Remdesivir (GS-5734) Impedes Enterovirus Replication Through Viral RNA Synthesis Inhibition

Figures

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  FIGURE 1. Remdesivir potently inhibits EV71 infection without significant cytotoxicity. (A) HeLa cells were infected with EV71 (MOI = 1) and treated with varying concentrations of remdesivir at 1 hpi. The viral RNA copy number was quantified by qRT-PCR at 24 hpi (mean ± SD, n = 3). (B) Cell viability after culture with different doses of remdesivir at 24 hpi assessed using a CCK8 assay (mean ± SD, n = 6). (C) Western blot of EV71 VP2 protein expression in HeLa cells. HeLa cells were infected with EV71 (MOI = 0.5). After adsorption, the cells were treated with the indicated concentrations of remdesivir and collected at 24 hpi. Then, the proteins were resolved by SDS-PAGE and western blotting. (D) Fluorescence microscopy examination of EV71-infected HeLa cells treated with different amounts of remdesivir (24 hpi). The dsRNA-specific antibody J2 (red) was used for staining viral dsRNA replication intermediates, and nuclei were labeled with Hoechst 33258 (blue). Bar = 100 μm. (E) Cell culture supernatants collected from (A) were used to infect HeLa cells, and after 1 h of adsorption at 37°C, fresh medium was added. At 24 hpi, cells were collected and subjected to viral RNA copy number…

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  FIGURE 2. Remdesivir inhibits EV71 replication post virus entry. (A) Schematic representation of the different treatment methods. Cells were pretreated with remdesivir (B), coadsorbed remdesivir and EV71 (C), remdesivir after EV71 adsorption that was removed after virus entry (D), or remdesivir after virus entry (E), and at 24 hpi, the EV71 viral RNA copy number was quantified by qRT-PCR (mean ± SD, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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  FIGURE 3. Remdesivir inhibits EV71 replication at the viral transcription stage. (A) Schematic representation of reverse transcription and PCR protocol for conventional real-time PCR (top) and tagged primer-mediated qRT-PCR (bottom). The tag within the tagged RT primer is shown in green, and the amplified products contained a portion of the tag and cDNA. (B,C) Synthesis of EV71 cRNA (normalized to the actin expression level) was inhibited by remdesivir (mean ± SD, n = 3). P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001.

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  FIGURE 4. Remdesivir exhibits broad-spectrum inhibition of enterovirus replication. (A) Phylogenetic analyses of the human enterovirus amino acid sequence. EV-A is marked in light blue, EV-B is marked in gold, EV-C is marked in orange, and EV-C is marked in green. CVB3 (B) and EV70 (C) RNA synthesis was inhibited by remdesivir (mean ± SD, n = 3). *P < 0.05, ***P < 0.001, and ****P < 0.0001.