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. 2021 Apr 30;12:651403. doi: 10.3389/fmicb.2021.651403

FIGURE 3.

FIGURE 3

MFQ inhibits the SARS-CoV-2 entry process. (A) SARS-CoV-2 life cycle. SARS-CoV-2 infection is initiated with virus attachment to the host cells that involves the cellular receptor, angiotensin converting enzyme 2 (ACE2), followed by the cleavage of viral Spike (S) proteins by either transmembrane serine protease (TMPRSS2) on the plasma membrane or cathepsins in the endosome/lysosome that induces fusion of viral and host membranes. Viral RNA is translated, processed and replicated to be assembled into progeny virus with viral structural proteins and released extracellularly. (B) Scheme of the time of addition analysis. Compounds were treated at three different times: (a) whole: throughout the assay for 25 h, (b) entry: for the initial 3 h to evaluate the effect on the viral entry process and (c) post-entry: for the last 22 h to evaluate the effect on viral replication/re-infection. Viral RNA levels in the culture supernatant are shown in the graph by setting that upon DMSO treatment as 100%. (C) Virus-cell attachment assay. VeroE6/TMPRSS2 cells were exposed to virus at an MOI of 0.001 at 4°C for 5 min with 50 μM MFQ or 100 U/mL Heparin, a SARS-CoV-2 attachment inhibitor used as a positive control. After washing the unbound virus, cell surface-attached virus was extracted and quantified by real-time RT-PCR. (D) Post-attachment assay. For evaluating the activity after virus attachment, from membrane fusion to virus secretion, VeroE6/TMPRSS2 cells preincubated with the virus at an MOI of 1.5 at 4°C for 1 h to allow virus attachment were treated with compounds for 6 h at 37°C. Extracellular viral RNA was quantified by RT-qPCR. E-64d, a cysteine protease inhibitor, was used as a positive control. (E) Pseudovirus assays carrying the SARS-CoV-2 Spike or hepatitis C virus (HCV) E1E2 envelope. In the SARS-CoV-2 pseudovirus assay, Camostat and E-64d were used as positive controls for inhibiting TMPRSS2 and cysteine protease, respectively (E, left). Bafilomycin A1 (BFA1), which reported to inhibit HCV entry, was used as a positive control for HCV pseudovirus assay (E, right). *p < 0.05 and **p < 0.01.