Studies have suggested that 5-ALA enhances aerobic energy metabolism, especially cytochrome c oxidase activity as well as protein expression in the mitochondria. 5-ALA also induces heme oxygenase-1 [HO-1] expression, a rate-limiting enzyme in heme metabolism, in the kidney and in cultured cells.
2.1. Cells and reagents
VeroE6 cells (donated by Dr. Ayato Takada, Hokkaido University, Japan) and Caco-2 cells (donated by Dr. Tetsuya Iida, Osaka University, Japan) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution. 5-amino leuvulinic acid (5-ALA) was donated by Neopharma Japan (Tokyo, Japan) and was dissolved to 100 mM in water. Sodium ferrous citrate (SFC) was also donated by Neopharma Japan and was dissolved to 25 mM in water with 1 M HCl. Remdesivir (Cayman Chemical, Ann Arbor, MI) was dissolved to 10 mM in DMSO. For immunostaining, goat serum (ThermoFisher Scientific, Waltham, MA), rabbit anti-SARS-CoV N antibody (NOVUS, Centennial, CO), Alexa Fluor 488 goat anti-rabbit IgG (ThermoFisher Scientific) and Hoechst33342 dye (ThermoFisher Scientific) was used.
2.2. Virus propagation
A JPN/NGS/IA-1/2020 strain of SARS-CoV-2 (GISAID accession no. EPI-ISL-481251), which was isolated from a Japanese patient, was propagated in VeroE6 cells. Culture supernatants were collected 4 days after infection, clarified by centrifugation at 2,000 × g for 15 min and stored at −80 °C until use. Virus titer was determined by a plaque assay using VeroE6 cells. After 1 h infection, the inoculum was washed out and the cells were incubated with 0.7% agarose gel in Minimum Essential Medium (MEM) supplemented with 2% FBS and 1% penicillin/streptomycin solution for 3 days. After virus inactivation using 4% paraformaldehyde (PFA) overnight, the cells were stained with crystal violet solution. The plaques were counted manually to calculate the virus titer. All experiments with replication competent SARS-CoV-2 were performed in a biosafety level 3 (BSL3) laboratory at Nagasaki University.
Then, the antiviral effect of 5-ALA was tested using this assay (Fig. 2A). We found that 72-hour pretreatment of VeroE6 cells with 5-ALA blocked SARS-CoV-2 infection (Fig. 2B).
However, 48-hour pretreatment with 5-ALA with and without SFC did not significantly affect SARS-CoV-2 infection (Fig. 2C), suggesting that a longer incubation time for 5-ALA treatment is required to make host cells resistant to the infection.
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