[논문]Vinyl-Stilbene Inhibits Human Norovirus RNA Replication by Activating Heat-Shock Factor-1

Lee, Ahrim; Sung, Jieun; Harmalkar, Dipesh S.; Kang, Hyeseul; Lee, Hwayoung; Lee, Kyeong; Lee, Choongho

doi:10.4062/biomolther.2021.153

Vinyl-Stilbene Inhibits Human Norovirus RNA Replication by Activating Heat-Shock Factor-1 원문보기

Biomolecules & therapeutics, v.30 no.1, 2022년, pp.64 - 71

Lee, Ahrim (College of Pharmacy, Dongguk University-Seoul) , Sung, Jieun (College of Pharmacy, Dongguk University-Seoul) , Harmalkar, Dipesh S. (College of Pharmacy, Dongguk University-Seoul) , Kang, Hyeseul (College of Pharmacy, Dongguk University-Seoul) , Lee, Hwayoung (College of Pharmacy, Dongguk University-Seoul) , Lee, Kyeong (College of Pharmacy, Dongguk University-Seoul) , Lee, Choongho (College of Pharmacy, Dongguk University-Seoul)

Abstract ▼ AI-Helper

Norovirus (NV) is the most common cause of viral gastroenteritis, with the potential to develop into a fatal disease in those who are immuno-compromised, and effective vaccines and treatments are still non-existent. In this study, we aimed to elucidate the molecular mechanism of the previously identified NV replication inhibitor utilizing a vinyl-stilbene backbone, AC-1858. First, we confirmed the inhibition of the NV RNA replication by a structural analog of AC-1858, AC-2288 with its exclusive cytoplasmic sub-cellular localization. We further validated the induction of one specific host factor, the phosphorylated form of heat shock factor (HSF)-1, and its increased nuclear localization by AC-1858 treatment. Finally, we verified the positive and negative impact of the siRNA-mediated downregulation and lentivirus-mediated overexpression of HSF-1 on NV RNA replication. In conclusion, these data suggest the restrictive role of the host factor HSF-1 in overall viral RNA genome replication during the NV life cycle.

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표/그림 (6)

그림 Fig. 1. Inhibition of NV RNA replication by AC-1858. (A) The chemical structure of a vinyl-stilbene compound, AC-1858, the most effective NV replication inhibitor. (B) HG23 cells were treated with increasing concentrations of AC-1858 for 72 h. Cell viability was measured by MTT assay. CC₅₀ is the concentration required for 50% cytotoxicity. NV and GAPDH RNA levels were quantitated by qRT-PCR. 2'-C-methyl cytidine (2CMC), a viral polymerase inhibitor, was used as a positive control. EC₅₀ is the concentration required for 50% inhibition of NV RNA replication. (C) An immunofluorescence assay was conducted to study the effects of AC-1858 on the expression of neomycin phosphotransferase. DAPI staining was used to indicate the locations of the nucleus in blue.
그림 Fig. 2. Inhibition of the NV RNA replication by AC-2288 via its cytoplasmic localization. (A) The chemical structure of AC-2288, an analog compound of AC-1858. (B) HG23 cells were treated with increasing concentrations of AC-2288 for 72 h. Cell viability was measured by MTT assay. CC₅₀ is the concentration required for 50% cytotoxicity. NV and GAPDH RNA levels were quantitated by qRT-PCR. 2'-C-methyl cytidine (2CMC), a viral polymerase inhibitor, was used as a positive control. EC₅₀ is the concentration required for 50% inhibition of NV RNA replication. (C) HG23 cells were treated with increasing concentrations of AC-2288 for 72 h. Subcellular localization of AC-2288 was examined by a confocal microscope. DAPI staining was used to indicate the locations of the nucleus in blue. (D) HG23 cells were treated with either 5 μM or 10 μM of AC-2288. Subcellular localization of AC-2288 was examined at 24 h, 48 h, and 72 h after treatment by a confocal microscope. DAPI staining was used to indicate the locations of the nucleus in blue. Calculated p values, which were less than 0.0001*** when compared with a control, were considered statistically.
그림 Fig. 3. Induction of the phosphorylated form of HSF-1 by AC- 1858. HG23 cells were treated with increasing concentrations of AC-1858 for 72 h. 2'-C-methyl cytidine (2CMC), a viral polymerase inhibitor, was used as a positive control. Expression levels of HSF- 1, phosphorylated HSF1, β-actin, and neomycin phosphotransferase proteins were analyzed by Western blotting with corresponding antibodies.
그림 Fig. 4. Increased nuclear localization of HSF-1 by AC-1858. The effects of AC-1858 on the subcellular localization of the endogenous HSF- 1 protein were studied in HG23 cells by immunofluorescent analysis. DAPI staining was used to indicate the locations of the nucleus in blue.
그림 Fig. 5. Reduced expression of HSF-1 leads to increased NV RNA replication. HG23 cells (A) and Huh7.5 cells (B) were transfected with siRNA-1, -2, and -3, which target HSF-1 for degradation. (A) Cell viability was measured by MTT assay. (B) Expression levels of HSF-1 protein and β-actin proteins were analyzed after 48 h of siRNA transfection. (C) The NV and GAPDH RNA levels were quantitated by qRT-PCR in HG23 cells.
그림 Fig. 6. Overexpression of HSF-1 leads to decreased NV RNA replication. HG23 cells were treated with 0.1, 1, and 10 μL of HSF- 1-encoding lentivirus-containing media. (A) Expression levels of HSF-1 after lentiviral infection were quantitated by Western blot using an anti-HSF-1 antibody. (B) The NV and GAPDH RNA levels were quantitated by qRT-PCR in HG23 cells.

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