Streptomyces Extract Inhibits Enterovirus 71 Replication by Activation of PKB/AKT Signaling

Abstract

Enterovirus 71 (EV71) is a main pathogen of hand-foot, and mouth disease (HFMD) in children and adults. HFMD is a disease that causes small blisters in the mouth and hands and feet. Although blisters are usually disappeared one to two weeks after infection, HFMD is a serious disease that can lead to encephalitis and manic diseases depending on the patient. However, a representative vaccine and treatment for HFMD have not been developed yet. In this study, we investigated the antiviral effect of Streptomyces sp. zx10-19 (KH29) extract (0.1 ~ 100 μg/㎖) using EV71 infected HeLa cells. KH29 extract (100 μg/㎖) treatment significantly inhibited expression of EV71 capsid protein VP1 and cleavage of translation initiation factor eIF4G1. In addition, PKB/AKT activity was significantly increased by KH29 extract treatment. In the reverse transcription-PCR, KH29 extract treatment significantly inhibited EV71 a positive and negative-strand RNA genome amplification at 100 ug/ml. Moreover, the downstream signal molecule GSK3-beta and NF-κB phosphorylation were significantly increased following AKT activation in KH29 extract treatment. These results suggest that KH29 extract may increases cell survival through AKT signaling and effectively inhibit the proliferation of EV71, which will be used as an effective substance for the development of therapeutic agents for EV71-induced HFMD. was cultured on HeLa cell monolayers. HeLa cells were grown for 16 h and infected with 10 7 plaque-forming units (PFU) of EV71. When the cytopathic effect (CPE) of the infected cells reached > 90%, the cells were subjected to three freeze-thaw cycles at -80℃. Virus stock concentrations were determined by tissue culture infectious dose 50 (TCID50). HeLa cells were cultured using Dulbecco’s modified eagle medium (DMEM, Welgene, Inc., Gyeongsan-si, Korea) with 5% fetal bovine serum (FBS), 1% penicillin-streptomycin sol. (Welgene, Inc) at 37℃ in a humidified 5% CO 2 incubator (16). VP1 Anti-sense 5'-TTGACAAAAACTGAGGGGTT-3' GAPDH Sense 5'-ATCAACGACCCCTTCATTGAC-3', and GAPDH Anti-sense 5'-CCAGTAGACTCCACGACATACTCAGC-3' with cDNA as template. Then, the PCR product was electrophoresed on 1.5% agarose gel, and viral VP1 gene positive and negative strands were quantified by semi-quantitative RT-PCR. All data were quantified by NIH-image J V1.45 software and normalized by GAPDH as described previously (16). EV71 replication is regulated by host cells signaling molecules such as GSK3β and NF-κB activity at the early stage of infection. Several well-characterized physiological substrates for Akt have been identified to date, including GSK-3 (11). GSK-3, a ubiquitously expressed protein–serine/threonine kinase, is inhibited by Akt phosphorylation in response to growth factor stimulation. These studies suggest that GSK3 is involved in multiple cellular processes, including metabolism, proliferation, and differentiation. We found that phosphorylation of GSK3β (Ser9) and NF-κB were significantly increased by KH29 extract treatment (Fig. 6). KH29 extract activates cell survival and proliferation through Akt signal-induced GSK3β (Ser9) and NF-κB phosphorylation during EV71 infection. Activated Akt phosphorylates and inactivates GSK3β which may improve cell survival in viral infection.