Laboratory of Virology, Department of Biochemistry, School of Science, University of Buenos Aires, Pabellón II, Piso 4to, Ciudad Universitaria, C1428BGA Buenos Aires, Argentina
Correspondence
Andrea A. Barquero
alecab{at}qb.fcen.uba.ar
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Many compounds of plant origin exhibiting antiviral activity have been described. These include alkaloids (Martin, 1987), flavonoids (Lin et al., 1999
), terpenes and polysaccharides (Bourne et al., 1999
), lignans (Charlton, 1998
), steroidal glycosides (Ikeda et al., 2000
) and proteins (Aoki et al., 1995
). Recently, we reported the isolation and purification of 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM) from leaf extracts of M. azedarach L., a tetranortriterpenoid with in vitro insecticidal properties (Lee et al., 1991
). We established that CDM inhibits VSV and herpes simplex virus type 1 (HSV-1) multiplication in vitro when added after infection, with low cytotoxicity (Alché et al., 2003
).
Since CDM exhibits antiviral activity against VSV and HSV-1, we hypothesized that it could be the molecule responsible for the broad spectrum of MA antiviral action previously shown. Therefore, the aim of the present paper was to unravel both the mechanism of action of CDM on the VSV multiplication cycle and to investigate its eventual effect on the induction of a refractory antiviral state.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antiviral compound.
CDM was purified from the leaves of M. azedarach L., as described by Alché et al. (2003), solubilized in MEM 1·5 % to a final concentration of 1 mg ml-1 and stored at -20 °C.
Acridine orange staining of living cells.
Vero cells grown on coverslips were stained with acridine orange (1 µg ml-1) for 15 min at 37 °C, washed twice with cold PBS, mounted in PBS, and visualized on a Zeiss Axioplan fluorescence microscope (magnification x400).
Analysis of VSV proteins by SDS-PAGE.
Vero cells were grown in 24-well plates and infected with VSV, then incubated in MEM 1·5 % until 5 h post-infection (p.i.). Protein labelling was carried out using 10 µCi [35S]methionine ml-1 (1175 Ci mmol-1; New England Nuclear) added from 5 to 7 h p.i. in methionine-free medium. The radiolabelled monolayers were dissolved in Laemmli buffer (Laemmli, 1970) and aliquots of each sample were heated at 90 °C for 5 min and analysed by 10 % SDS-PAGE.
Preparation of [35S]methionine-labelled VSV.
Vero cells cultured in 750 cm2 flasks were infected with VSV at an m.o.i. of 0·01 p.f.u. per cell. At 15 h p.i., the maintenance medium was replaced with methionine-free medium containing 10 µCi [35S]methionine ml-1 (1175 Ci mmol-1; New England Nuclear). After an 810 h incubation period, the medium was collected and cellular debris was removed. The virus was concentrated by centrifugation at 26 000 r.p.m. for 2 h at 4 °C in a Beckman SW 28 rotor. Further purification was achieved by centrifugation at 46 000 r.p.m. for 2 h at 4 °C in a Beckman SW 55 Ti rotor in a sucrose discontinuous gradient. After centrifugation, the virus band was collected, pelleted and titrated. The virus titre obtained was 4·6x109 p.f.u. ml-1, corresponding to 1500 p.f.u. c.p.m.-1
Binding and uptake assays.
To measure binding, 1·4x104 and 3x104 c.p.m. of 35S-labelled VSV diluted in binding medium (serum-free MEM containing 0·5 mg BSA ml-1 and 3 mM HEPES) were allowed to bind to Vero cells at 0 °C and 37 °C, respectively, with gentle agitation. At indicated times, the total cell-associated radioactivity was measured by lysing the cells with 0·1 M NaOH/1 % SDS and directly adding a detergent-based scintillation fluid. The internalized radioactivity was counted after treating the cells with 1 mg proteinase K ml-1 to remove surface-associated virus.
Endocytosis and degradation were studied by binding 3x104 c.p.m. 35S-labelled VSV to cells at 0 °C and then raising the temperature to 37 °C. At different times p.i., the internalized radioactivity was counted as described above. The appearance of TCA-soluble [35S]methionine in the medium was determined by precipitation with an equal volume of 10 % TCA on ice for 45 min and centrifugation for 5 min at 10 000 g. The supernatant was then counted for radioactivity. In all cases, radioactivity was counted in a liquid scintillation counter (240 CL/D Packard).
Indirect immunofluorescence assay (IFI).
Vero cells grown on coverslips to 70 % confluence were infected with VSV. For surface and internal staining, Vero cells were fixed with methanol for 10 min at -20 °C. Fixed cells were then incubated for 30 min at 37 °C with rabbit anti-G protein polyclonal antibodies or mouse anti-M protein monoclonal antibodies (mAbs) (kindly provided by Pablo Grigera, CEVAN, Buenos Aires, Argentina). Cells were then incubated with goat anti-rabbit or anti-mouse IgG secondary antibodies, respectively, conjugated to FITC or TRITC (Sigma) for 30 min at 37 °C. For surface IFI staining, the addition of primary antibodies to VSV-infected cells for 30 min at 4 °C was done prior to fixation with methanol. In both cases, coverslips were rinsed and mounted. Cells were photographed with a Zeiss microscope with epifluorescence optics.
Confocal microscopy.
Vero cells grown on coverslips in a 24-well plate were transfected with a cDNA encoding galactosyltransferase T2 fused with the enhanced green fluorescent protein (GalT2GFP) (Giraudo et al., 2001), provided by Hugo Maccioni. Lipofectin reagent (Gibco) was used for transfection of Vero cells with 2 µg of plasmid DNA per well. After 24 h transfection, cells were infected with VSV at an m.o.i of 1 p.f.u. per cell. At 6 h p.i., cells were processed for G protein staining using an anti-rabbit TRITC-conjugated antibody. Coverslips were mounted and analysed with an Olympus FB300 confocal microscope. Images were collected and processed using Fluoview version 3.2 and Adobe Photoshop software.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Initially, we established that CDM prevented VSV multiplication in Vero cells when added 2 h before infection, exhibiting 50 % inhibition of virus yield at a concentration of 0·75 µM and 50 % cytotoxicity at a concentration above 520 µM (data not shown).
The effect of CDM on both virus multiplication and endosomal pH was examined in Vero cell monolayers treated with 7·5 µM CDM for 2 h at 37 °C and infected with 200 p.f.u. VSV per well at 0, 2, 4, 8, 10 and 24 h post-treatment (p.t.). A control set of uninfected cells grown on coverslips was stained with acridine orange at the same time intervals p.t. When Vero cells were infected immediately after treatment or as long as 10 h p.t., there was more than 90 % inhibition in a plaque reduction assay. In contrast, susceptibility of cells to virus infection was restored by 24 h p.t. (Fig. 1A).
|
In conclusion, CDM induces a refractory state in Vero cell monolayers that is maintained for at least 10 h p.t., as well as causing the pH of intracellular vesicles to become alkaline, as is also the case with MA.
Effect of CDM on VSV protein expression
We have previously demonstrated that CDM inhibits VSV multiplication when added after infection in a multicycle growth experiment (Alché et al., 2003). A CDM dose-dependent inhibitory effect was also observed under single-step growth conditions. Vero cells infected with VSV (m.o.i.=1) for 1 h at 37 °C were treated with different concentrations of CDM and at 6 h p.i., supernatants from treated and untreated cultures were titrated in a plaque assay. VSV titres after treatment with 0·75, 1·5, 7·5, 15 and 75 µM CDM were 10x105, 8x105, 4·2x105, 1x105 and 0·83x105 p.f.u. ml-1, respectively, whereas an infectivity of 1·5x106 p.f.u. ml-1 was obtained in infected but untreated cells. Virus replication was also suppressed by 90 and 91 % when 75 µM CDM was added to the cultures for 03 and 36 h p.i., respectively.
In view of these results, we decided to investigate whether the antiviral activity of CDM targets unique or different targets in the VSV multiplication cycle by analysing the expression of viral proteins.
Vero cells were treated with different concentrations of CDM before or after infection with VSV at an m.o.i. of 1 p.f.u. per cell. At 5 h p.i., cells were pulse-labelled with [35S]methionine for 2 h and the labelled polypeptides were analysed by SDS-PAGE. At this time post-infection, host translation has been shut-down and infected cells mostly synthesize VSV proteins. When added before infection, CDM decreased the levels of VSV proteins in a dose-dependent manner and restored the expression of cellular proteins (Fig. 2A). The inhibition observed suggested that CDM blocks the synthesis of viral proteins or an initial event in VSV infection in pre-treated cells.
|
Effect of CDM on the early steps of VSV multiplication
To assess the effect of CDM pre-treatment on the initial stages of the infection cycle, radioactively labelled VSV was used to determine the interaction of VSV and cells at 0 °C and 37 °C. Vero cells were treated with 75 µM CDM for 2 h at 37 °C, then infected with [35S]VSV at 0 °C. Less than 10 % of the added virus became cell-associated after 1 h at 0 °C, indicating that CDM pre-treatment did not interfere with VSV binding at 0 °C (data not shown).
To determine the effect of CDM on virus internalization, the amount of both total cell-associated and proteinase K-resistant radioactivity was measured. A partial inhibition (44 %) of total cell-associated VSV in pre-treated cells was observed (Table 1). However, the internalized radioactivity did not differ significantly from that obtained in the untreated culture (Table 1
). These experiments indicated that CDM diminishes the amount of surface-associated VSV at 37 °C.
|
|
|
Effect of CDM on low-pH-induced VSV fusion entry
Since the uncoating of VSV depends on a membrane fusion event and VSV can be made to fuse directly with the plasma membrane by lowering the pH of the medium, we decided to examine the effect of CDM on the acid-induced virus fusion activity. For this purpose, Vero cells infected with VSV at an m.o.i. of 10 p.f.u. per cell for 1 h at 0 °C were incubated with MEM pH 5 for 5 min at 37 °C. The medium was then replaced with MEM pH 7·4 and virus yield was measured at 20 h p.i. No difference in virus titre corresponding to infected cells treated either with MEM pH 5 or MEM pH 7·4 was observed. However, whereas CDM pre-treatment reduced VSV production by 3 logs in cells maintained in MEM pH 7·4, only a 1 log reduction in VSV yield was found in cells briefly exposed to MEM pH 5 (Fig. 5A, i).
|
Additional data supporting the impairment of the VSV endocytic pathway by CDM were provided by morphological analysis of the M protein, which was expressed in untreated Vero cells at 2 h p.i. independently of the pH of the medium (Fig. 5C, i and ii). However, in CDM pre-treated infected cultures, M protein was only synthesized when VSV fusion was induced at pH 5 (Fig. 5C
, iv), whereas a restricted and punctuate fluorescence pattern was observed at pH 7·4 (Fig. 5C
, iii).
Therefore, VSV inhibition due to CDM pre-treatment can be by-passed by inducing virus fusion at the cell surface.
Intracellular localization of M and G VSV proteins in CDM post-treated cells
Since VSV proteins are synthesized in the presence of CDM when it is added after infection (Fig. 2B), we decided to visualize the intracellular localization of M and G proteins in infected and treated cells by total or surface IFI staining.
No difference in M protein intracellular localization in infected control cells and CDM post-treated infected cells were observed (Fig. 6B and C, respectively). With respect to G protein cytoplasmic expression, no significant difference between the number of fluorescent cells from untreated and CDM-treated cultures was detected (Fig. 6E and F
). Strikingly, whereas the G protein was distributed throughout the cytoplasm and the plasma membrane of untreated infected cells (Fig. 6E
), it appeared to be associated with the perinuclear region in the majority of cells treated with CDM (Fig. 6F
). The antiviral treatment caused an inhibition of 67 % in the number of fluorescent cells expressing G protein at their surface (Fig. 6I
). These results demonstrated that the intracellular transport of the VSV G protein is affected by CDM.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CDM was shown to induce cytoplasmic alkalinization. We strongly suggest that the establishment of the refractory state is associated with this alteration, since the anti-VSV activity correlated closely with the increase in pH for as long as 10 h (Fig. 1).
Since the acidification of intracellular endosomes is required for virus endocytosis, the inhibition of entry of VSV nucleocapsids into Vero cells by the endocytic pathway was expected. In this sense, the first clue for the involvement of an early step in the VSV multiplication cycle affected by CDM was the lack of viral protein synthesis in pre-treated infected cells (Fig. 2A). Further results discounted adsorption as being affected by CDM, although virus internalization was partially inhibited (Fig. 3
). This partial inhibition did not account for the lack of viral protein synthesis observed and perhaps could be a consequence of an eventual inhibitory effect of CDM on membrane and/or receptor recycling. Similar results were reported by Pérez & Carrasco (1994)
in BHK-21 cells infected with Semliki forest virus and treated with bafilomycin A1, which interferes with virus uncoating.
The antiviral effect of CDM was partially reversed when VSV entered cells by direct fusion at the plasma membrane, revealing that VSV was able to enter a multiplication cycle, despite the refractory antiviral state of the cells. At pH 5·7, only 10 % of infecting virus directly fuses with cellular membrane (Puri et al., 1988), which would explain the partial reversion of the inhibitory effect of CDM.
It is well known that M protein becomes soluble in the cytoplasm after virus internalization (Rigaut et al., 1991). Since we found a pattern of punctuate fluorescence in CDM pre-treated cells (Fig. 5C
, iii), we inferred that M protein was confined to the endosomes due to the inability of virus particles to uncoat, even 2 h post-internalization.
Taken together, these findings suggest that delivery of VSV nucleocapsids into the cytoplasm was hampered in CDM pre-treated cells, though this is not enough to explain the anti-VSV activity of CDM when added after infection in a single-cycle growth experiment. The fact that VSV protein synthesis was not affected (Fig. 2B) suggests that a late step in the virus multiplication cycle is hindered by CDM post-treatment.
IFI staining results clearly showed that the G protein did not appear at the surface of CDM-treated infected cells (Fig. 6I) and the cytoplasmic fluorescent pattern observed corroborated that G protein accumulated somewhere along the exocytic route (Fig. 6F
), even when CDM was added from 3 to 6 h p.i. (data not shown). Confocal microscopy revealed that glycoprotein G was confined to the Golgi complex after CDM treatment (Fig. 7
). Hence, CDM appears to be affecting the endocytic and exocytic pathways of VSV as a consequence of its action on the pH of intracellular organelles.
Acidification of vacuolar compartments plays an important role in a variety of cellular processes. Perturbation of the acidic pH of various intracellular organelles by acidotrophic agents, such as weak bases and ammonium chloride, ionophores such as monensin and specific inhibitors of vacuolar (V-) ATPase such as bafilomycin, affects both viral endocytic and exocytic pathways (Sidhu et al., 1999). Although the precise mechanism by which CDM exerts its effects is still unresolved, it is the first tetranortriterpenoid with antiviral activity described responsible for the alkalinization of intracellular compartments.
Palokangas et al. (1994) have shown that bafilomycin A1 blocks VSV glycoprotein transport in BHK-21 cells and suggested that normally the Golgi complex is acidified by a vacuolar-type H+-ATPase. Likewise, the inhibition of transport of the VSV G protein by IFN-
may be related to the inhibition of V-ATPase-mediated acidification of the trans-Golgi network (Sidhu et al., 1999
). An eventual relationship between the CDM pleiotropic effect and V-ATPase should be investigated to elucidate further whether the antiviral activity observed is a consequence of CDM modulation of V-ATPase activity.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alché, L. E., Ferek, G. A., Meo, M., Coto, C. E. & Maier, M. S. (2003). An antiviral meliacarpin from leaves of Melia azedarach L. Z Naturforsch Redaktion Sect C 58c, 215219.
Andrei, G. M., Damonte, E. B., de Torres, R. A. & Coto, C. E. (1988). Induction of a refractory state to viral infection in mammalian cells by a plant inhibitor isolated from leaves of Melia azedarach L. Antivir Res 9, 221231.[CrossRef][Medline]
Andrei, G. M., Couto, A. S., Lederkremer, R. M. & Coto, C. E. (1994). Purification and partial characterization of an antiviral active peptide from Melia azedarach L. Antivir Chem Chemother 5, 105110.
Aoki, H., Akaike, T., Abe, K., Kuroda, M., Arai, S., Okamura, R. I., Negi, A. & Maeda, H. (1995). Antiviral effect of oryzacystatin, a proteinase inhibitor in rice, against herpes simplex virus type 1 in vitro and in vivo. Antimicrob Agents Chemother 39, 846849.[Abstract]
Benencia, F., Courrèges, M. C. & Coulombié, F. C. (1997). Antiviral activity of crude polysaccharides from Trichilia glabra leaves. Fitoterapia LXVIII, 173175.
Bohnenstengel, F. I., Wray, V., Witte, L., Srivastava, R. P. & Proksch, P. (1999). Insecticidal meliacarpins (C-seco limonoids) from Melia azedarach. Phytochemistry 50, 977982.[CrossRef]
Bourne, K. Z., Bourne, N., Reising, S. F. & Stanberry, L. R. (1999). Plant products as topical microbicide candidates: assessment of in vitro and in-vivo activity against herpes simplex virus type 2. Antivir Res 42, 219226.[CrossRef][Medline]
Castilla, V., Barquero, A. A., Mersich, S. E. & Coto, C. E. (1998). In vitro anti-Junín virus activity of a peptide isolated from Melia azedarach L. leaves. Int J Antimicrob Agents 10, 6775.[CrossRef][Medline]
Charlton, J. L. (1998). Antiviral activity of lignans. J Nat Prod 61, 14471451.[CrossRef][Medline]
Córdoba, M. A., Coto, C. E. & Damonte, E. B. (1991). Virucidal activity in aqueous extracts obtained from Cedrela tubiflora leaves. Phytother Res 5, 250253.
Descalzo, A. & Coto, C. E. (1989). Inhibición del virus de pseudorrabia (Suid herpesvirus 1) por acción de un antiviral aislado de hojas de Melia azedarach L. Rev Argent Microbiol 21, 133140.[Medline]
Giraudo, C., Daniotti, J. & Maccioni, H. (2001). Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus. Proc Natl Acad Sci U S A 98, 16251630.
Hattori, M., Nakabayashi, T., Lim, Y., Miyashiro, H., Kurokawa, M., Shiraki, K., Gupta, M., Correa, M. & Pilapitiya, U. (1995). Inhibitory effects of various Ayurvedic and panamanian medicinal plants on the infection of herpes simples virus-1 in vitro and in vivo. Phytother Res 9, 270276.
Ikeda, T., Ando, J., Miyazono, A., Zhu, X. H., Tsumagari, H., Nohara, T., Yokomizo, K. & Uyeda, M. (2000). Anti-herpes virus activity of Solanum steroidal glycosides. Biol Pharm Bull 23, 363364.[Medline]
Kim, M., Kim, S. K., Park, B. N. & 7 other authors (1999). Antiviral effects of 28-deacetylsendanin on herpes simplex virus-1 replication. Antivir Res 43, 103112.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Lee, S. M., Klocke, J. A., Barnby, M. A., Yamasaki, R. B. & Balandrin, M. F. (1991). Insecticidal constituents of Azadirachta indica and Melia azedarach (Meliaceae). In Naturally Occurring Pest Bioregulators, ACS Symposium Series no 449, pp. 293304. Edited by P. A. Hedin. Washington, DC: American Chemical Society.
Lin, Y. M., Flavin, M. T., Schure, R., Chen, F. C., Sidwell, R., Barnard, D. L., Huffman, J. H. & Kern, E. R. (1999). Antiviral activities of biflavonoids. Planta Med 65, 120125.[CrossRef][Medline]
Mantani, N., Andoh, T., Kawamata, H., Terasawa, K. & Ochiai, H. (1999). Inhibitory effect of Ephedrae herba, an oriental traditional medicine, on the growth of influenza A/PR/8 virus in MDCK cells. Antivir Res 44, 193200.[CrossRef][Medline]
Martin, S. F. (1987). The Amaryllidaceae alkaloids. In The Alkaloids, vol. 30, pp. 251253. Edited by A. Brossi. New York: Academic Press.
Palokangas, H., Metsikkö, K. & Väänänen, K. (1994). Active vacuolar H+-ATPase is required for both endocytic and exocytic processes during viral infection of BHK-21 cells. J Biol Chem 269, 1757717585.
Pérez, L. & Carrasco, L. (1994). Involvement of the vacuolar H+-ATPase in animal virus entry. J Gen Virol 75, 25952606.[Abstract]
Puri, A., Winick, J., Lowy, R. J., Covell, D., Eidelman, O., Walter, A. & Blumenthal, R. (1988). Activation of vesicular stomatitis virus fusion with cells by pretreatment at low pH. J Biol Chem 263, 47494753.
Rigaut, K. D., Birk, D. E. & Lenard, J. (1991). Intracellular distribution of input vesicular stomatitis virus proteins after uncoating. J Virol 65, 26222628.[Medline]
Sidhu, G., Singh, A., Sundarrajan, R. N., Sundar, S. V. & Maheshwari, R. K. (1999). Role of vacuolar H+-ATPase in interferon-induced inhibition of viral glycoprotein transport. J Interferon Cytokine Res 19, 12971303.[CrossRef][Medline]
Simoes, C. M., Falkenberg, M., Auler Mentz, L., Schenkel, E. P. & Amoros, M. (1999). Antiviral activity of South brazilian medicinal plant extracts. Phytomedicine 6, 205214.[Medline]
Wachsman, M. B., Martino, V., Gutkind, G. O., Coussio, J. D., Coto, C. E. & de Torres, R. A. (1982). Antiviral activity of a Melia azedarach L. plant extract. Fitoterapia 53, 167170.
Wachsman, M. B., Damonte, E. B., Coto, C. E. & de Torres, R. A. (1987). Antiviral effects of Melia azedarach L. leaves extracts on Sindbis virus-infected cells. Antivir Res 8, 112.[CrossRef][Medline]
Wachsman, M. B., Castilla, V. & Coto, C. E. (1998). Inhibition of foot and mouth disease virus (FMDV) uncoating by a plant-derived peptide isolated from Melia azedarach L. leaves. Arch Virol 143, 581590.[CrossRef][Medline]
Yukawa, T. A., Kurokawa, M., Sato, H. & 7 other authors (1996). Prophylactic treatment of citomegalovirus infection with traditional herbs. Antivir Res 32, 6370.[CrossRef][Medline]
Received 8 May 2003;
accepted 8 October 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |