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. 2013 May 1;8(5):e62482.
doi: 10.1371/journal.pone.0062482. Print 2013.

Inhibition of enveloped viruses infectivity by curcumin

Tzu-Yen Chen  1 , Da-Yuan Chen, Hsiao-Wei Wen, Jun-Lin Ou, Shyan-Song Chiou, Jo-Mei Chen, Min-Liang Wong, Wei-Li Hsu
Affiliations

Inhibition of enveloped viruses infectivity by curcumin

Tzu-Yen Chen et al. PLoS One. .

Abstract

Curcumin, a natural compound and ingredient in curry, has antiinflammatory, antioxidant, and anticarcinogenic properties. Previously, we reported that curcumin abrogated influenza virus infectivity by inhibiting hemagglutination (HA) activity. This study demonstrates a novel mechanism by which curcumin inhibits the infectivity of enveloped viruses. In all analyzed enveloped viruses, including the influenza virus, curcumin inhibited plaque formation. In contrast, the nonenveloped enterovirus 71 remained unaffected by curcumin treatment. We evaluated the effects of curcumin on the membrane structure using fluorescent dye (sulforhodamine B; SRB)-containing liposomes that mimic the viral envelope. Curcumin treatment induced the leakage of SRB from these liposomes and the addition of the influenza virus reduced the leakage, indicating that curcumin disrupts the integrity of the membranes of viral envelopes and of liposomes. When testing liposomes of various diameters, we detected higher levels of SRB leakage from the smaller-sized liposomes than from the larger liposomes. Interestingly, the curcumin concentration required to reduce plaque formation was lower for the influenza virus (approximately 100 nm in diameter) than for the pseudorabies virus (approximately 180 nm) and the vaccinia virus (roughly 335 × 200 × 200 nm). These data provide insights on the molecular antiviral mechanisms of curcumin and its potential use as an antiviral agent for enveloped viruses.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Treatment of curcumin reduces infectivity of enveloped viruses.
(A) 4 HA units of Newcastle disease viruses (NDV) were incubated with 2-fold serially diluted curcumin or DMSO (vehicle control) and the hemagglutination inhibitory activity of curcumin was tested by incubation with chicken RBC at room temperature for 30 minutes. (B) time-of-drug addition test: 10 µM of curcumin or DMSO (as solvent control) was included into culture medium at various time points of Japanese encephalitis virus (JEV) or Dengue virus (DV-II) infection (200 pfu), for instance: (1) full time treatment: curcumin was added to vero cells at 8-hour prior to infection and included throughout the time of infection, (2) co-treatment: curcumin mixed with virus in the infection medium was added simultaneously to the cells and left on the cells throughout; (3) after-entry: curcumin was added to cells at 2 hpi and remained throughout the time of infection. Small-sized plaque of DV-II was indicated by arrowhead. Consistent results were observed from at least three independent experiments.
Figure 2
Figure 2. Pre-treatment of curcumin strongly inhibited enveloped viruses, but does not affect plaque formation of enterovirus 71 (EV 71).
(A) 2,000 pfu of Pseudorabies virus (PRV), Japanese encephalitis virus (JEV), and Dengue virus serotype II (DVII) were pre-treated with 30 µM of curcumin for one hour and remaining viral infectivity was measured by standard plaque assay. To count plaque numbers, after one hour incubation, the mixture of virus and drug was further diluted into 10−1, 10−2, 10−3 with medium without serum followed by standard plaque assay. White spots indicate viral plaques. (B–C) To measure the effect of curcumin, 2,000 pfu of EV71, JEV, and influenza virus, strain PR8 were pre-treated with a serial dilutions of curcumin (30, 20, 10, 5, 1, 0.5, 0.1 µM to 0 µM) for one hour and the plaque formation ability was measured by standard plaque assay. Plaque formation ability of EV71 as not inhibited by curcumin, whereas infectivity of influenza viruses was strongly affected (B). Pre-treatment of curcumin inhibit plaque formation of JEV and Influenza to a similar extent, whereas EV71 remained unaffected (C). The results from Fig. 2C were plotted based on three independent experiments.
Figure 3
Figure 3. Curcumin affects the DNA transfection and structure of liposomes.
The effect of curcumin on membrane structure was tested in two systems, commercial liposome-based transfection reagent (A) and Sulforhodamine B (SRB)-loaded liposome (B and C). (A) Curcumin was incubated with the mixture of eGFP plasmid (Clontech) and Cellfectin (Invitrogen) at room temperature for 40 min before added to the cell monolayer. At 24-hour post transfection, the transfection efficiency and the intensity of GFP in individual cells were recorded by fluorescent microscopy (upper panel) and flow cytometry analysis (lower panel). The images were taken under the same setting. (B) SRB-Liposomes were incubated with various concentrations of curcumin (30 µM, 60 µM), or DMSO (the solvent control) at room temperature for one hour followed by detection of SRB fluorescence (C) SRB-Liposome was incubated with two different doses of PR8 influenza viruses (2,000 and 10,000 pfu) and curcumin (60 µM), or DMSO at room temperature for one hour. The leakage of SRB fluorescence was detected by SpectraMax M2e Microplate Reader (Molecular Devices, Inc., California, United States) at excitation wavelength of 490 nm. The results from Fig. 3B and 3C were plotted based on three independent experiments.
Figure 4
Figure 4. Effect of curcumin on liposomes and viruses with different sizes.
(A) Three different diameters of SRB-Liposome i.e. 300 nm, 220 nm, 120 nm were incubated with curcumin (60 µM), DMSO (the solvent control), or 15 mM n-octylglucoside (n-OG), a detergent serving as positive control, at room temperature for one hour. The leakage of SRB fluorescence was detected by SpectraMax M2e Microplate Reader (Molecular Devices, Inc., California, United States) at excitation wavelength of 490 nm. (B) Effects of curcumin on plaque formation of enveloped viruses with different sizes. 2,000 pfu of influenza virus (strain PR8) and two DNA viruses, i.e. pseudorabies virus (PRV) and vaccinia viruses (VAC) were pre-treated with 30 µM of curcumin for one hour. The plaque formation ability was measured by standard plaque assay and plotted as a percentage of the untreated controls. Dash lines indicate reduction of plaque formation by 50% relative to the control group. Data are presented as mean values ± standard deviation (SD) from three independent experiments.
Figure 5
Figure 5. Effect of curcumin on inhibition of viral plaque formation and infectivity is irreversible.
A time course treatment was conducted to determine the time required for plaque reduction (A). Influenza virus (50 pfu) was mixed with curcumin (30 µM) or DMSO (solvent control). At different periods of times, i.e. 0, 5, 10, 20, 40, 60 min of incubation, the virus-test drug mixtures were added to cells followed by standard plaque assay. To evaluate whether curcumin-induced inhibition of viral plaque formation can be restored, one hour after curcumin (30 µM) treatment, the influenza virus (2000 pfu of PR8) and curcumin mixture was subsequently diluted to final concentrations of curcumin at 6 µM, 3 µM, and 1.5 µM, followed by standard plaque assay (B). To test whether the curcumin-induced loss of virus infectivity is irreversible, 50 pfu of PR8 viruses were incubated with 30 µM of curcumin or DMSO in a total volume of 500 µl infectious medium. One hour after incubation, inoculums were injected into the allantoic sac of 4 embryonated hen’s eggs. Treated eggs were incubated in 37°C incubator for 18 hours, the yield of virus progeny was determined by HA test (C). Consistent results were observed from at least three independent experiments.
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This work was supported by grants from National Science Council (98-2313-B-005-015-MY3, 101-2321-B-005-005) and National Chung-Hsing University (TCVGH-NCHU1017612). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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