Oligonol a low molecular weight polyphenol of lychee fruit extract inhibits proliferation of influenza virus by blocking reactive oxygen species-dependent ERK phosphorylation.
The emergence of resistance to anti-influenza drugs calls for the search for new antiviral molecules with different resistance profiles. Polyphenolic compounds are found in various plants and have antiviral and antioxidative properties. We tested the hypothesis that oligonol, a lychee fruit-derived low molecular weight polyphenol, possesses anti-influenza effects by inhibiting phosphorylation of extracellular-signal-regulated kinases (ERK). Real time PCR, plaque assay, and immunofluorescence techniques were used to study the effects of oligonol on proliferation of influenza virus. Oligonol inhibits influenza virus proliferation by blocking attachment of the virus to MDCK cells and by suppression of nuclear export of influenza virus ribonucleoprotein (RNP). Infection of MDCK cells with influenza virus leads to an increase in production of reactive oxygen species (ROS) and induction of a ROS-dependent ERK phosphorylation. Inhibition of ERK activation by a dominant negative mutant of ERK or N-acetyl-cysteine (NAC) leads to inhibition of influenza RNP nuclear export. Phorbol 12-myristate 13-acetate (PMA) induces ROS production, ERK phosphorylation and enhances influenza proliferation in MDCK cells. Oligonol and NAC inhibit PMA-induced ERK phosphorylation and ROS production. Our studies suggest that the underlying mechanism for the inhibitory effect of oligonol on influenza virus RNP nuclear export is blocking of ROS-dependent induction of ERK phosphorylation.
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Influenza is a leading cause of morbidity, mortality, and economic loss throughout the world. Prevention and treatment of influenza currently rely on inactivated vaccines and antiviral agents. Two classes of antiviral agents are currently available for influenza management in the event of an influenza epidemic/pandemic. M2 channel blockers (e.g. Amantadine) and neuraminidase inhibitor (e.g. oseltamivir) both targeting the virus proteins. However, use of M2 blockers, is limited by a lack of inhibitory effect against influenza B viruses, side effects, and a rapid emergence of antiviral resistance (Hayden 2006). Although widespread drug resistance has not risen to date for neuraminidase inhibitor class, several viruses have been isolated from clinical trials and from in vitro passage studies that are resistant to one or more neuraminidase inhibitors (Meijer et al. 2009; Stephenson et al. 2009). The recent emergence of H1N1 "swine" flu emphasizes the urgent need for continued search for new compounds with different resistance profiles relative to the currently marketed agents. Influenza virus, like any other virus, exploits the cellular machinery to replicate. Blocking of cellular mechanisms required for viral replication may, thus, be an alternative approach to inhibit virus proliferation. Inhibition of virus-induced intracellular signaling cascades that may support virus replication is a novel approach for development of anti-influenza agent. The advantage of this strategy is that the virus cannot replace the missing cellular function and, thus, emergence of resistance should not easily occur. The naturally occurring antiviral nutrients may be of special interest because they are widely available and can be used as part of the diet to combat diseases, including influenza infection (Wang et al. 2006; Naithani et al. 2008; Choi et al. 2009). Plant-derived flavonoid polyphenolic compound, found in fruits, leaves, and vegetables have recently been the focus of many studies because of their beneficial health effects in several disease models and their antioxidative properties (Pietta 2000; Cazarolli et al. 2008). The anti-influenza effects of polyphenol and antioxidant compounds have been reported previously (Nakayama et al. 1993). However, the underlying molecular mechanisms have not been determined yet. Oligonol is an optimized phenolic product containing catechin-type monomers and oligomers of proanthocyanidin (Sakurai et al. 2008). Fruits and plants are rich sources of phenolic compounds such as (+) catechin and (-) epicatechin and their extracts have long been recognized to possess a wide range of properties, including antioxidant, anti-bacterial, anti-inflammatory, antiallergic, hepatoprotective, anti-thrombotic, antiviral, anti-carcinogenic and vasodilatory actions (Aruoma et al. 2006). The polyphenol oligomers which constitute a small portion of the total polyphenols in the plant are responsible for the biologic effect of polyphenols. The concentration of plant monomeric and oligomeric compounds diminishes as the plant grows and ripens because of continuing polymerization of polyphenols so that bioactive components give way to polymers with high molecular weights. It has been reported that lychee fruit has a high level of monomeric and oligomeric polyphenol (Sarni-Manchado et al. 2000). Oligonol is extracted from lychee fruit and its phenolic composition is reflective of the phenolic composition of lychee fruit. As indicated in Table 1 the difference between oligonol and other procyanidin preparations (e.g. lychee) is that oligonol provides a higher level of low molecular weight polyphenols produced with a novel manufacturing process based on fragmentation of polyphenol polymers (Tanaka et al. 2007). The contents of oligomers in a typical polyphenol polymer can be less than 10%. However, based on HPLC analysis the constituents of oligonol are 15.7% monomer, 13.3% dimer, and 4% trimer (Table 1). Oligonol is produced by the oligomerization of polyphenol polymers, typically proanthocyanidins, thus oligonol delivers higher levels of the oligomeric proanthocyanidins compared to fruits and plant sources that contain mostly large molecular weight proanthocyanidins. As fruits and plants contain lower levels of oligomeric proanthocyanidins, the easily absorbed forms, oligonol bridges this gap in that it contains higher levels of these compounds and therefore is more bioavailable than the polymeric forms. Many of the biological functions of flavonoids and phenolic compounds have been attributed to their antioxida-tive activities. There is increasing evidence that the oxidoreductive balance of cells is involved in viral infections and that certain antioxidant molecules exert potent antiviral activities.
Table 1 HPLC analysis for components of oligonol and lychee fruit polyphenol. Name Oligonol Lychee fruit polyphenol Monomer (+)-catechin 0.1%> 0.1%> (-)-epicatechin 7.5% 6.4% (-)-ECG 2.1% n.d. (-)-EGCG 6.4% n.d. Dimer Procyanidin A1 4.2% 4.0% Procyanidin A2 5.1% 3.3% Procyanidin B1 1.4% 0.8% Procyanidin B2 2.9% 1.7% EC-EGCG 0.3% n.d. Trimer (-)-Epicatechin-(4[beta] 4.0% 3.6% [right arrow] 8, 2[beta] [right arrow] o-7)-epicatechin- (4[beta] [right arrow] 8)-epicatechin
In this study we sought to determine the molecular basis of a link between antioxidative and anti-influenza properties of polyphenol compounds. It is well-documented that ERK phosphorylation is crucial for proliferation of a large number of viruses (Pleschka 2008). Recently, the inhibition of ERK phosphorylation by oligonol has been reported (Kundu et al. 2009). We tested this hypothesis that oligonol blocks influenza virus proliferation through inhibition of ERK phosphorylation.
Materials and methods
Oligonol (>95% purity) (Amino Up Chemical Company, Sapporo, Japan) was obtained from a patented technology process (WO 2004/103988 Al). Briefly, the process involves the extraction of powdered dried fruits with aqueous ethanol. The concentrate was subjected to a DIAION HP-20 column and eluted with aqueous. This was washed and evaporated to dryness yielding a dark brown powder consisting of a mixture of proanthocyanidins. The resulting mixture was combined with L-cysteine hydrochloride monohydrate and L-ascorbic acid in [H.sub.2]O and heated at 60 [degrees] for 48 h. The reaction mixture was passed through a DIAION HP-20 column, washed with [H.sub.2]O and eluted with 40% (v/v) ethanol. Evaporation of the eluate yielded a reddish brown powder, the oligomeric proanthocyanidin complexes.
Oligonol powder was dissolved in DMSO (10mg/ml, stock). MDCK cells were purchased from ATCC (Manassas, VA). Hoechst 33342, N-Acetyl Cystein, phorbol 12-myristate 13-acetate and Trizol reagent were purchased from Sigma-Aldrich (St. Louis, MO). U0126 was purchased from Calbiochem (La Jolla, CA). Anti-influenza monoclonal antibody against nucleoprotein (NP) protein was purchased from Chemicon International Inc. (Temec-ula, CA). Anti-ERK-1/2 (mouse monoclonal, p44/p42, clone MK12) and anti-phospho-specific ERK-1/2 (mouse monoclonal anti-phospho-ERK-1/2 (Thr202/Tyr204, Thr185/Tyr187), recombinant clone AW39R) were purchased from Millipore (Billerica, MA). Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IGg (secondary) was purchased from Abcam (Cambridge, MA). Chicken red blood cells (RBCs) were obtained from Lampire Biological Laboratories (Pipersville, PA). Adenovirus particles including Null control, GFP and dominant negative mutant of ERK and ViraDuctin adenovirus transduction reagent were purchased from Cell Biolabs (San Diego, CA).
Viral stock and cell culture
Influenza virus A/Hong Kong/2/68; H3N2 [A/HK (H3N2)] was used as the primary influenza virus strain in our experiments. MDCK cells were cultured in Dulbecco's modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Invitrogen).
Cell viability assay
The effect of oligonol and other chemicals on the viability of MDCK cells were determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT) assay as described (Haidari et al. 2009).
When 90% confluent, MDCK cells were washed twice with PBS to remove residual FBS and infected with virus at multiplicity of infection (MOI) of 10 or 0.01 to allow single-cycle (8-10h after infection) or multicycle (24 h after infection) replication, respectively. Viral stock was used in serum-free DMEM for 60 min in 37[degrees] C to inoculate the cells (adsorption phase). Cells were then washed with PBS and cultured in DMEM + 2% FBS, and tolylsulfonyl pheny-lalanyl chloromethyl ketone (TPCK) trypsin (4 [micro]g/ml, Worthington Biochemicals, Tryp-MEM) either with or without drug treatment, for 8-24 h (post-infection phase).
Extracellular virus yield reduction assay
The extracellular and intracellular virus yield reduction assays were performed in 24-well plates containing confluent MDCK monolayers. Four experiments were conducted for each assay. For the extracellular assays drugs were added to cells in 24-well plates, and the plates were incubated for 16h at 37[degrees]C. The cells were then inoculated with virus, first for 1 h, and then after washing for 24 h at 37[degrees]C in DMEM + 2% FBS, and TPCK trypsin. The medium was removed and centrifuged at 3200 x g for 5 min to remove the floating cells and used for ribonucleic acid (Palamara et al. 2005) extraction and quantification of virus using (Real Time-Polymerase Chain Reaction) RT-PCR technique. The media were also used for plaque assay. For the qualifying the intracellular production of virus RNA, after the exposure of cells to the drugs for the indicated incubation time the cells were washed twice with PBS and were exposed to Trizol reagent. The RNA were extracted and used for RT-PCR as previously described (Haidari et al. 2009).
Real time PCR assay
TaqMan real time PCR was used to quantify the presence of virus after infection with influenza virus as described (Haidari et al. 2009). The primer and probe sequences for detection of influenza A was already optimized (Ward et al. 2004). The primers (forward) 5'-AAG ACC AAT CCT GTC ACC TCT GA-3'and (reverse) 5'-CAA AGC GTC TAC GCT GCA GTC C-3' amplify a 104-base pair fragment in the M1 gene of influenza A. The influenza A specific probe FAM (6-carboxyfluorescein)-5'-TTT GTG TTC ACG CTC ACC GT-3' TAMRA (6-carboxytetramethylrhodamine) annealed to part of the sequence amplified by the two primers.
Measurement of ROS levels
MDCK cells were infected with MOI of 10, 1 and 0.1 of influenza virus and after 4 h were exposed to 10 [micro]M of 2',7'-dichiorodihydrofluorescein (DCF) for 30 min. The cells were washed, trypsinized, fixed and the intensity of DCF fluorescence was quantified using flow cytometry technique. Cells were analyzed by a Beckman Coulter FC-500 instrument (Beckman Coulter, Fullerton, CA). At least 10,000 cells were analyzed for each sample.
ERK phosphorylation assay
MDCK cells were cultured on six-well plates until 80-90% confluent. Where indicated, the cells were preincubated with oligonol, NAC or H202 for 1 h. Cells were extracted in RIPA buffer, which contains 0.1% SDS, 1% deoxycholate, 1% NP-40, 10mM sodium phosphate, 150mM NaCl, 2 mM EDTA, 50 mM NaF, 5 mM sodium pyrophosphate, 0.1 mM sodium vanadate, 2 mM PMSF, 0.1 mg/ml leupeptin, and 100 KIU/ml aprotinin. Equal amounts of each extract were then subjected to SDS-PAGE on 10% slabs, electrotransferred to polyvinylidene difluoride membranes, and probed for phosphorylated ERK-1/2 and total ERK-1/2 by immunoblot analysis.
Adenovirus-mediated gene transfer
Pre-confluent cells were infected with adenoviral vectors (null, GFP and DN-ERK) with a MOI of 100 plaque-forming units per cell in presence of ViraDuctin. Cells were incubated for 48 h in the presence of viral particles and were assessed for the expression of the transferred genes.
The techniques for plaque assay, immunofluorescence staining and hemagglutination inhibition assays were already described (Haidari et al. 2009).
Oligonol inhibits replication of influenza virus
The viability test demonstrated that oligonol concentrations of less than 10 [micro]g/ml are not toxic for MDCK cells. Therefore, oligonol with concentrations of 0-10 ([micro]g/ml were used for the experiments. It is known that DMSO depending on the concentration and cell type can increase influenza virus release (Scholtissek and Muller 1988). Therefore, in the control experiments we used the same amount of DMSO that was used for dissolving the substances with a maximum amount of 0.2% DMSO in the solutions. MDCK cells were pre-exposed to oligonol for 16 h and were infected with influenza virus. After 1 h the cells were washed with PBS, exposed to oligonol and the media were collected after 24 h. As depicted in Fig. 1A and B the proliferation of influenza virus was inhibited in the multiple cycles of influenza growth using plaque assay and RT-PCR techniques.
[FIGURE 1 OMITTED]
An early phase in influenza virus life cycle is inhibited by oligonol
We first attempted to identify the step(s) of the influenza virus life cycle that were affected by oligonol. When the MDCK cells underwent a 24-h preinfection treatment with oligonol, with drug washout right before viral challenge, the inhibitory effect of oligonol disappeared (Fig. 1C). While, when we exposed the cells to oligonol only during the viral adsorption phase (1 h in 37 [degrees]C) the virus proliferation was completely suppressed (Fig. 1D). This argues for a very early step in the virus life cycle that is affected by oligonol. To study whether oligonol would prevent the ability of the virus particles to bind to cell surface receptors, we employed hemagglutination inhibition (HI) assays.
Influenza A viruses are able to agglutinate red blood cells (RBC) via the viral glycoprotein, the hemagglutinin, that binds to N-acetylneuraminic acid at the cell surface. The RBCs get crosslinked by the virus and will form a type of lattice in this case. Our study showed that oligonol inhibits agglutination of chicken red blood cells by influenza virus (Fig. 2A). Indicating that oligonol is capable of directly interfering with the viral HA or N-acetylneuraminic acid to block binding of HA to the cellular receptors. On the other hand when isolated influenza viruses were exposed to oligonol for 30 min in a cell free tube their ability to replicate in MDCK cells dramatically reduced, suggesting that oligonol directly interact with molecules of influenza virus (Fig. 2B).
[FIGURE 2 OMITTED]
Oligonol inhibits influenza RNP nuclear export
Next we explored if oligonol inhibits replication of influenza virus independent of blocking the early phase of influenza life cycle. Exposure of MDCK cells to oligonol after the attachment of influenza to the cells (after viral adsorption phase) inhibited the virus proliferation in the multiple growth cycles (Fig. 2C). Suggesting that oligonol not only blocks the early stage of influenza proliferation but also blocks a late stage of the proliferation as well. We previously used immunofluorescent microscopic technique to study the intracellular trafficking of influenza virus by labeling nucleoprotein (NP) of influenza virus. During influenza virus replication, viral RNAs are packaged into helical ribonucleoprotein (RNP) complexes with polymerase and NP in the host-cell nucleus and are subsequently exported into the cytosol to be assembled with the other structural proteins (Cros and Palese 2003). Our previous studies demonstrated that in MDCK cells a single life cycle of influenza A [A/HK (H3N2)] is around 8 h. Influenza RNPis detectable in the nucleus of infected cells between 2 and 3 h after inoculation and between 6 and 8 h influenza RNP is exported from nucleus to cytoplasm (Haidari et al. 2009). We first studied the effect of oligonol on intracellular replication of influenza RNA in a single life cycle. The MDCK cells were infected with influenza virus and 2 h after the adsorption phase we added oligonol to the cells. After 8 h the media were removed and the cells were washed twice with PBS, lysed with Trizol reagent. The total RNA was extracted and the levels of intracellular influenza RNA were measured. RT-PCR showed a reduction in influenza RNA from the oligonol treated MDCK compare to the non-treated cells (Fig. 2D). Then we studied the intracellular trafficking of influenza RNP using immunofluores-cent microscopic technique to determine which step in influenza life cycle is inhibited by oligonol. Addition of oligonol 2 h after the exposure of MDCK cells to influenza lead to inhibition of influenza RNP export from nucleus to cytoplasm (Fig. 3).
[FIGURE 3 OMITTED]
Oligonol inhibits nuclear export of influenza RNP by blocking ERK phosphorylation
The role of ERK phosphorylation in nuclear export of influenza virus has been documented (Pleschka et al. 2001). To determine the underlying mechanism for the inhibitory effect of oligonol on nuclear translocation of influenza RNP the impact of oligonol on ERK phosphorylation was tested. Because the basal level of ERK 1/2 phosphorylation was low, a protein kinase C activator, PMA was used to induce ERK phosphorylation in MDCK cells. As shown in Fig. 4A PMA increased ERK 1/2 phosphorylation dose-dependently. Fig. 4B indicates that infection of MDCK cells with influenza virus also increased ERK-1/2 phosphorylation. Exposure of MDCK cells to oligonol inhibited PMA-induced ERK-1/2 phosphorylation (Fig. 5). To confirm the previous findings that suggested a crucial role for ERK phosphorylation in nuclear export of influenza (Pleschka et al. 2001), we used an adenovirus dominant negative mutant of ERK and a chemical inhibitor of MEK (U0126) to block ERK phosphorylation. Exposure of MDCK cells to recombinant Ad-based vector at a MOI of 100 reproducibly resulted in a transduction efficiency of 90% or greater as determined by the percentage of cells exhibiting fluorescence following transduction with Ad vector encoding GFP. Inhibition of ERK phosphorylation by transduction of MDCK cells with dominant negative mutant of ERK was confirmed by Western blot technique (data not shown). Both dominant negative mutant of ERK and U0126 blocked influenza RNP nuclear export (Fig. 6A). In addition, pre-exposure of MDCK cells to PMA enhanced the proliferation of influenza virus (Fig. 6B). These experiments confirmed that ERK phosphorylation is crucial for nuclear export of influenza RNP.
[FIGURE 4 OMITTED]
[FIGURE 5 OMITTED]
[FIGURE 6 OMITTED]
ROS production is critical for influenza-induced ERK phosphorylation
Since PMA is known for induction of intracellular ROS production we studied if antioxidative properties of oligonol plays role in its anti-influenza effect. Infection of MDCK cells with influenza increased production of ROS using flow cytometry technique (Fig. 7Aand B). Oligonol reduced PMA-induced increase in ROS production (Fig. 8A and B). In addition as depicted in Fig. 7C the proliferation of influenza virus was inhibited when MDCK cells were exposed to an antioxidant, NAC. This inhibition was due to the blocking of nuclear export of influenza RNP by NAC (Fig. 3). To test the effects of ROS on ERK phosphorylation we exposed MDCK cells to hydrogen peroxide. Exposure of MDCK cells to hydrogen peroxide leads to an increase in ERK phosphorylation (Fig. 8C). NAC inhibited PMA-induced ERK phosphorylation (Fig. 5A) indicating the critical role of ROS in ERK phosphorylation. As shown in Figs. 4, 5 and 8 there is a reverse correlation between ERK phosphorylation and ERK protein expression.
[FIGURE 7 OMITTED]
[FIGURE 8 OMITTED]
Our studies demonstrate that oligonol inhibits proliferation of influenza virus in two stages of the virus's life cycle: the early stage of influenza proliferation and the export of influenza RNP from nucleus to cytoplasm. Our studies suggest that the mechanism behind the inhibitory effect of oligonol on the nuclear export of RNP is inhibition of ERK phosphorylation. Our experiments showed that exposure of cells to an antioxidant, NAC, blocks influenza proliferation and ERK phosphorylation suggesting that anti-influenza effects of oligonol can be attributable to its antioxidative properties.
The safety and toxicology of oligonol has been extensively studied and found to be safe with LD50 in rats of 5.0 g/kg body weight. This corresponds to a dose of 300 g for an average human with a 60 kg body weight demonstrating that oligonol is safe enough for use as a supplement (Aruoma et al. 2006; Fujii et al. 2007, 2008). Studies conducted on 30 healthy volunteers consuming oligonol at doses of 100 and 200 mg/day for 92 days showed that continuous intake of oligonol allows sustainable levels of total polyphenols in the serum (Fujii et al. 2007). It has been shown that oligonol has a stronger protective effect against the [KBrO.sub.3]-induced renal toxicity than epigallocatechin gallate (EGCG). In comparison with ECGC, Oligonol elicits the stronger antioxidant capacity following in vivo supplementation to rats. Therefore, it was speculated that the superior effect of oligonol over EGCG against the [KBrO.sub.3]-induced renal toxicity is due to the catechin-type monomers and the content of more bioavailable oligomeric proanthocyanidins (Nishioka et al. 2006).
In our previous study, we demonstrated that an extract of pomegranate, a fruit that contains a substantial amount of polyphenol also inhibits RBC agglutination by influenza (Haidari et al. 2009). Other plant extracts that contain polyphenols such as green tea, CYSTUS052 and Chaenomeles sinensis have been reported to inhibit agglutination of red blood cells by influenza virus (Song et al. 2005; Ehrhardt et al. 2007; Sawai et al. 2008). On a molecular basis oligonol appears to interfere with the virus surface proteins and inhibits binding of the virus particle to the cellular receptors. As we showed the exposure of isolated influenza virus to oligonol for 30 min in a cell free tube blocks the virus proliferation, suggesting a direct interaction between oligonol and influenza virus. This interaction might lead to influenza virus aggregation, which itself may result in reduced infectivity. However, direct binding of oligonol to cell surface molecules such as sialic acids that are receptors for influenza virus cannot be ruled out by our study. It is well known that polyphenols exhibit protein-binding capacity, suggesting that oligonol may interact with influenza virus via such a physical and unspecific interaction. Further studies on the molecular level are needed to determine the mechanisms by which oligonol or other polyphenolic compounds inhibit the early phase of influenza proliferation. Several viruses, including influenza, induce an imbalance of intracellular redox state toward pro-oxidant conditions (Schwarz 1996; Stehbens 2004). Through different mechanisms these alterations contribute both to influenza virus proliferation and to the pathogenesis of virus-induced diseases. At the same time, influenza virus activates several intracellular signaling pathways such as Ras/Raf/Mek/ERK that are involved in important physiological functions of the cell (Pleschka 2008; Ramos 2008). Interestingly, many of these pathways are finely regulated by small changes in intracellular redox state, and the virus-induced redox imbalance might also control viral replication through this mechanism. The role of ERK phosphorylation in proliferation of viruses has been reported previously (Pleschka 2008). The mitogen-activated protein (MAP) kinase extracellular-signal-regulated kinases (ERKs) are widely expressed protein kinase intracellular signaling molecules which are involved in functions including the regulation of cell proliferation and differentiation. Many different stimuli, including growth factors, cytokines, virus infections, ligands for heterotrimeric G protein-coupled receptors, transforming agents, and carcinogens activate the ERK pathway (Ramos 2008). Our findings are in agreement with the findings of Pleschka et al. demonstrating that ERK phosphorylation increases following influenza infection. The authors revealed that the inhibition of ERK phosphorylation blocks export of influenza RNP from nucleus to cytoplasm of infected cells (Pleschka et al. 2001). Our study and others demonstrated that oligonol inhibits ERK phosphorylation (Kundu et al. 2009). We demonstrated that following influenza infection, the production of ROS increases in the cells. Previously, the effect of influenza infection in increasing ROS production has been reported (Uchide et al. 2002). We showed that exposure of cells to an antioxidant, NAC inhibits influenza proliferation by blocking the virus RNP nuclear export. The protective effect of NAC in the reduction of mortality between mice infected with influenza virus has been reported previously (Ungheri et al. 2000). These findings are in line with other studies reported that antioxidants can act as anti-influenza agents (Cai et al. 2003; Sokmen et al. 2005; Furuya et al. 2008).
The mechanisms by which antioxidants inhibit influenza proliferation have not been fully explored. Activation of transcription factor NF-kappaB is critical for influenza proliferation (Ludwig and Planz 2008). This process is mediated by oxidative radicals because treatment of cells with pyrrolidine dithiocarbamate, a scavenger of such radicals, abolished the NF-kappaB activation (Flory et al. 2000). One mechanism that can explain the anti-influenza properties of antioxidant is inhibition of influenza-induced activation of NF-kappaB. In support of this Kundu et al. (2009) reported that oligonol inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced NF-kappaB activation. However, the role of NF-kappaB activation in nuclear export of influenza RNP has not been explored. Besides to activation of NF-kappaB, other signaling cascades are activated during influenza infection and their activation are mediated by ROS. Therefore, anti-influenza effects of antioxidant can be attributed to inhibition of ERK which is also crucial for influenza RNP nuclear export (Pleschka et al. 2001). Resveratrol a polyphenol that has antioxidant properties also inhibits influenza proliferation by blocking nuclear translocation of influenza RNP (Palamara et al. 2005). These findings suggest a crucial role for ROS in export of influenza RNP from nucleus to cytoplasm. Protein kinase C activator, PMA has been reported to increase both ROS production and ERK phosphorylation (Atten et al. 1998; Lee et al. 2004). Exposure of MDCK cells to PMA enhanced influenza proliferation. This finding provides further evidence to support this hypothesis that ROS or/and ERK phosphorylation are critical for nuclear export of influenza RNP. ERK phosphorylation is regulated through activation of RAS/RAF/MEK signaling cascade. Besides to RAS/RAF/MEK pathway PKC-[alpha] activation leads to ERK phosphorylation (Wen-Sheng and Jun-Ming 2005). The role of PKC-[alpha] in nuclear export of influenza RNP has been demonstrated (Bui et al. 2002). Marjuki et al. (2006) demonstrated that influenza infection triggers PKC-[alpha] activation which leads to ERK phosphorylation and nuclear export of influenza RNP. Our studies and others demonstrated that hydrogen peroxide increases ERK phosphorylation (Lee et al. 2005). Oligonol and NAC both inhibit PMA-induced ERK phosphorylation, indicating that ERK phosphorylation depends on ROS production. The molecular mechanisms by which antioxidants inhibit ERK phosphorylation merits further exploration. ROS involve in both RAS/RAF/MEK and PKC activation (Wu et al. 2006). Therefore, the inhibition ERK phosphorylation by oligonol or NAC can be attributable to inhibition of RAS/RAF/MEK and/or PKC.
The underlying mechanism for the critical role of ERK phosphorylation in influenza RNP nuclear export is yet to be determined. The influenza virus Nonstructural Protein 2 (NS2) protein was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell (O'Neill et al. 1998). It has been shown that inhibition of RAF signaling results in impaired function of influenza NS2 protein (Pleschka et al. 2001). Sawai et al. (2008) demonstrated that a high molecular weight polyphenols compound extracted from a traditional Chinese medicine inhibits influenza proliferation by suppressing expression of influenza NS2 protein. Therefore, one can speculate that oligonol and other polyphenol compounds modulate nuclear export of influenza RNP by inhibiting NS2 protein expression via inhibition of ERK phosphorylation. Further investigations are needed to test this hypothesis.
Our studies suffer from some limitations. The quantification of viral RNA by RT-PCR, as used by our studies does not indicate the actual amount of infectious virions. However, it is a good indicator for comparison of released virus between drug-treated and non-treated conditions. As shown in figures A and B there is a good concordance between the plaque assay and RT-PCR techniques. The physiologic relevance of our in vitro data should be interpreted with caution. Investigating the impacts of oligonol on influenza infection using the animal models will shed light on the in vivo role of this polyphenolic compound in inhibiting influenza proliferation. Taken together, our studies provide evidence that oligonol inhibits proliferation of influenza virus by blocking ROS-dependent ERK phosphorylation. With further studies, oligonol will prove to be beneficial with a potential in therapeutic and prophylactic applications in influenza epidemics and pandemics.
Dr. Haidari has received research support from Amino Up Chemical Co, Japan.
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Leila Gangehei (a), Muzammil Ali (a), Wei Zhang (b), Zhenping Chen (b), Koji Wakame (c), Mehran Haidari (a), (b), *
(a) University of Texas Health Science Center at Houston, Houston, TX, USA
(b) Texas Heart Institute, Houston, TX, USA
(c) Amino Up Chemical Co., Japan
* Corresponding author at: 6770 Bertner Ave., C1000, Houston, TX 77030, USA. Tel.: +1 832 355 9077; fax: +1 832 355 9595.
E-mail address: Mehran.Haidari@uth.tmc.edu (M. Haidari).
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