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doi:10.1016/j.ijantimicag.2003.07.022 
[FONT=Verdana,Arial,Helvetica,sans-serif] Copyright ? 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. [/FONT] Antiviral activity of antimicrobial cationic peptides against Junin virus and herpes simplex virus
Vanesa C. Albiol Matanic and Viviana Castilla <sup>
</sup><sup>, </sup><sup>
</sup>
Laboratorio de Virolog?a, Departamento de Qu?mica Biol?gica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabell?n II, Piso 4, C1428BGA, Buenos Aires, Argentina
Received 22 May 2003; accepted 24 July 2003. Available online 16 March 2004.
Abstract
The in vitro antiviral activity of antimicrobial cationic peptides: cecropin A, melittin, magainin I and II and indolicidin against the arenavirus Junin virus (JV), and herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) was evaluated. Cecropin A effectively inhibited JV multiplication and failed to affect HSV replication whereas melittin impeded the multiplication of JV and HSV, but was highly toxic for the host cell. Magainins I and II exhibited inhibitory action toward HSV-1 and HSV-2 but were inactive against JV. Only indolicidin showed a direct inactivation effect on cell-free virus stocks. Besides its inhibitory effect on JV replication cecropin A also was active against the arenaviruses Tacaribe and Pichinde, mainly affecting late events of arenavirus multiplication cycle by preventing viral morphogenesis and egress from infected cells.
Author Keywords: Author Keywords: Arenavirus; Herpesvirus; Antimicrobial peptides; Antiviral activity
Article Outline
<dl><dt>1. Introduction</dt><dt>2. Materials and methods</dt><dl><dt>2.1. Cells and viruses</dt><dt>2.2. Antimicrobial peptides</dt><dt>2.3. Cytotoxicity assay</dt><dt>2.4. Antiviral activity</dt><dt>2.5. Virucidal assay</dt><dt>2.6. Analysis of radiolabelled proteins</dt><dt>2.7. Effect of time of cecropin A addition or removal on JV replication</dt><dt>2.8. Indirect immunofluorescence (IF) assay</dt></dl><dt>3. Results</dt><dl><dt>3.1. Antiviral activity of cationic peptides against JV, HSV-1 and HSV-2</dt><dt>3.2. Effect of cecropin A on Tacaribe virus and Pichinde virus multiplication</dt><dt>3.3. Virucidal action of cationic peptides</dt><dt>3.4. Effect of cecropin A on N protein synthesis</dt><dt>3.5. Effect of time of addition or removal of cecropin A at different times after infection on JV production</dt><dt>3.6. Effect of cecropin A on G1 intracellular transport</dt></dl><dt>4. Discussion</dt><dt>Acknowledgements</dt><dt>References</dt></dl>
1. Introduction
Natural cationic peptides have a variety of interesting biological activities including antibacterial, antifungal, antiparasitic, antitumoural, and antiviral activities [1, 2, 3 and 4]. The most common classes of cationic peptides are β-sheet peptides stabilised by disulphide bridges and unstructured peptides that fold into amphipathic α-helices upon contact with membranes. Less common are extended peptides with a predominance of one or two amino acids [4].
Although antibacterial activity of cationic peptides has been extensively studied, little is known about their ability to act as antiviral agents. Besides, the mechanism of action of those peptides that exhibit antiviral activity is still poorly understood.
The antiviral activity of human defensins, a group of β-sheet peptides, against enveloped viruses such as herpes simplex virus type 1 (HSV-1) and 2 (HSV-2), immunodeficiency virus type 1 (HIV-1), vesicular stomatitis virus (VSV), influenzavirus and cytomegalovirus has been ascribed to direct inactivation of viral particles [5]. On the other hand, it has been reported that α-helical peptides can act as virucidal agents or as inhibitors of virus multiplication. Two major types of α-helical peptides have been isolated from insects: melittin, a 26 amino acid peptide which is the major component of bee venom and a family of antibacterial peptides, 35?39 amino acids in length, named cecropins [3]. While melittin, at high concentrations, is lytic for blood red cells, cecropins appear to be non toxic for mammalian cells [6]. Antiviral activity of melittin against several enveloped viruses has been attributed to direct lysis of viral membranes, however, at lower non-virolytic concentrations, melittin exhibits inhibitory action against HSV-1 and HIV-1 [6, 7 and 8]. Among cecropins, it has been demonstrated that cecropin A inhibits HIV-1 multiplication in acutely and chronically infected cells [6 and 7]. Magainins I and II are 23-residue α-helical peptides isolated from the skin of the frog Xenopus laevis, that differ by substitutions in positions 10 and 22 [1 and 9] and it has been reported that synthetic analogues of magainins display virucidal action towards HSV-1 [10].
Direct viral inactivation against HIV-1 has been also described for indolicidin, a 13 amino acid peptide with extended structure and a high content of tryptophan, isolated from bovine neutrophils [4, 9 and 11].
Arenavirus are rodent-associated viruses that can cause severe human haemorrhagic fevers. In particular, Junin virus (JV) is the aetiological agent of Argentine haemorrhagic fever (AHF), an endemo-epidemic disease geographically restricted to the most fertile areas of the country. Though several compounds inhibit in vitro arenavirus multiplication [12], to date ribavirin is the only compound that has shown partial efficacy against JV infection in animal models and humans, but with a high level of undesirable secondary reactions [13 and 14]. Hence, the current therapy for AHF patients is the early administration of immune plasma, however, this therapy is not effective when it is initiated after 8 days illness [15].
Since there are no reports about the effect of cationic peptides on arenavirus multiplication, in the present study we tested the in vitro antiviral activity of cecropin A, melittin, magainins I and II and indolicidin against JV. In addition, to determine the specificity of action of these compounds, we also evaluated their antiviral action against HSV-1 and HSV-2, taking into account that among the selected peptides used here only melittin has been proved to be active against HSV-1 [8].
2. Materials and methods
2.1. Cells and viruses
Vero and BHK-21 cells were grown in Eagle?s minimal essential medium (MEM, GIBCO) containing 5% inactivated calf serum and 50 μg/ml gentamicin. Maintenance medium (MM), pH 7.5, consisted of MEM supplemented with 1.5% inactivated calf serum and gentamicin. IV<sub>4454</sub>, an attenuated strain of JV isolated from a mild human case of AHF [16], was propagated on BHK-21 cells. Tacaribe virus (strain TRLV<sub>11573</sub>), Pichinde virus (strain AN<sub>3739</sub>), HSV-1 (strain F) and HSV-2 (strain G) were propagated on Vero cells. Virus stocks were plaque-assayed on Vero cells.
2.2. Antimicrobial peptides
Stock solutions of cecropin A, melittin, magainins I and II and indolicidin (Sigma?Aldrich) were prepared in MEM at 250 μM and stored at −20 ?C until use.
2.3. Cytotoxicity assay
Vero cells grown in 96-well tissue culture plates were incubated with different concentrations of each compound for 24 h at 37 ?C. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) procedure [17]. The cytotoxicity of each compound was expressed as the 50% cytotoxic concentration (CC<sub>50</sub>), which is the concentration required to reduce cell viability to 50% of the control.
2.4. Antiviral activity
Antiviral activity was determined by a virus yield inhibition assay. Vero cell monolayers were infected at a multiplicity of infection (moi) of 0.1 PFU/cell. After 1 h adsorption at 37 ?C, MM containing various concentrations of the compounds was added. After 24 h of incubation at 37 ?C, culture supernatants were harvested and extracellular virus yields were determined by plaque assay on Vero cell monolayers.
The 50% effective concentration (EC<sub>50</sub>) and the 90% effective concentration (EC<sub>90</sub>) were calculated as the compound concentration that reduces virus yield by 50 and 90%, respectively.
2.5. Virucidal assay
Viral suspensions were incubated in the presence or absence of various concentrations of each compound for 2 h at 37 ?C. After the incubation period, the mixtures were placed on ice, serially diluted in MM and remaining infectivity was determined by plaque assay.
2.6. Analysis of radiolabelled proteins
Mock-infected or JV-infected Vero cells (moi = 5 PFU/cell) were incubated with MM containing or not cecropin A (5 or 10 μM) for 48 h at 37 ?C. Then cultures were washed with phosphate buffer saline (PBS), incubated with methionine-free medium for 1.5 h and then labelled with <sup>35</sup>S-methionine (100 μCi/ml). After a labelling period of 4 h, cells were washed three times with cold PBS.
To analyse cell protein synthesis treated and untreated mock-infected cultures were resuspended in sample buffer (PAGE) containing 5% SDS, 2% 2-mercaptoethanol, 10% glycerol and 0.005% bromophenol blue in 0.0625 M Tris?HCl, pH 6.8. Cell lysates were heated for 2 min in boiling water and SDS-PAGE was performed on 10% acrylamide gel. Radiolabelled proteins were visualised by fluorography on AGFA films.
To analyse viral protein synthesis mock-infected or JV-infected cells treated or not with cecropin A were lysed in radioimmune precipitation assay buffer (RIPA: 10 mM Tris?HCl pH 7.4, 0.15 M NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate and 0.4 mM PMSF) and incubated with monoclonal antibody (NA05AG12) reactive against viral nucleocapsid protein N [18] for 30 min at 37 ?C and further incubated at 0 ?C for 4 h. Antibody?antigen complexes were collected with protein A-sepharose (Sigma?Aldrich) and proteins were separated on 10% SDS-PAGE and visualised as described above.
2.7. Effect of time of cecropin A addition or removal on JV replication
Monolayers of confluent Vero cells were allowed to adsorb JV at a moi of 0.1 PFU/cell for 1 h at 37 ?C. After removal of the inocula, MM containing cecropin A (25 μM) was added to infected cells at 1, 3, 5 or 8 h post-infection (p.i.) and cultures were further incubated at 37 ?C. Another set of cell cultures was infected with JV in the presence of cecropin A (25 μM) and the compound was removed by medium change at 3, 5, 8 or 24 h p.i. In all cases, at 24 h p.i., extracellular virus yields were measured from supernatants by plaque assay. To determine cell-associated infectivity cultures were subjected to two cycles of freeze-thawing followed by centrifugation at low speed and the supernatants obtained were titrated by plaque assay.
2.8. Indirect immunofluorescence (IF) assay
Vero cell monolayers grown in coverslips were infected with JV at a moi of 0.1 PFU/cell and after 1 h adsorption, cultures were incubated in MM containing or not cecropin A (25 μM) at 37 ?C. At 18 h p.i. supernatants were collected and extracellular virus production was determined by plaque assay. The remaining cells were washed three times with cold PBS and the IF assays were performed as previously described [19] with minor modifications. For cytoplasmic IF assay, cells were fixed in methanol for 10 min at −20 ?C and then incubated with the monoclonal antibody GB03-BE08, reactive against G1 viral glycoprotein [18], for 30 min at 37 ?C. To perform the surface IF assay, cells were incubated with the monoclonal antibody for 30 min at 4 ?C and then fixed with methanol as described above. The indirect staining was carried out by using goat anti-mouse antibody conjugated to fluorescein isothiocyanate (Sigma?Aldrich). The number of fluorescent cells was counted from 20 randomly selected fields using a Zeiss Axioplan microscope.
3. Results
3.1. Antiviral activity of cationic peptides against JV, HSV-1 and HSV-2
We first examined the effect of cationic peptides on cell viability after incubating Vero cell monolayers with different concentrations of each compound at 37 ?C for 24 h. Cytotoxicity was evaluated using the MTT method and by examining cell morphology by light microscopy. While cecropin A, magainins I and II and indolicidin did not show cytotoxicity up to a concentration of 100 μM (the highest concentration assayed), treatment with melittin at concentrations above 5 μM caused cell rounding and monolayer detachment. As it is shown in Table 1, melittin CC<sub>50</sub> value, obtained by the MTT method, was 8.51 μM.
A dose dependent inhibition of JV multiplication was observed in cecropin A treated cultures. The EC<sub>50</sub> value of cecropin A against JV was 3.24 μM and virus yield was inhibited by more than 90% in cultures treated with 40 μM of cecropin A, compared with untreated ones (Table 1 and Fig. 1). In contrast, this peptide did not exhibit inhibitory effect on the production of HSV-1 and HSV-2 infectious particles, even at a concentration of 40 μM (Table 1).
On the other hand, magainins I and II, that produced a dose dependent inhibition of HSV-1 and HSV-2 multiplication with similar EC<sub>50</sub> values for both viruses, were inactive against JV (Table 1).
Finally, indolicidin, the only peptide with extended structure assayed, exhibited a strong inhibitory action against HSV. At the highest concentration tested (50 μM), indolicidin reduced HSV-1 and HSV-2 yields by 99%. However, from the EC<sub>50</sub> values obtained it appears that indolicidin is about five times more potent against HSV-2 than against HSV-1 (Table 1). On the contrary, no significant differences on JV yield were observed in indolicidin treated cultures compared with untreated ones.
3.2. Effect of cecropin A on Tacaribe virus and Pichinde virus multiplication
Although both cecropin A and melittin were active toward JV and the EC<sub>50</sub> for melittin was lower than the EC<sub>50</sub> for cecropin A, the selectivity index of melittin is low due to its high toxicity to the host cell (Table 1). Since cecropin A showed antiviral activity toward JV without affecting HSV multiplication, this compound was further examined for its inhibitory action against other arenaviruses. For this purpose, Vero cells were infected with Tacaribe virus or Pichinde virus (moi=0.1) and after 1 h adsorption at 37 ?C cultures were incubated in MM containing or not different concentrations of cecropin A and at 24 h p.i. virus yield was determined by plaque assay. As it is shown in Fig. 1, cecropin A also affected Tacaribe virus and Pichinde virus multiplication in a dose dependent manner. The EC<sub>50</sub> for Tacaribe virus and Pichinde virus were 1.96 and 6.56 μM, respectively, and those values were similar to the EC<sub>50</sub> obtained for JV (Table 1). In addition, the EC<sub>90</sub> values for JV, Tacaribe virus and Pichinde virus were of 28.57, 33.39 and 39 μM, respectively.
3.3. Virucidal action of cationic peptides
In order to determine whether cationic peptides had a direct inactivating effect on virus infectious particles, an assay of virucidal activity was performed. To this end, aliquots of JV, HSV-1 or HSV-2 suspensions were exposed for 2 h at 37 ?C to each compound using the highest peptide concentration tested in the virus yield inhibition assay described above. Then samples were chilled and diluted to reduce the peptide concentration below the corresponding effective antiviral concentration EC<sub>50</sub> and residual infectivity was determined by a plaque formation assay.
As can be seen in Table 2, in the experimental conditions assayed, indolicidin was the only peptide that displayed virucidal action. A potent inactivating effect was observed against HSV-1 and HSV-2, since infectivity was reduced by more than 99% for both viruses. Less degree of inactivation was observed for indolicidin treated JV suspension, since residual JV infectivity was reduced by 50% compared with untreated control sample (Table 2).
Only five proteins are expressed from the two genome fragments of JV: the S RNA encodes the major structural proteins, the nucleocapsid protein (N) and two envelope glycoproteins (G1 and G2), whereas the L RNA encodes the RNA polymerase protein (L) and an 11 kDa protein with regulatory functions called Z [20].
In order to analyse further the selective inhibitory action of cecropin A against JV in vitro multiplication, the effect of this peptide on both cellular and viral protein synthesis was examined. First, non infected Vero cells were treated with cecropin A for 48 h at 37 ?C and after a 4 h pulse labelling with <sup>35</sup>S-methionine, cells were lysed and radiolabelled cell proteins were analysed by SDS-PAGE. As can be seen in Fig. 2, treatment with 5 or 10 μM of cecropin A had no effect on cell protein synthesis. The other set of infected labelled cultures, treated or not with cecropin A (10 μM) for 48 h, was immunoprecipitated with a monoclonal antibody reactive against viral protein N and further analysed by SDS-PAGE. In untreated infected cultures (Fig. 2, lane 5) the N protein (63 kDa) and a polypeptide band with apparent molecular weight of approximately 45 kDa, corresponding to an N degradation product previously described [18], were observed. A reduction in the amount of both N and the N derived polypeptide of 45 kDa was detected in infected cultures treated with cecropin A (Fig. 2, lane 4). Thus, these results indicate that cecropin A is able to selectively inhibit viral protein synthesis without affecting host cell macromolecular synthesis. Although in this experiment a high multiplicity of infection was employed, the low efficiency of JV adsorption process does not insure the synchronisation of the infection. Moreover, the labelling period with <sup>35</sup>S-methionine was performed at 48 h p.i., due to the intrinsically poor efficiency of JV protein synthesis that impedes its analysis at earlier times after infection. The time required for a complete JV multiplication cycle ranges from 12 to 16 h, thus, as several multiplication cycles take place within a period of 48 h, inhibition of viral protein synthesis might be an indirect consequence of the inhibitory action of cecropin A on any other step of the virus replication cycle.
To further characterise the antiviral mode of action of cecropin A, we next examined the effect of time of cecropin A addition on JV multiplication. Cecropin A (25 μM) was added to JV-infected cells at different times after adsorption and at 24 h p.i. the extracellular and cell-associated virus yields were determined. As it is shown in Fig. 3a, within the first 8 h of infection, inhibition of JV multiplication is not dependent on the time of addition of the peptide. On the other hand, a differential effect of cecropin A on extracellular and cell-associated infectivity was observed. Whereas extracellular virus production was highly inhibited (90%), cell-associated infectivity was only reduced by 65%. Another set of cell cultures was infected with JV in the presence of cecropin A (25 μM). In this case, the compound was removed at different times after infection and cell-associated and extracellular virus yields were quantified at 24 h p.i. The presence of cecropin A during the first 5 h of infection did not inhibit viral production indicating that this peptide has no effect on early steps of JV multiplication (Fig. 3b). However, when the compound was removed at 8 h p.i. virus infectivity released to the culture supernatant was decreased by 90% with respect to untreated control, whereas cell-associated infectivity was reduced by 47%. These results suggest that cecropin A exerts its inhibitory action when it is present during late events of viral multiplication cycle, mainly affecting the release of infectious viral particles to the extracellular medium.
JV glycoproteins are synthesised in the endoplasmic reticulum as a precursor called GPC (62?65 kDa), and after post-translational modifications and cleavage in the distal trans Golgi network, mature glycoproteins named G1 (38 kDa) and G2 (45?50 kDa) are transported to the cell surface where virus assembly takes place [21]. G1, which has been claimed to induce neutralising antibodies, seems to be the only glycoprotein exposed on the virion surface as it was shown by in vitro iodination of intact purified virions [22].
The results described above (Fig. 3) suggest that cecropin A affects late steps of JV multiplication, so we decided to evaluate the action of this compound on the intracellular transport of the main JV glycoprotein G1. To this end, the expression of G1 in the cytoplasm and membrane of infected cells, treated or not with cecropin A for 18 h, was analysed by total and surface indirect immunofluorescence assays. Measurement of virus yield obtained from culture supernatants showed that under these experimental conditions a 90% inhibition of extracellular virus titre was achieved in the presence of the peptide. Total IF assay revealed that the number of fluorescent cells in cecropin A treated cultures was reduced only by 26% compared with untreated ones, indicating that the inhibitory action of the compound is not due to the interference with viral antigen synthesis. However, cecropin A produced a notable alteration on G1 intracellular localisation since a different pattern of staining was observed in untreated cells from that in cecropin A treated ones. While control cultures displayed intense perinuclear fluorescence and a more diffuse and homogeneous cytoplasmic staining, cecropin A treated cells exhibited mainly a marked fluorescence in the perinuclear region (Fig. 4a and b). On the other hand, cecropin A caused not only a decrease of 50% on the number of cells that exhibited membrane fluorescence but also a reduction on fluorescence intensity of positive cells (Fig. 4c and d). Inhibition of membrane G1 expression caused by cecropin A indicates that this compound might interfere either with G1 intracellular transport to the cell surface or with proper insertion of this glycoprotein into the cell membrane.
Some antimicrobial cationic peptides display antiviral activity against several enveloped viruses, but the lack of sequence homology among the different peptides isolated up today makes it difficult to predict their antiviral and cytotoxic activities [5, 6, 7, 8, 10, 11 and 23]. In the present study we evaluated the inhibitory and virucidal activities of the α-helical peptides cecropin A, melittin, magainin I and magainin II, and of indolicidin, an extended peptide, against JV, HSV-1 and HSV-2.
Besides the inhibitory action of melittin against HIV-1 and HSV-1 previously reported [6 and 8], here we demonstrate its antiviral activity toward JV and HSV-2 ( Table 1). It has been proved that melittin suppresses cell fusion mediated by HSV-1 syncytial mutants probably by interfering with the activity of the Na<sup>+</sup>, K<sup>+</sup> ATPase, cellular enzyme involved in the membrane fusion process [8]. Even though the mechanism of action of melittin has not been completely elucidated, its broad spectrum of antiviral action might be related to the ability of this peptide to affect ion gradients across the cell membrane. However, this compound is too toxic to consider it as a valuable antiviral agent.
On the other hand, cecropin A and magainins I and II did not affect cell viability in the experimental conditions tested. Cecropin A, which effectively inhibited JV multiplication, was inactive against HSV (Table 1). The anti-JV activity of cecropin A was comparable with its antiretroviral effect against HIV-1, with EC<sub>50</sub> values in the range of 2?3 μM [6]. In contrast, magainins were active against HSV-1 and HSV-2 but failed to inhibit JV replication ( Table 1).
Though the antiviral activity of several synthetic derivatives of magainin against HSV-1 has been attributed to their virucidal action [10], in our experimental conditions no direct inactivating effect was detected for the α-helical peptides tested ( Table 2). In accordance with our results, it was reported that magainin II does not reduce infectivity of cell-free HSV-1 stocks [23].
Studies performed in order to evaluate antiviral activity of indolicidin against HIV-1 indicated that this peptide causes the direct inactivation of virus particles [11]. In accordance with this report, indolicidin also showed a strong virucidal action against HSV-1 and HSV-2, whereas a less potent inactivating effect was detected against JV ( Table 2). Virucidal action of indolicidin against JV and HIV-1 appears to be similar since Robinson et al. [11] proved that a 50% reduction in residual infectivity of HIV-1 stocks is achieved after treatment with indolicidin at concentrations in the range 30?50 μM. On the other hand, though the presence of indolicidin during JV multiplication in Vero cells did not affect virus production, a strong inhibitory effect of indolicidin was demonstrated in HSV-1 and HSV-2 infected cultures (Table 1). The reduction of HSV-1 and HSV-2 yields caused by indolicidin can be ascribed to a direct inactivation of progeny virus released from indolicidin treated cells, however, an inhibitory effect of indolicidin on HSV multiplication can not be excluded.
Cecropin A showed a selective inhibitory action against JV and other species of arenavirus (Fig. 1), at concentrations that did not affect cell viability or HSV production, so we analysed in more detail the ability of this peptide to interfere with arenavirus multiplication. Cecropin A treatment reduced JV protein synthesis under conditions where the synthesis of host cell proteins remained unaffected ( Fig. 2). The analysis of the time-related cecropin A effect indicated that the inhibitory action of the compound is mainly exerted at late stages of JV multiplication cycle ( Fig. 3). The addition of cecropin A at 8 h p.i. inhibited the formation of both cell-associated and cell-free virus, however, the production of extracellular virus was significantly more sensitive to the peptide. Since cecropin A did not affect infectivity of virus particles by direct contact (Table 2), these experiments indicate that cecropin A treatment results in the accumulation of progeny virus in the cell. In addition, the partial reduction of cell-associated infectivity obtained in cecropin A treated cultures also suggests an inhibitory effect of the compound on JV maturation process. It has been demonstrated that the proteolytic processing of JV glycoprotein precursor, GPC, together with the presence of mature glycoprotein G1 at the cell membrane is required for the morphogenesis of infectious JV particles [21]. Cecropin A caused an altered intracellular distribution and membrane expression of the viral glycoprotein G1, as it was shown by membrane and cytoplasmic IF assays ( Fig. 4). These findings indicate that the inhibition of G1 transport and/or insertion into the cell membrane might prevent the formation of infectious particles leading to the reduction of cell-associated infectivity ( Fig. 3). Though late events on arenavirus replication are poorly known it is accepted that virus envelopment occurs at the plasma membrane and mature virions are released by a budding process [24]. In contrast, HSV morphogenesis takes place at cytoplasmic compartments and the egress of infectious particles occurs by the exocytic pathway [25]. It is possibly that these differences in morphogenesis and viral egress are associated with the lack of antiviral activity of cecropin A against HSV.
The understanding of the mechanisms underlying antiviral action of cationic peptides is necessary to consider this type of compound as potential useful antiviral agents. The data presented here indicate that, at non cytotoxic concentrations, α-helical peptides melittin, cecropin A and magainins I and II lack of virucidal effect and exhibit a differential inhibitory action against JV and HSV. On the contrary, indolicidin seems to exert its antiviral activity by direct inactivation of virus particles. In addition, we established that the inhibitory action of cecropin A against JV is related with the ability of this compound to interfere with virus morphogenesis and release of viral particles from the infected cell. Further studies must be done in order to investigate the action of cecropin A on glycoprotein processing and membrane fusion events involved in JV assembly and budding steps.
Acknowledgements
We thank Dr. C.E. Coto for her critical reading of the manuscript and Dr. A. Sanchez for his generous gift of monoclonal antibodies. This work was supported by grants from the Universidad de Buenos Aires (JX88, X051).
[FONT=Verdana,Arial,Helvetica,sans-serif] Copyright ? 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. [/FONT] Antiviral activity of antimicrobial cationic peptides against Junin virus and herpes simplex virus
Vanesa C. Albiol Matanic and Viviana Castilla <sup>
</sup><sup>, </sup><sup></sup> Laboratorio de Virolog?a, Departamento de Qu?mica Biol?gica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabell?n II, Piso 4, C1428BGA, Buenos Aires, Argentina
Received 22 May 2003; accepted 24 July 2003. Available online 16 March 2004.
Abstract
The in vitro antiviral activity of antimicrobial cationic peptides: cecropin A, melittin, magainin I and II and indolicidin against the arenavirus Junin virus (JV), and herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) was evaluated. Cecropin A effectively inhibited JV multiplication and failed to affect HSV replication whereas melittin impeded the multiplication of JV and HSV, but was highly toxic for the host cell. Magainins I and II exhibited inhibitory action toward HSV-1 and HSV-2 but were inactive against JV. Only indolicidin showed a direct inactivation effect on cell-free virus stocks. Besides its inhibitory effect on JV replication cecropin A also was active against the arenaviruses Tacaribe and Pichinde, mainly affecting late events of arenavirus multiplication cycle by preventing viral morphogenesis and egress from infected cells.
Author Keywords: Author Keywords: Arenavirus; Herpesvirus; Antimicrobial peptides; Antiviral activity
Article Outline
<dl><dt>1. Introduction</dt><dt>2. Materials and methods</dt><dl><dt>2.1. Cells and viruses</dt><dt>2.2. Antimicrobial peptides</dt><dt>2.3. Cytotoxicity assay</dt><dt>2.4. Antiviral activity</dt><dt>2.5. Virucidal assay</dt><dt>2.6. Analysis of radiolabelled proteins</dt><dt>2.7. Effect of time of cecropin A addition or removal on JV replication</dt><dt>2.8. Indirect immunofluorescence (IF) assay</dt></dl><dt>3. Results</dt><dl><dt>3.1. Antiviral activity of cationic peptides against JV, HSV-1 and HSV-2</dt><dt>3.2. Effect of cecropin A on Tacaribe virus and Pichinde virus multiplication</dt><dt>3.3. Virucidal action of cationic peptides</dt><dt>3.4. Effect of cecropin A on N protein synthesis</dt><dt>3.5. Effect of time of addition or removal of cecropin A at different times after infection on JV production</dt><dt>3.6. Effect of cecropin A on G1 intracellular transport</dt></dl><dt>4. Discussion</dt><dt>Acknowledgements</dt><dt>References</dt></dl>
1. Introduction
Natural cationic peptides have a variety of interesting biological activities including antibacterial, antifungal, antiparasitic, antitumoural, and antiviral activities [1, 2, 3 and 4]. The most common classes of cationic peptides are β-sheet peptides stabilised by disulphide bridges and unstructured peptides that fold into amphipathic α-helices upon contact with membranes. Less common are extended peptides with a predominance of one or two amino acids [4].
Although antibacterial activity of cationic peptides has been extensively studied, little is known about their ability to act as antiviral agents. Besides, the mechanism of action of those peptides that exhibit antiviral activity is still poorly understood.
The antiviral activity of human defensins, a group of β-sheet peptides, against enveloped viruses such as herpes simplex virus type 1 (HSV-1) and 2 (HSV-2), immunodeficiency virus type 1 (HIV-1), vesicular stomatitis virus (VSV), influenzavirus and cytomegalovirus has been ascribed to direct inactivation of viral particles [5]. On the other hand, it has been reported that α-helical peptides can act as virucidal agents or as inhibitors of virus multiplication. Two major types of α-helical peptides have been isolated from insects: melittin, a 26 amino acid peptide which is the major component of bee venom and a family of antibacterial peptides, 35?39 amino acids in length, named cecropins [3]. While melittin, at high concentrations, is lytic for blood red cells, cecropins appear to be non toxic for mammalian cells [6]. Antiviral activity of melittin against several enveloped viruses has been attributed to direct lysis of viral membranes, however, at lower non-virolytic concentrations, melittin exhibits inhibitory action against HSV-1 and HIV-1 [6, 7 and 8]. Among cecropins, it has been demonstrated that cecropin A inhibits HIV-1 multiplication in acutely and chronically infected cells [6 and 7]. Magainins I and II are 23-residue α-helical peptides isolated from the skin of the frog Xenopus laevis, that differ by substitutions in positions 10 and 22 [1 and 9] and it has been reported that synthetic analogues of magainins display virucidal action towards HSV-1 [10].
Direct viral inactivation against HIV-1 has been also described for indolicidin, a 13 amino acid peptide with extended structure and a high content of tryptophan, isolated from bovine neutrophils [4, 9 and 11].
Arenavirus are rodent-associated viruses that can cause severe human haemorrhagic fevers. In particular, Junin virus (JV) is the aetiological agent of Argentine haemorrhagic fever (AHF), an endemo-epidemic disease geographically restricted to the most fertile areas of the country. Though several compounds inhibit in vitro arenavirus multiplication [12], to date ribavirin is the only compound that has shown partial efficacy against JV infection in animal models and humans, but with a high level of undesirable secondary reactions [13 and 14]. Hence, the current therapy for AHF patients is the early administration of immune plasma, however, this therapy is not effective when it is initiated after 8 days illness [15].
Since there are no reports about the effect of cationic peptides on arenavirus multiplication, in the present study we tested the in vitro antiviral activity of cecropin A, melittin, magainins I and II and indolicidin against JV. In addition, to determine the specificity of action of these compounds, we also evaluated their antiviral action against HSV-1 and HSV-2, taking into account that among the selected peptides used here only melittin has been proved to be active against HSV-1 [8].
2. Materials and methods
2.1. Cells and viruses
Vero and BHK-21 cells were grown in Eagle?s minimal essential medium (MEM, GIBCO) containing 5% inactivated calf serum and 50 μg/ml gentamicin. Maintenance medium (MM), pH 7.5, consisted of MEM supplemented with 1.5% inactivated calf serum and gentamicin. IV<sub>4454</sub>, an attenuated strain of JV isolated from a mild human case of AHF [16], was propagated on BHK-21 cells. Tacaribe virus (strain TRLV<sub>11573</sub>), Pichinde virus (strain AN<sub>3739</sub>), HSV-1 (strain F) and HSV-2 (strain G) were propagated on Vero cells. Virus stocks were plaque-assayed on Vero cells.
2.2. Antimicrobial peptides
Stock solutions of cecropin A, melittin, magainins I and II and indolicidin (Sigma?Aldrich) were prepared in MEM at 250 μM and stored at −20 ?C until use.
2.3. Cytotoxicity assay
Vero cells grown in 96-well tissue culture plates were incubated with different concentrations of each compound for 24 h at 37 ?C. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) procedure [17]. The cytotoxicity of each compound was expressed as the 50% cytotoxic concentration (CC<sub>50</sub>), which is the concentration required to reduce cell viability to 50% of the control.
2.4. Antiviral activity
Antiviral activity was determined by a virus yield inhibition assay. Vero cell monolayers were infected at a multiplicity of infection (moi) of 0.1 PFU/cell. After 1 h adsorption at 37 ?C, MM containing various concentrations of the compounds was added. After 24 h of incubation at 37 ?C, culture supernatants were harvested and extracellular virus yields were determined by plaque assay on Vero cell monolayers.
The 50% effective concentration (EC<sub>50</sub>) and the 90% effective concentration (EC<sub>90</sub>) were calculated as the compound concentration that reduces virus yield by 50 and 90%, respectively.
2.5. Virucidal assay
Viral suspensions were incubated in the presence or absence of various concentrations of each compound for 2 h at 37 ?C. After the incubation period, the mixtures were placed on ice, serially diluted in MM and remaining infectivity was determined by plaque assay.
2.6. Analysis of radiolabelled proteins
Mock-infected or JV-infected Vero cells (moi = 5 PFU/cell) were incubated with MM containing or not cecropin A (5 or 10 μM) for 48 h at 37 ?C. Then cultures were washed with phosphate buffer saline (PBS), incubated with methionine-free medium for 1.5 h and then labelled with <sup>35</sup>S-methionine (100 μCi/ml). After a labelling period of 4 h, cells were washed three times with cold PBS.
To analyse cell protein synthesis treated and untreated mock-infected cultures were resuspended in sample buffer (PAGE) containing 5% SDS, 2% 2-mercaptoethanol, 10% glycerol and 0.005% bromophenol blue in 0.0625 M Tris?HCl, pH 6.8. Cell lysates were heated for 2 min in boiling water and SDS-PAGE was performed on 10% acrylamide gel. Radiolabelled proteins were visualised by fluorography on AGFA films.
To analyse viral protein synthesis mock-infected or JV-infected cells treated or not with cecropin A were lysed in radioimmune precipitation assay buffer (RIPA: 10 mM Tris?HCl pH 7.4, 0.15 M NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate and 0.4 mM PMSF) and incubated with monoclonal antibody (NA05AG12) reactive against viral nucleocapsid protein N [18] for 30 min at 37 ?C and further incubated at 0 ?C for 4 h. Antibody?antigen complexes were collected with protein A-sepharose (Sigma?Aldrich) and proteins were separated on 10% SDS-PAGE and visualised as described above.
2.7. Effect of time of cecropin A addition or removal on JV replication
Monolayers of confluent Vero cells were allowed to adsorb JV at a moi of 0.1 PFU/cell for 1 h at 37 ?C. After removal of the inocula, MM containing cecropin A (25 μM) was added to infected cells at 1, 3, 5 or 8 h post-infection (p.i.) and cultures were further incubated at 37 ?C. Another set of cell cultures was infected with JV in the presence of cecropin A (25 μM) and the compound was removed by medium change at 3, 5, 8 or 24 h p.i. In all cases, at 24 h p.i., extracellular virus yields were measured from supernatants by plaque assay. To determine cell-associated infectivity cultures were subjected to two cycles of freeze-thawing followed by centrifugation at low speed and the supernatants obtained were titrated by plaque assay.
2.8. Indirect immunofluorescence (IF) assay
Vero cell monolayers grown in coverslips were infected with JV at a moi of 0.1 PFU/cell and after 1 h adsorption, cultures were incubated in MM containing or not cecropin A (25 μM) at 37 ?C. At 18 h p.i. supernatants were collected and extracellular virus production was determined by plaque assay. The remaining cells were washed three times with cold PBS and the IF assays were performed as previously described [19] with minor modifications. For cytoplasmic IF assay, cells were fixed in methanol for 10 min at −20 ?C and then incubated with the monoclonal antibody GB03-BE08, reactive against G1 viral glycoprotein [18], for 30 min at 37 ?C. To perform the surface IF assay, cells were incubated with the monoclonal antibody for 30 min at 4 ?C and then fixed with methanol as described above. The indirect staining was carried out by using goat anti-mouse antibody conjugated to fluorescein isothiocyanate (Sigma?Aldrich). The number of fluorescent cells was counted from 20 randomly selected fields using a Zeiss Axioplan microscope.
3. Results
3.1. Antiviral activity of cationic peptides against JV, HSV-1 and HSV-2
We first examined the effect of cationic peptides on cell viability after incubating Vero cell monolayers with different concentrations of each compound at 37 ?C for 24 h. Cytotoxicity was evaluated using the MTT method and by examining cell morphology by light microscopy. While cecropin A, magainins I and II and indolicidin did not show cytotoxicity up to a concentration of 100 μM (the highest concentration assayed), treatment with melittin at concentrations above 5 μM caused cell rounding and monolayer detachment. As it is shown in Table 1, melittin CC<sub>50</sub> value, obtained by the MTT method, was 8.51 μM.
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[SIZE=-1]Table 1. Cytotoxicity and antiviral activity of cationic peptides against JV, HSV-1 and HSV-2
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[SIZE=-1]Data represent the mean values from two independent experiments. CC<sub>50</sub>: compound concentration required to reduce cell viability by 50%, as determined by the MTT method. EC<sub>50</sub>: compound concentration required to reduce virus yield by 50%.
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Antiviral activity of the compounds was examined by a virus yield inhibition assay. Vero cells infected with JV, HSV-1 or HSV-2 (moi=0.1) were incubated, immediately after virus adsorption, in MM containing different non cytotoxic concentrations of each compound. At 24 h p.i. virus yield was quantified by plaque assay and compared with untreated infected cultures.
[SIZE=-1]Table 1. Cytotoxicity and antiviral activity of cationic peptides against JV, HSV-1 and HSV-2
[SIZE=-1]Data represent the mean values from two independent experiments. CC<sub>50</sub>: compound concentration required to reduce cell viability by 50%, as determined by the MTT method. EC<sub>50</sub>: compound concentration required to reduce virus yield by 50%.
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A dose dependent inhibition of JV multiplication was observed in cecropin A treated cultures. The EC<sub>50</sub> value of cecropin A against JV was 3.24 μM and virus yield was inhibited by more than 90% in cultures treated with 40 μM of cecropin A, compared with untreated ones (Table 1 and Fig. 1). In contrast, this peptide did not exhibit inhibitory effect on the production of HSV-1 and HSV-2 infectious particles, even at a concentration of 40 μM (Table 1).
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[SIZE=-1]Fig. 1. Inhibition of arenavirus multiplication by cecropin A. Vero cells were infected with JV (black bars), Tacaribe virus (open bars) or Pichinde virus (shaded bars) at a moi of 0.1 PFU/cell. After 1 h adsorption at 37 ?C, inocula were removed and cultures were incubated in MM containing different concentrations of cecropin A. At 24 h p.i., extracellular virus yield was determined. Results are expressed as the percentage of virus yield inhibition in cecropin A treated cultures with respect to untreated controls. Data are mean values from two separate experiments. [/SIZE]
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Melittin, within the non-toxic range of concentrations (0.5?3 μM), showed inhibitory action against the three viruses assayed. At a concentration of 3 μM, this peptide reduced HSV-1 and HSV-2 yields by 80%, whereas JV was more sensitive to melittin inhibitory action since a 99% reduction of virus infectivity was achieved at this concentration of melittin. In addition, EC<sub>50</sub> against JV was lower than EC<sub>50</sub> values obtained for both HSV-1 and HSV-2 (Table 1).
[SIZE=-1]Fig. 1. Inhibition of arenavirus multiplication by cecropin A. Vero cells were infected with JV (black bars), Tacaribe virus (open bars) or Pichinde virus (shaded bars) at a moi of 0.1 PFU/cell. After 1 h adsorption at 37 ?C, inocula were removed and cultures were incubated in MM containing different concentrations of cecropin A. At 24 h p.i., extracellular virus yield was determined. Results are expressed as the percentage of virus yield inhibition in cecropin A treated cultures with respect to untreated controls. Data are mean values from two separate experiments. [/SIZE]
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On the other hand, magainins I and II, that produced a dose dependent inhibition of HSV-1 and HSV-2 multiplication with similar EC<sub>50</sub> values for both viruses, were inactive against JV (Table 1).
Finally, indolicidin, the only peptide with extended structure assayed, exhibited a strong inhibitory action against HSV. At the highest concentration tested (50 μM), indolicidin reduced HSV-1 and HSV-2 yields by 99%. However, from the EC<sub>50</sub> values obtained it appears that indolicidin is about five times more potent against HSV-2 than against HSV-1 (Table 1). On the contrary, no significant differences on JV yield were observed in indolicidin treated cultures compared with untreated ones.
3.2. Effect of cecropin A on Tacaribe virus and Pichinde virus multiplication
Although both cecropin A and melittin were active toward JV and the EC<sub>50</sub> for melittin was lower than the EC<sub>50</sub> for cecropin A, the selectivity index of melittin is low due to its high toxicity to the host cell (Table 1). Since cecropin A showed antiviral activity toward JV without affecting HSV multiplication, this compound was further examined for its inhibitory action against other arenaviruses. For this purpose, Vero cells were infected with Tacaribe virus or Pichinde virus (moi=0.1) and after 1 h adsorption at 37 ?C cultures were incubated in MM containing or not different concentrations of cecropin A and at 24 h p.i. virus yield was determined by plaque assay. As it is shown in Fig. 1, cecropin A also affected Tacaribe virus and Pichinde virus multiplication in a dose dependent manner. The EC<sub>50</sub> for Tacaribe virus and Pichinde virus were 1.96 and 6.56 μM, respectively, and those values were similar to the EC<sub>50</sub> obtained for JV (Table 1). In addition, the EC<sub>90</sub> values for JV, Tacaribe virus and Pichinde virus were of 28.57, 33.39 and 39 μM, respectively.
3.3. Virucidal action of cationic peptides
In order to determine whether cationic peptides had a direct inactivating effect on virus infectious particles, an assay of virucidal activity was performed. To this end, aliquots of JV, HSV-1 or HSV-2 suspensions were exposed for 2 h at 37 ?C to each compound using the highest peptide concentration tested in the virus yield inhibition assay described above. Then samples were chilled and diluted to reduce the peptide concentration below the corresponding effective antiviral concentration EC<sub>50</sub> and residual infectivity was determined by a plaque formation assay.
As can be seen in Table 2, in the experimental conditions assayed, indolicidin was the only peptide that displayed virucidal action. A potent inactivating effect was observed against HSV-1 and HSV-2, since infectivity was reduced by more than 99% for both viruses. Less degree of inactivation was observed for indolicidin treated JV suspension, since residual JV infectivity was reduced by 50% compared with untreated control sample (Table 2).
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[SIZE=-1]Table 2. Evaluation of virucidal activity of cationic peptides against JV, HSV-1 and HSV-2
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[SIZE=-1]Viral suspensions were treated with MM (control) or MM containing the indicated concentration of each compound for 2 h at 37 ?C and remaining infectivity was titrated by plaque assay. The data represent the average values from duplicate independent experiments?S.D.
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3.4. Effect of cecropin A on N protein synthesis
[SIZE=-1]Table 2. Evaluation of virucidal activity of cationic peptides against JV, HSV-1 and HSV-2
[SIZE=-1]Viral suspensions were treated with MM (control) or MM containing the indicated concentration of each compound for 2 h at 37 ?C and remaining infectivity was titrated by plaque assay. The data represent the average values from duplicate independent experiments?S.D.
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Only five proteins are expressed from the two genome fragments of JV: the S RNA encodes the major structural proteins, the nucleocapsid protein (N) and two envelope glycoproteins (G1 and G2), whereas the L RNA encodes the RNA polymerase protein (L) and an 11 kDa protein with regulatory functions called Z [20].
In order to analyse further the selective inhibitory action of cecropin A against JV in vitro multiplication, the effect of this peptide on both cellular and viral protein synthesis was examined. First, non infected Vero cells were treated with cecropin A for 48 h at 37 ?C and after a 4 h pulse labelling with <sup>35</sup>S-methionine, cells were lysed and radiolabelled cell proteins were analysed by SDS-PAGE. As can be seen in Fig. 2, treatment with 5 or 10 μM of cecropin A had no effect on cell protein synthesis. The other set of infected labelled cultures, treated or not with cecropin A (10 μM) for 48 h, was immunoprecipitated with a monoclonal antibody reactive against viral protein N and further analysed by SDS-PAGE. In untreated infected cultures (Fig. 2, lane 5) the N protein (63 kDa) and a polypeptide band with apparent molecular weight of approximately 45 kDa, corresponding to an N degradation product previously described [18], were observed. A reduction in the amount of both N and the N derived polypeptide of 45 kDa was detected in infected cultures treated with cecropin A (Fig. 2, lane 4). Thus, these results indicate that cecropin A is able to selectively inhibit viral protein synthesis without affecting host cell macromolecular synthesis. Although in this experiment a high multiplicity of infection was employed, the low efficiency of JV adsorption process does not insure the synchronisation of the infection. Moreover, the labelling period with <sup>35</sup>S-methionine was performed at 48 h p.i., due to the intrinsically poor efficiency of JV protein synthesis that impedes its analysis at earlier times after infection. The time required for a complete JV multiplication cycle ranges from 12 to 16 h, thus, as several multiplication cycles take place within a period of 48 h, inhibition of viral protein synthesis might be an indirect consequence of the inhibitory action of cecropin A on any other step of the virus replication cycle.
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[SIZE=-1]Fig. 2. Effect of cecropin A on cellular and JV protein synthesis. (a) Vero cells were incubated in MM (lane 1) or MM containing 5 μM (lane 2) or 10 μM (lane 3) cecropin A for 48 h at 37 ?C. Then cells were pulse-labelled with <sup>35</sup>S-methionine for 4 h, resuspended in lysis buffer and labelled cell proteins were analysed by SDS-PAGE. (b) Mock-infected (lane 6) or JV-infected Vero cells (lanes 4 and 5) were incubated in the presence (lane 4) or absence (lanes 5 and 6) of 10 μM cecropin A. At 48 h p.i., after a 4 h pulse labelling with <sup>35</sup>S-methionine, cells were lysed and N protein was immunoprecipitated and analysed by SDS-PAGE. Arrows show the position of bands corresponding to N protein and its degradation product. [/SIZE]
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3.5. Effect of time of addition or removal of cecropin A at different times after infection on JV production
[SIZE=-1]Fig. 2. Effect of cecropin A on cellular and JV protein synthesis. (a) Vero cells were incubated in MM (lane 1) or MM containing 5 μM (lane 2) or 10 μM (lane 3) cecropin A for 48 h at 37 ?C. Then cells were pulse-labelled with <sup>35</sup>S-methionine for 4 h, resuspended in lysis buffer and labelled cell proteins were analysed by SDS-PAGE. (b) Mock-infected (lane 6) or JV-infected Vero cells (lanes 4 and 5) were incubated in the presence (lane 4) or absence (lanes 5 and 6) of 10 μM cecropin A. At 48 h p.i., after a 4 h pulse labelling with <sup>35</sup>S-methionine, cells were lysed and N protein was immunoprecipitated and analysed by SDS-PAGE. Arrows show the position of bands corresponding to N protein and its degradation product. [/SIZE]
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To further characterise the antiviral mode of action of cecropin A, we next examined the effect of time of cecropin A addition on JV multiplication. Cecropin A (25 μM) was added to JV-infected cells at different times after adsorption and at 24 h p.i. the extracellular and cell-associated virus yields were determined. As it is shown in Fig. 3a, within the first 8 h of infection, inhibition of JV multiplication is not dependent on the time of addition of the peptide. On the other hand, a differential effect of cecropin A on extracellular and cell-associated infectivity was observed. Whereas extracellular virus production was highly inhibited (90%), cell-associated infectivity was only reduced by 65%. Another set of cell cultures was infected with JV in the presence of cecropin A (25 μM). In this case, the compound was removed at different times after infection and cell-associated and extracellular virus yields were quantified at 24 h p.i. The presence of cecropin A during the first 5 h of infection did not inhibit viral production indicating that this peptide has no effect on early steps of JV multiplication (Fig. 3b). However, when the compound was removed at 8 h p.i. virus infectivity released to the culture supernatant was decreased by 90% with respect to untreated control, whereas cell-associated infectivity was reduced by 47%. These results suggest that cecropin A exerts its inhibitory action when it is present during late events of viral multiplication cycle, mainly affecting the release of infectious viral particles to the extracellular medium.
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[SIZE=-1]Fig. 3. Effect of time of cecropin A addition or removal on JV replication. Vero cells were infected with VJ and at different times after infection cecropin A (25 μM) was added and maintained till 24 h p.i. at 37 ?C (a). Another set of cultures was infected with JV in the presence of cecropin A (25 μM). At different times p.i. the compound was removed and cells were further incubated at 37 ?C up to 24 h p.i. (b). In both cases time 0 was defined as the time when virus was added to start infection. At 24 h p.i. extracellular virus titer from treated (▪) or untreated cultures (
) were determined. Cells were harvested and infectious cell-associated virus was also assayed from treated (?) or untreated cells (○). Data are mean values from two separate experiments. [/SIZE]
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3.6. Effect of cecropin A on G1 intracellular transport
[SIZE=-1]Fig. 3. Effect of time of cecropin A addition or removal on JV replication. Vero cells were infected with VJ and at different times after infection cecropin A (25 μM) was added and maintained till 24 h p.i. at 37 ?C (a). Another set of cultures was infected with JV in the presence of cecropin A (25 μM). At different times p.i. the compound was removed and cells were further incubated at 37 ?C up to 24 h p.i. (b). In both cases time 0 was defined as the time when virus was added to start infection. At 24 h p.i. extracellular virus titer from treated (▪) or untreated cultures (
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JV glycoproteins are synthesised in the endoplasmic reticulum as a precursor called GPC (62?65 kDa), and after post-translational modifications and cleavage in the distal trans Golgi network, mature glycoproteins named G1 (38 kDa) and G2 (45?50 kDa) are transported to the cell surface where virus assembly takes place [21]. G1, which has been claimed to induce neutralising antibodies, seems to be the only glycoprotein exposed on the virion surface as it was shown by in vitro iodination of intact purified virions [22].
The results described above (Fig. 3) suggest that cecropin A affects late steps of JV multiplication, so we decided to evaluate the action of this compound on the intracellular transport of the main JV glycoprotein G1. To this end, the expression of G1 in the cytoplasm and membrane of infected cells, treated or not with cecropin A for 18 h, was analysed by total and surface indirect immunofluorescence assays. Measurement of virus yield obtained from culture supernatants showed that under these experimental conditions a 90% inhibition of extracellular virus titre was achieved in the presence of the peptide. Total IF assay revealed that the number of fluorescent cells in cecropin A treated cultures was reduced only by 26% compared with untreated ones, indicating that the inhibitory action of the compound is not due to the interference with viral antigen synthesis. However, cecropin A produced a notable alteration on G1 intracellular localisation since a different pattern of staining was observed in untreated cells from that in cecropin A treated ones. While control cultures displayed intense perinuclear fluorescence and a more diffuse and homogeneous cytoplasmic staining, cecropin A treated cells exhibited mainly a marked fluorescence in the perinuclear region (Fig. 4a and b). On the other hand, cecropin A caused not only a decrease of 50% on the number of cells that exhibited membrane fluorescence but also a reduction on fluorescence intensity of positive cells (Fig. 4c and d). Inhibition of membrane G1 expression caused by cecropin A indicates that this compound might interfere either with G1 intracellular transport to the cell surface or with proper insertion of this glycoprotein into the cell membrane.
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[SIZE=-1]Fig. 4. Intracellular localisation of G1 glycoprotein in cecropin A treated cells. Cytoplasmic IF staining of JV-infected Vero cells in the absence (a) or in the presence (b) of 25 μM cecropin A. Cell surface IF staining of JV infected cells in the absence (c) or in the presence (d) of 25 μM cecropin A. Magnification 1000?. [/SIZE]
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4. Discussion
[SIZE=-1]Fig. 4. Intracellular localisation of G1 glycoprotein in cecropin A treated cells. Cytoplasmic IF staining of JV-infected Vero cells in the absence (a) or in the presence (b) of 25 μM cecropin A. Cell surface IF staining of JV infected cells in the absence (c) or in the presence (d) of 25 μM cecropin A. Magnification 1000?. [/SIZE]
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Some antimicrobial cationic peptides display antiviral activity against several enveloped viruses, but the lack of sequence homology among the different peptides isolated up today makes it difficult to predict their antiviral and cytotoxic activities [5, 6, 7, 8, 10, 11 and 23]. In the present study we evaluated the inhibitory and virucidal activities of the α-helical peptides cecropin A, melittin, magainin I and magainin II, and of indolicidin, an extended peptide, against JV, HSV-1 and HSV-2.
Besides the inhibitory action of melittin against HIV-1 and HSV-1 previously reported [6 and 8], here we demonstrate its antiviral activity toward JV and HSV-2 ( Table 1). It has been proved that melittin suppresses cell fusion mediated by HSV-1 syncytial mutants probably by interfering with the activity of the Na<sup>+</sup>, K<sup>+</sup> ATPase, cellular enzyme involved in the membrane fusion process [8]. Even though the mechanism of action of melittin has not been completely elucidated, its broad spectrum of antiviral action might be related to the ability of this peptide to affect ion gradients across the cell membrane. However, this compound is too toxic to consider it as a valuable antiviral agent.
On the other hand, cecropin A and magainins I and II did not affect cell viability in the experimental conditions tested. Cecropin A, which effectively inhibited JV multiplication, was inactive against HSV (Table 1). The anti-JV activity of cecropin A was comparable with its antiretroviral effect against HIV-1, with EC<sub>50</sub> values in the range of 2?3 μM [6]. In contrast, magainins were active against HSV-1 and HSV-2 but failed to inhibit JV replication ( Table 1).
Though the antiviral activity of several synthetic derivatives of magainin against HSV-1 has been attributed to their virucidal action [10], in our experimental conditions no direct inactivating effect was detected for the α-helical peptides tested ( Table 2). In accordance with our results, it was reported that magainin II does not reduce infectivity of cell-free HSV-1 stocks [23].
Studies performed in order to evaluate antiviral activity of indolicidin against HIV-1 indicated that this peptide causes the direct inactivation of virus particles [11]. In accordance with this report, indolicidin also showed a strong virucidal action against HSV-1 and HSV-2, whereas a less potent inactivating effect was detected against JV ( Table 2). Virucidal action of indolicidin against JV and HIV-1 appears to be similar since Robinson et al. [11] proved that a 50% reduction in residual infectivity of HIV-1 stocks is achieved after treatment with indolicidin at concentrations in the range 30?50 μM. On the other hand, though the presence of indolicidin during JV multiplication in Vero cells did not affect virus production, a strong inhibitory effect of indolicidin was demonstrated in HSV-1 and HSV-2 infected cultures (Table 1). The reduction of HSV-1 and HSV-2 yields caused by indolicidin can be ascribed to a direct inactivation of progeny virus released from indolicidin treated cells, however, an inhibitory effect of indolicidin on HSV multiplication can not be excluded.
Cecropin A showed a selective inhibitory action against JV and other species of arenavirus (Fig. 1), at concentrations that did not affect cell viability or HSV production, so we analysed in more detail the ability of this peptide to interfere with arenavirus multiplication. Cecropin A treatment reduced JV protein synthesis under conditions where the synthesis of host cell proteins remained unaffected ( Fig. 2). The analysis of the time-related cecropin A effect indicated that the inhibitory action of the compound is mainly exerted at late stages of JV multiplication cycle ( Fig. 3). The addition of cecropin A at 8 h p.i. inhibited the formation of both cell-associated and cell-free virus, however, the production of extracellular virus was significantly more sensitive to the peptide. Since cecropin A did not affect infectivity of virus particles by direct contact (Table 2), these experiments indicate that cecropin A treatment results in the accumulation of progeny virus in the cell. In addition, the partial reduction of cell-associated infectivity obtained in cecropin A treated cultures also suggests an inhibitory effect of the compound on JV maturation process. It has been demonstrated that the proteolytic processing of JV glycoprotein precursor, GPC, together with the presence of mature glycoprotein G1 at the cell membrane is required for the morphogenesis of infectious JV particles [21]. Cecropin A caused an altered intracellular distribution and membrane expression of the viral glycoprotein G1, as it was shown by membrane and cytoplasmic IF assays ( Fig. 4). These findings indicate that the inhibition of G1 transport and/or insertion into the cell membrane might prevent the formation of infectious particles leading to the reduction of cell-associated infectivity ( Fig. 3). Though late events on arenavirus replication are poorly known it is accepted that virus envelopment occurs at the plasma membrane and mature virions are released by a budding process [24]. In contrast, HSV morphogenesis takes place at cytoplasmic compartments and the egress of infectious particles occurs by the exocytic pathway [25]. It is possibly that these differences in morphogenesis and viral egress are associated with the lack of antiviral activity of cecropin A against HSV.
The understanding of the mechanisms underlying antiviral action of cationic peptides is necessary to consider this type of compound as potential useful antiviral agents. The data presented here indicate that, at non cytotoxic concentrations, α-helical peptides melittin, cecropin A and magainins I and II lack of virucidal effect and exhibit a differential inhibitory action against JV and HSV. On the contrary, indolicidin seems to exert its antiviral activity by direct inactivation of virus particles. In addition, we established that the inhibitory action of cecropin A against JV is related with the ability of this compound to interfere with virus morphogenesis and release of viral particles from the infected cell. Further studies must be done in order to investigate the action of cecropin A on glycoprotein processing and membrane fusion events involved in JV assembly and budding steps.
Acknowledgements
We thank Dr. C.E. Coto for her critical reading of the manuscript and Dr. A. Sanchez for his generous gift of monoclonal antibodies. This work was supported by grants from the Universidad de Buenos Aires (JX88, X051).