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Apigenin inhibits enterovirus-71 infection by disrupting viral RNA association with trans-acting factors.

Zhang W, Qiao H, Lv Y, Wang J, Chen X, Hou Y, Tan R, Li E - PLoS ONE (2014)

Bottom Line: We found that apigenin treatment did not affect EV71-induced nucleocytoplasmic redistribution of hnRNP A1 and A2 proteins.As a positive sense, single strand RNA virus, EV71 has a type I internal ribosome entry site (IRES) that cooperates with host factors and regulates EV71 translation.We found that apigenin treatment selectively suppressed the expression of GFP, but not a control gene.

View Article: PubMed Central - PubMed

Affiliation: Medical School and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, Jiangsu, China.

ABSTRACT
Flavonoids are widely distributed natural products with broad biological activities. Apigenin is a dietary flavonoid that has recently been demonstrated to interact with heterogeneous nuclear ribonucleoproteins (hnRNPs) and interferes with their RNA editing activity. We investigated whether apigenin possessed antiviral activity against enterovirus-71 (EV71) infection since EV71 infection requires of hnRNP proteins. We found that apigenin selectively blocks EV71 infection by disrupting viral RNA association with hnRNP A1 and A2 proteins. The estimated EC50 value for apigenin to block EV71 infection was determined at 10.3 µM, while the CC50 was estimated at 79.0 µM. The anti-EV71 activity was selective since no activity was detected against several DNA and RNA viruses. Although flavonoids in general share similar structural features, apigenin and kaempferol were among tested compounds with significant activity against EV71 infection. hnRNP proteins function as trans-acting factors regulating EV71 translation. We found that apigenin treatment did not affect EV71-induced nucleocytoplasmic redistribution of hnRNP A1 and A2 proteins. Instead, it prevented EV71 RNA association with hnRNP A1 and A2 proteins. Accordingly, suppression of hnRNP A1 and A2 expression markedly reduced EV71 infection. As a positive sense, single strand RNA virus, EV71 has a type I internal ribosome entry site (IRES) that cooperates with host factors and regulates EV71 translation. The effect of apigenin on EV71 infection was further demonstrated using a bicistronic vector that has the expression of a GFP protein under the control of EV71 5'-UTR. We found that apigenin treatment selectively suppressed the expression of GFP, but not a control gene. In addition to identification of apigenin as an antiviral agent against EV71 infection, this study also exemplifies the significance in antiviral agent discovery by targeting host factors essential for viral replication.

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Antiviral activity evaluation of flavonoids against EV71 infection. (A) Names and chemical structures of flavonoids tested in this study.The compounds were evaluated for their antiviral activity against EV71 infection. Apigenin and kaempferol were the only compounds that showed antiviral activity at 30 µM concentration. (B, C) Confirmation of kaempferol antiviral activity by titration for infectious virion production (B) and viral VP1 protein expression (C). RD cells were untreated or pretreated with kaempferol along with apigenin, naringenin and hesperetin at 30 µM for 2 hr and then infected with EV71 (MOI = 0.10) for 36 hr. Infectious virion production was titrated using a secondary infection assay (B) and VP1 expression was detected by immunoblotting (C). Que: quercetin; Api: apigenin; Kae: kaempferol; Hes: hesperetin; Nar: naringenin. GAPDH expression was used as a loading control. Both studies were performed twice independently. Data are presented as mean ± SD of triplicate samples. An unpaired t test was performed for statistical analysis. *: p≤0.05. (D) Anti-oxidative agent N-acetyl cysteine (NAC) treatment does not affect EV71 infection. RD cells were untreated or pretreated with NAC at varying concentrations (1, 3, and 10 mM) or apigenin at 30 µM for 2 hr. The cells were then infected with EV71 (MOI = 0.10 TCID50 per cell) for 36 hr. EV71 VP1 protein expression were determined by immunoblotting analysis. The experiment was performed twice independently. GAPDH was used as a loading control.
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pone-0110429-g004: Antiviral activity evaluation of flavonoids against EV71 infection. (A) Names and chemical structures of flavonoids tested in this study.The compounds were evaluated for their antiviral activity against EV71 infection. Apigenin and kaempferol were the only compounds that showed antiviral activity at 30 µM concentration. (B, C) Confirmation of kaempferol antiviral activity by titration for infectious virion production (B) and viral VP1 protein expression (C). RD cells were untreated or pretreated with kaempferol along with apigenin, naringenin and hesperetin at 30 µM for 2 hr and then infected with EV71 (MOI = 0.10) for 36 hr. Infectious virion production was titrated using a secondary infection assay (B) and VP1 expression was detected by immunoblotting (C). Que: quercetin; Api: apigenin; Kae: kaempferol; Hes: hesperetin; Nar: naringenin. GAPDH expression was used as a loading control. Both studies were performed twice independently. Data are presented as mean ± SD of triplicate samples. An unpaired t test was performed for statistical analysis. *: p≤0.05. (D) Anti-oxidative agent N-acetyl cysteine (NAC) treatment does not affect EV71 infection. RD cells were untreated or pretreated with NAC at varying concentrations (1, 3, and 10 mM) or apigenin at 30 µM for 2 hr. The cells were then infected with EV71 (MOI = 0.10 TCID50 per cell) for 36 hr. EV71 VP1 protein expression were determined by immunoblotting analysis. The experiment was performed twice independently. GAPDH was used as a loading control.

Mentions: Finally, we investigated whether flavonoids with similar structural features possessed antiviral activity against EV71 infection. Monolayers of RD cells were treated with a panel of flavonoids (Fig. 4A) at 30 µM prior to EV71 infection. Interestingly, apigenin along with kaempferol, a compound recently reported to inhibit EV71 infection by altering trans-acting factor binding [11], were the only compounds showed activity against EV71 infection. The antiviral effect of kaempferol was nevertheless validated by titration of infectious virion production (Fig. 4B) and by viral protein expression (Fig. 4C). In addition to reduction of virus titers, treatment with kaempferol, but not naringenin, quercetin, or hesperetin, resulted in significant reduction of VP1 protein expression (Fig. 4C). Although the anti-oxidant activity of polyphenols has commonly be attributed to their antiviral property, the fact that apigenin and kaempferol were among the only compounds with antiviral activity against EV71 infection argues strongly against anti-oxidative property for apigenin antiviral activity. Indeed, N-acetyl cysteine, a commonly used anti-oxidative agent, showed no effect against EV71 infection (Fig. 4D).


Apigenin inhibits enterovirus-71 infection by disrupting viral RNA association with trans-acting factors.

Zhang W, Qiao H, Lv Y, Wang J, Chen X, Hou Y, Tan R, Li E - PLoS ONE (2014)

Antiviral activity evaluation of flavonoids against EV71 infection. (A) Names and chemical structures of flavonoids tested in this study.The compounds were evaluated for their antiviral activity against EV71 infection. Apigenin and kaempferol were the only compounds that showed antiviral activity at 30 µM concentration. (B, C) Confirmation of kaempferol antiviral activity by titration for infectious virion production (B) and viral VP1 protein expression (C). RD cells were untreated or pretreated with kaempferol along with apigenin, naringenin and hesperetin at 30 µM for 2 hr and then infected with EV71 (MOI = 0.10) for 36 hr. Infectious virion production was titrated using a secondary infection assay (B) and VP1 expression was detected by immunoblotting (C). Que: quercetin; Api: apigenin; Kae: kaempferol; Hes: hesperetin; Nar: naringenin. GAPDH expression was used as a loading control. Both studies were performed twice independently. Data are presented as mean ± SD of triplicate samples. An unpaired t test was performed for statistical analysis. *: p≤0.05. (D) Anti-oxidative agent N-acetyl cysteine (NAC) treatment does not affect EV71 infection. RD cells were untreated or pretreated with NAC at varying concentrations (1, 3, and 10 mM) or apigenin at 30 µM for 2 hr. The cells were then infected with EV71 (MOI = 0.10 TCID50 per cell) for 36 hr. EV71 VP1 protein expression were determined by immunoblotting analysis. The experiment was performed twice independently. GAPDH was used as a loading control.
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Related In: Results  -  Collection

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pone-0110429-g004: Antiviral activity evaluation of flavonoids against EV71 infection. (A) Names and chemical structures of flavonoids tested in this study.The compounds were evaluated for their antiviral activity against EV71 infection. Apigenin and kaempferol were the only compounds that showed antiviral activity at 30 µM concentration. (B, C) Confirmation of kaempferol antiviral activity by titration for infectious virion production (B) and viral VP1 protein expression (C). RD cells were untreated or pretreated with kaempferol along with apigenin, naringenin and hesperetin at 30 µM for 2 hr and then infected with EV71 (MOI = 0.10) for 36 hr. Infectious virion production was titrated using a secondary infection assay (B) and VP1 expression was detected by immunoblotting (C). Que: quercetin; Api: apigenin; Kae: kaempferol; Hes: hesperetin; Nar: naringenin. GAPDH expression was used as a loading control. Both studies were performed twice independently. Data are presented as mean ± SD of triplicate samples. An unpaired t test was performed for statistical analysis. *: p≤0.05. (D) Anti-oxidative agent N-acetyl cysteine (NAC) treatment does not affect EV71 infection. RD cells were untreated or pretreated with NAC at varying concentrations (1, 3, and 10 mM) or apigenin at 30 µM for 2 hr. The cells were then infected with EV71 (MOI = 0.10 TCID50 per cell) for 36 hr. EV71 VP1 protein expression were determined by immunoblotting analysis. The experiment was performed twice independently. GAPDH was used as a loading control.
Mentions: Finally, we investigated whether flavonoids with similar structural features possessed antiviral activity against EV71 infection. Monolayers of RD cells were treated with a panel of flavonoids (Fig. 4A) at 30 µM prior to EV71 infection. Interestingly, apigenin along with kaempferol, a compound recently reported to inhibit EV71 infection by altering trans-acting factor binding [11], were the only compounds showed activity against EV71 infection. The antiviral effect of kaempferol was nevertheless validated by titration of infectious virion production (Fig. 4B) and by viral protein expression (Fig. 4C). In addition to reduction of virus titers, treatment with kaempferol, but not naringenin, quercetin, or hesperetin, resulted in significant reduction of VP1 protein expression (Fig. 4C). Although the anti-oxidant activity of polyphenols has commonly be attributed to their antiviral property, the fact that apigenin and kaempferol were among the only compounds with antiviral activity against EV71 infection argues strongly against anti-oxidative property for apigenin antiviral activity. Indeed, N-acetyl cysteine, a commonly used anti-oxidative agent, showed no effect against EV71 infection (Fig. 4D).

Bottom Line: We found that apigenin treatment did not affect EV71-induced nucleocytoplasmic redistribution of hnRNP A1 and A2 proteins.As a positive sense, single strand RNA virus, EV71 has a type I internal ribosome entry site (IRES) that cooperates with host factors and regulates EV71 translation.We found that apigenin treatment selectively suppressed the expression of GFP, but not a control gene.

View Article: PubMed Central - PubMed

Affiliation: Medical School and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, Jiangsu, China.

ABSTRACT
Flavonoids are widely distributed natural products with broad biological activities. Apigenin is a dietary flavonoid that has recently been demonstrated to interact with heterogeneous nuclear ribonucleoproteins (hnRNPs) and interferes with their RNA editing activity. We investigated whether apigenin possessed antiviral activity against enterovirus-71 (EV71) infection since EV71 infection requires of hnRNP proteins. We found that apigenin selectively blocks EV71 infection by disrupting viral RNA association with hnRNP A1 and A2 proteins. The estimated EC50 value for apigenin to block EV71 infection was determined at 10.3 µM, while the CC50 was estimated at 79.0 µM. The anti-EV71 activity was selective since no activity was detected against several DNA and RNA viruses. Although flavonoids in general share similar structural features, apigenin and kaempferol were among tested compounds with significant activity against EV71 infection. hnRNP proteins function as trans-acting factors regulating EV71 translation. We found that apigenin treatment did not affect EV71-induced nucleocytoplasmic redistribution of hnRNP A1 and A2 proteins. Instead, it prevented EV71 RNA association with hnRNP A1 and A2 proteins. Accordingly, suppression of hnRNP A1 and A2 expression markedly reduced EV71 infection. As a positive sense, single strand RNA virus, EV71 has a type I internal ribosome entry site (IRES) that cooperates with host factors and regulates EV71 translation. The effect of apigenin on EV71 infection was further demonstrated using a bicistronic vector that has the expression of a GFP protein under the control of EV71 5'-UTR. We found that apigenin treatment selectively suppressed the expression of GFP, but not a control gene. In addition to identification of apigenin as an antiviral agent against EV71 infection, this study also exemplifies the significance in antiviral agent discovery by targeting host factors essential for viral replication.

Show MeSH
Related in: MedlinePlus