COVID‐19, a global pandemic, has caused over 750,000 deaths worldwide as of August 2020. A vaccine or remedy for SARS‐CoV‐2, the virus responsible for COVID‐19, is necessary to slow down the spread and lethality of COVID‐19. However, there is currently no effective treatment available against SARS‐CoV‐2. In this report, we demonstrated that EGCG and theaflavin, the main active ingredients of green tea and black tea, respectively, are potentially effective to inhibit SARS‐CoV‐2 activity. Coronaviruses require the 3CL‐protease for the cleavage of its polyprotein to make individual proteins functional. EGCG and theaflavin showed inhibitory activity against the SARS‐CoV‐2 3CL‐protease in a dose‐dependent manner, and the half inhibitory concentration (IC₅₀) was 7.58 μg/ml for EGCG and 8.44 μg/ml for theaflavin. In addition, we did not observe any cytotoxicity for either EGCG or theaflavin at the concentrations tested up to 40 μg/ml in HEK293T cells. These results suggest that upon further study, EGCG and theaflavin can be potentially useful to treat COVID‐19.

AMP-activated protein kinase (AMPK) regulates autophagy initiation when intracellular ATP level decreases. However, the role of AMPK during autophagosome maturation is not fully understood. Here, we report that AMPK contributes to efficient autophagosome maturation and lysosomal fusion. Using CRISPR-Cas9 gene editing, we generated AMPK α1 knockout HEK293T cell lines, in which starvation-induced autophagy is impaired. Compound C, an AMPK-independent autophagy inducer, and trehalose, an mTOR-independent autophagy inducer were used to examine the role of AMPK in autophagosome maturation and lysosomal fusion. While the treatment of control cells with either compound C or trehalose induces activation of autophagosomes as well as autolysosomes, the treatment of AMPK α1 knockout cells with compound C or trehalose induces mainly activation of autophagosomes, but not autolysosomes. We demonstrate that this effect is due to interference with the fusion of autophagosomes with lysosomes in AMPK α1 knockout cells. The transient expression of AMPK α1 can rescue autophagosome maturation. These results indicate that AMPK α1 is required for efficient autophagosome maturation and lysosomal fusion.

The PKA-CREB signaling pathway is involved in many cellular processes including autophagy. Recent studies demonstrated that PKA-CREB inhibits autophagy in yeast; however, the role of PKA-CREB signaling in mammalian cell autophagy has not been fully characterized. Here, we report that the integral membrane protein ITM2A expression is positively regulated by PKA-CREB signaling and ITM2A expression interferes with autophagic flux by interacting with vacuolar ATPase (v-ATPase). The ITM2A promoter contains a CRE element, and mutation at the CRE consensus site decreases the promoter activity. Forskolin treatment and PKA expression activate the ITM2A promoter confirming that ITM2A expression is dependent on the PKA-CREB pathway. ITM2A expression results in the accumulation of autophagosomes and interferes with autolysosome formation by blocking autophagic flux. We demonstrated that ITM2A physically interacts with v-ATPase and inhibits lysosomal function. These results support the notion that PKA-CREB signaling pathway regulates ITM2A expression, which negatively regulates autophagic flux by interfering with the function of v-ATPase.

The COVID-19 pandemic has resulted in a huge number of deaths from 2020 to 2021; however, effective antiviral drugs against SARS-CoV-2 are currently under development. Recent studies have demonstrated that green tea polyphenols, particularly EGCG, inhibit coronavirus enzymes as well as coronavirus replication in vitro. Herein, we examined the inhibitory effect of green tea polyphenols on coronavirus replication in a mouse model. We used epigallocatechin gallate (EGCG) and green tea polyphenols containing more than 60% catechin (GTP60) and human coronavirus OC43 (HCoV-OC43) as a surrogate for SARS-CoV-2. Scanning electron microscopy analysis results showed that HCoV-OC43 infection resulted in virion particle production in infected cells. EGCG and GTP60 treatment reduced coronavirus protein and virus production in the cells. Finally, EGCG- and GTP60-fed mice exhibited reduced levels of coronavirus RNA in mouse lungs. These results demonstrate that green tea polyphenol treatment is effective in decreasing the level of coronavirus in vivo.