Mt–I and Mt-II isolation 2.9. Post-infection model Crude venom of Bothrops asper was a pool obtained from more than 20 specimens kept at the Serpentarium facility of Instituto Clodomiro Picado, University of Costa Rica. The lyophilized venom (200 mg) was dissolved in 0.1 M Tris, 0.1 M KCl (pH 7.0) buffer and fractionated by cation-exchange chromatography on a CM-Sephadex C25 column (20 × 2 cm) equilibrated with the same buffer. Elution was carried out at a flow of 0.4 mL/min with a linear gradient from 0.1 M to 0.75 M KCl in 0.1 M Tris (pH 7.0). Proteins were monitored at 280 nm with a BioLogic chromatograph (Bio-Rad), and peaks corresponding to Mt–I and Mt-II, respectively, were collected and further purified by RP-HPLC as described [24], using a semi-preparative C8 column (Vydac, 10 × 250 mm, 5 μm particle) eluted at 2.5 mL/min with a gradient from water to acetonitrile, both containing 0.1% trifluoroacetic acid, in a model 1220 Agilent chromatograph monitored at 215 nm. The
Cells monolayers were adsorbed with the virus for 1 h at 37 °C and 5% CO2, to be later covered with Dulbecco medium containing 50 μg/ mL of Mt–I or Mt-II. Virus quantification proceeded as previously indicated. 3. Results and discussion 3.1. Bothrops asper sPLA2s display antiviral action against Flaviviruses An initial screening for the activity of Mt–I and Mt-II against various enveloped viruses, as well as against the Sabin vaccine strains of Polioviruses (Table 1), showed that members of Flaviviridae were highly susceptible to inhibition when exposed to either of the two 2
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Table 1 Virus strains tested in the inhibition activity experiments. Virus
Strain
Family
Enveloped
Dengue DENV-1 Dengue DENV-2 Dengue DENV-3 Dengue DENV-4 Yellow Fever YFV Herpes simplex HSV-1 Herpes simplex HSV-2 Influenza A H3N2 Sabin Poliovirus 1 Sabin Poliovirus 2 Sabin Poliovirus 3 Vesicular stomatitis VSV Vesicular stomatitis VSV
Angola Jamaica Nicaragua Dominica vaccine 17D ATCC VR733 ATCC VR734 vaccine Alice vaccine Sabin vaccine Sabin vaccine Sabin Indiana New Jersey
Flaviviridae Flaviviridae Flaviviridae Flaviviridae Flaviviridae Herpesviridae Herpesviridae Orthomyxoviridae Picornaviridae Picornaviridae Picornaviridae Rhabdoviridae Rhabdoviridae
yes yes yes yes yes yes yes yes no no no yes yes
Table 2 Antiviral potencies of Mt–I and Mt-II against DENV2, expressed as inhibitory concentrations, IC50 and IC90. Parameter
Mt–I
Mt-II
IC50 (ng/mL) IC90 (ng/mL)
1.5 1.7
2768 3058
procedure resulted in ~97% inactivation of Mt–I PLA2 activity on the NOBA substrate. When the p-BPB-treated Mt–I was comparatively titrated for virus inhibition activity, the IC50 and IC90 potency values (0.37 and 0.56 ng/mL, respectively) increased by nearly two orders of magnitude (90 and 317 ng/mL, respectively), supporting the relevance of enzymatic action in the virus inhibiting mechanism of Mt–I. Previous studies on Mt-II have shown that this PLA2-like protein has the ability to disrupt liposomes, as well as to induce lysis of various cell types in culture and local myonecrosis in vivo, in spite of lacking enzymatic activity [7]. Its C-terminal region encompassing amino acid residues 115–129 was identified as responsible for the membrane-permeabilizing effect of the protein [27] and furthermore shown to reproduce its bactericidal activity [11]. Therefore, it was of interest to explore if the synthetic peptide 115–129 of Mt-II would be capable of inhibiting Dengue viruses. As seen in Fig. 2, this peptide lacked antiviral effect even at concentrations as high as 1 mg/mL. This result is in agreement with the observed weak inhibitory effect of intact Mt-II, which we interpret as being caused by trace contamination with the enzymatically active Mt–I. Both types of proteins co-exist in the venom of B. asper, as well as in many viperid species, and their absolute chromatographic separation is often difficult to achieve [24]. Aiming to explore the mechanism of the antiviral effect of Mt–I, additional experiments were performed by pre-exposing the target cells to the protein, followed by extensive washing, prior to virus adsorption
proteins and subsequently tested for infectivity by the plaque-forming technique (Fig. 1). At a protein concentration of 50 μg/mL, the inhibition observed toward YFV and DENV1-4 strains was complete for both Mt–I and Mt-II, while effects recorded against other viruses were moderate to negligible. Following this, the potency of Mt–I and Mt-II against DENV2 was tested by titration at various protein dilutions, to estimate median (IC50) and 90% (IC90) inhibitory concentrations. Results revealed a striking difference between the effect of the two proteins, the catalytically active PLA2 (Mt–I) being about three orders of magnitude more potent than the catalytically inactive PLA2-like (Mt-II) (Table 2). The large difference in the potency of Mt–I and Mt-II suggested that the enzymatic activity of the former could be playing a role in the inhibitory effect expressed toward DENV. To evaluate this possibility, Mt–I was treated with the alkylating agent p-BPB, which modifies the single histidine residue in the catalytic center of this enzyme [24]. This
Fig. 1. Antiviral action of Bothrops asper sPLA2s (Mt–I and Mt-II) against Flaviviruses. The antiviral effect was estimated by pre-incubation of viruses with 50 μg/mL of each protein for 1 h at 37 °C, and subsequent plaque titration in cell cultures. As controls, viruses were incubated identically with medium alone (MEM 2%). (A) Titers of viruses preincubated with Mt–I or Mt-II (filled bars) or with medium (empty bars). Results are expressed as mean ± range (n = 2). (B) Expression of results as means of the titer reduction in comparison with the corresponding control. Abbreviations for viruses are as in Table 1.
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disrupt the viral envelope, in line with the previous findings of Muller et al. [18]. On the other hand, the catalytically-inactive Mt-II displayed only a weak antiviral effect (three orders of magnitude lower than Mt–I), which might either be intrinsic, or alternatively, attributable to possible trace contamination with Mt–I during chromatographic isolation. On the basis of evidence for the involvement of enzymatic activity in the action of Mt–I, added to the observation that the synthetic peptide 115–129 of Mt-II is unable to express antiviral action, we favor the second hypothesis. A previous study [19] reported a similar antiviral action against Dengue viruses for both an Asp49 PLA2 and a Lys49 PLA2-like protein, isolated from the venom of the Brazilian snake Bothrops leucurus. These two proteins have homology with Mt–I and Mt-II, respectively, evaluated in the present study. Therefore, our findings partly differ from those previously reported [19] in the sense that the virucidal activity of Mt-II (Lys49) was about three orders of magnitude lower than that of Mt–I. The question arises as to why the Bothrops asper PLA2, Mt–I, shows a marked preference for the enveloped viruses of Flaviviridae, as compared to other enveloped virus species (Fig. 1). Factors such as phospholipid composition, fluidity, and curvature of bilayers, among others, are known to influence the catalytic activity of secreted PLA2 enzymes [1]. It is reasonable to speculate that the structural organization and physico-chemical properties of the envelope of the Flaviviridae family members could favor the catalytic action of Mt–I, in contrast to envelopes of other virus families. In support of this assumption, it was observed that depending on the cell type in which a virus was propagated, its susceptibility to the viral action of Mt–I changed dramatically. DENV2 grown in human cells was much more resistant to Mt–I than DENV2 grown in mosquito cells (Fig. 4). This finding suggests that differences in envelope bilayer composition or structural organization, possibly related to the cell type or even cell compartment providing the envelope, may be crucially involved in the virucidal action of PLA2 enzymes, and warrants further studies to explore how the different replicative hosts of the virions influence the virucidal effect. Finally, the structural homology of viperid venom PLA2s such as B. asper Mt–I (present study), B. leucurus B/D PLA2 [19], or C. d. terrificus CB-PLA2 [22] with the human secreted gIIAPLA2 [3] suggest the possibility that the latter enzyme could play a relevant role in the innate immune response during Dengue infections, a hypothesis that would be interesting to investigate.
Fig. 2. Lack of antiviral action of the synthetic peptide ‘p115’ of Bothrops asper Mt-II against DENV1. The antiviral effect was determined by pre-incubation of the virus with 1000 μg/mL or 100 μg/mL of the peptide, or 50 μg/ mL of Mt–I as a positive control, for 1 h at 37 °C, and subsequent plaque titration in cell cultures (BHK-21). Bars represent mean ± range of duplicate cell cultures.
Fig. 3. Mt–I acts by a direct virucidal mechanism. DENV1 was co-incubated with Mt–I (50 μg/mL) for 1 h at 37 °C before infecting the cell (BHK-21) monolayer to quantify the virus by plaque technique. Complete abrogation of plaques demonstrates a direct virucidal mechanism. In the pre-infection model, the cell monolayer was treated for 1 h at 37 °C with Mt–I (50 μg/mL), then washed 5 times with PBS and infected with the virus. In the post-infection model, the cell monolayer was first infected with the virus, and then the Dulbecco's medium added contained Mt–I (50 μg/mL). Bars represent mean ± range of duplicate cell cultures.
(pre-infection assay), or adding the protein to cells after the adsorption of the virus (post-infection assay). Only the direct interaction between DENV1 virus and Mt–I led to the complete abrogation of the viral plaques (Fig. 3), indicating that this protein acts by a virucidal mechanism. The partial reduction in viral plaques observed in the preinfection experiment model is likely to be explained by a slight cytotoxic action of Mt–I on cells, since although this protein was tested at a sub-cytolytic concentration (50 μg/mL) it is possible that a minor cell damage may occur [29]. Altogether, our findings support the conclusion that Mt–I is capable of exerting a potent virus inhibitory activity against members of the Flaviviridae family. The mechanism involved in this effect is directly virucidal, and relies on the expression of PLA2 enzymatic activity, which would presumably hydrolyze viral envelope phospholipids and
Fig. 4. DENV2 grown in human cells is more resistant to Mt–I action than if grown in mosquito cells. DENV2 was replicated in C636 (mosquito) and HepG2 (human) cell lines, and the equivalent to 20 PFU of each virus was treated with varying Mt–I dilutions for 1 h at 37 °C before virus quantification by the plaque technique. DENV2 propagated in HepG2 was detectable at lower Mt–I dilutions, indicating that it was much more resistant to the virucidal effect of Mt–I, in comparison to DENV2 propagated in mosquito cells, which was highly susceptible. Quantification was made in BHK-21 cells. Bars represent mean ± range of duplicate cell cultures. 4
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Author contributions
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GDL and BL conceived the study; HB performed experiments supervised by GDL; HB, GDL, and BL analyzed results; HB drafted the manuscript; GDL and BL reviewed and edited the final version. Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Acknowledgements Dr Julián Fernández and Dr Steve Quirós are gratefully acknowledged for valuable discussions on this study. We thank Vicerrectoría de Investigación, Universidad de Costa Rica (project B-8115) for financial support, and CYTED (Red BUDE PAV-AM) for promoting Iberoamerican collaboration on the study of antimicrobial agents. References [1] Berg OG, Gelb MH, Tsai MD, Jain MK. Interfacial enzymology: the secreted phospholipase A2-paradigm. Chem Rev 2001;101:2613–53. [2] Murakami M, Kudo I. Phospholipase A2. J Biochem 2002;131:285–92. [3] Dennis EA, Cao J, Hsu YH, Magrioti V, Kokotos G. Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention. Chem Rev 2011;111:6130–85. [4] Kini RM. Phospholipase A2: a complex multifunctional protein puzzle. In: Kini RM, editor. Venom phospholipase A2 enzymes: structure, function, and mechanism. England: John Wiley & Sons; 1997. p. 321–52. [5] Krizaj I. Roles of secreted phospholipases A₂ in the mammalian immune system. Protein Pept Lett 2014;21:1201–8. [6] Montecucco C, Gutiérrez JM, Lomonte B. Cellular pathology induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action. Cell Mol Life Sci 2008;65:2897–912. [7] Lomonte B, Rangel J. Snake venom Lys49 myotoxins: from phospholipases A2 to non-enzymatic membrane disruptors. Toxicon 2012;60:520–30. [8] Fernandes CA, Borges RJ, Lomonte B, Fontes MR. A structure-based proposal for a comprehensive myotoxic mechanism of phospholipase A2-like proteins from viperid snake venoms. Biochim Biophys Acta 2014;1844:2265–76. [9] Weiss J, Inada M, Elsbach P, Crowl RM. Structural determinants of the action against Escherichia coli of a human inflammatory fluid phospholipase A2 in concert with polymorphonuclear leukocytes. J Biol Chem 1994;269:26331–7. [10] Dubouix A, Campanac C, Fauvel J, Simon MF, Salles JP, Roques C, Chap H, Marty N. Bactericidal properties of group IIa secreted phospholipase A2 against Pseudomonas aeruginosa clinical isolates. J Med Microbiol 2003;52:1039–45. [11] Páramo L, Lomonte B, Pizarro-Cerdá J, Bengoechea JA, Gorvel JP, Moreno E. Bactericidal activity of Lys49 and Asp49 myotoxic phospholipases A2 from Bothrops asper snake venom: synthetic Lys49 myotoxin II-(115-129)-peptide identifies its bactericidal region. Eur J Biochem 1998;253:452–61. [12] Chioato L, Aragão EA, Ferreira TL, Ward RJ. Active site mutants of human secreted group IIA phospholipase A2 lacking hydrolytic activity retain their bactericidal effect. Biochimie 2012;94:132–6.
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