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Mechanism of Action of the Suppression of Influenza Virus Replication

by Ko-Ken Tang through Inhibition of the Phosphatidylinositol 3-kinase/Akt

Signaling Pathway and Viral RNP Nuclear Export

 

Ming-Sian Wu, Hung-Rong Yen, Chia-Wen Chang, Tsui-Yi Peng, Chung-Fan Hsieh, Chi-Jene Chen,

Tzou-Yien Lin, Jim-Tong Horng

 

Abstract

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Aims of the study: Ko-Ken Tang (KKT, aka kakkon-to), a conventional Chinese herbal medicine, has been used for the treatment of the common cold, fever and influenza virus infection. However, the underlying mechanism of its activity against influenza virus infection remains elusive. In this study, the antiviral effect and its underlying mechanism was evaluated, including the investigation of anti-influenza virus activity of KKT on MDCK cells and corresponding mechanism related to phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and its consecutive viral RNP nuclear export.

 

Materials and methods: The anti viral activity of non-toxic concentration of KKT was examined against various strains of influenza virus and enterovirus 71 by neutralization assay. PI3K/Akt signaling activated by influenza virus was inspected in A549 cells by western blot. Inhibition of influenza polymerase activity by KKT was measured with plasmid-based reverse genetics using primer extension assay and luciferase reporter assay. Inhibition of viral vRNP nuclear export was demonstrated by laser confocal microscopy and interspecies heterokaryon assay.

 

Results: KKT inhibits influenza virus replication but not entry, and it exhibits abroad spectrum inhibitory activity against human influenza A viruses and enterovirus 71. KKT does not inhibit viral polymerase activity but directly blocks the virus-induced phosphatidylinositol3-kinase/Akt signaling pathway, which in turns causes retention of viral nucleoprotein in the nucleus, thereby interfering with virus propagation. The inhibition by KKT of the nuclear export of viral protein was further confirmed by heterokaryon assay.

 

Conclusions: The results obtained in this study give scientific support to KKT for the treatment of influenza virus infection. KKT could be of potential use in the management of seasonal pandemic influenza virus infection in addition to other clinically available drugs.

Key words: Cytopathic effect, Influenza virus, Kakkon-to, Ko-Ken Tang, Nucleoprotein, Phosphatidylinositol 3-kinase/Akt

1. Introduction
2. Materials and Methods
   1. Cell culture, viruses and reagents
   2. Preparation of crude herb extract: KKT was purchased from Sun Ten Pharmaceutical Co., Ltd., (Taipei, Taiwan). Each 7.5g contains 4g of dry extract (from 6g pueraria root, 4.5g ma huang, 3g cinnamon twigs, 3g peony root, 4g jujube date, licorice and 4.5g ginger) and 3.5g corn starch.
   3. Cytotoxicity assay, antiviral effectiveness assay and selectivity index (SI)
   4. Time course (time-of-addition) assay and multicycle growth inhibition assay
   5. RNP activity measured by reporter assay
   6. RNP activity measured by primer extension assay
   7. Viral infection, solubilization, electrophoresis and Western blot analysis
   8. Fluorescence confocal microscopy
   9. Heterokaryon assay
3. Results
   1. Antiviral efficacy and cytotoxicity assay
   2. Time course assay in influenza A virus-infected cells
   3. KKT inhibits PI3K activation induced by influenza A/WSN/33 infection
   4. KKT impairs viral propagation and viral protein synthesis
   5. KKT does not inhibit viral polymerase activity
   6. KKT treatment results in NP retention in the nucleus
4. Discussion

 

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