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Amphotericin B Inhibits Enterovirus 71 Replication by Impeding Viral Entry

View Article: PubMed Central - PubMed

ABSTRACT

Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease that leads to cardiopulmonary complications and death in young children. There is thus an urgent need to find new treatments to control EV71 infection. In this study, we report potent inhibition of EV71 by a polyene antibiotic Amphotericin B. Amphotericin B profoundly diminished the expression of EV71 RNA and viral proteins in the RD cells and the HEK293 cells. As a result, EV71 production was inhibited by Amphotericin B with an EC50 (50% effective concentration) of 1.75 μM in RD cells and 0.32 μM in 293 cells. In addition to EV71, EV68 was also strongly inhibited by Amphotericin B. Results of mechanistic studies revealed that Amphotericin B targeted the early stage of EV71 infection through impairing the attachment and internalization of EV71 by host cells. As an effective anti-fungi drug, Amphotericin B thus holds the promise of formulating a novel therapeutic to treat EV71 infection.

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Amphotericin B impedes the binding and internalization of EV71 virions to host cells.EV71 was incubated with Amphotericin B pretreated RD (a) and 293 cells (b). Then background, bound, internal cells (described in materials and methods) were lysed for RNA extraction. Viral RNA was quantified by semi-RT PCR (upper panel) and viral RNA copy number was quantified by quantitative RT-PCR (lower panel). The results are plotted relative to virus background in DMSO treated cells. The data represent 3 independent experiments, and error bars represent SD (*P < 0.05, **P < 0.01, t test). (c) Levels of SCARB2 expression in background, bound, internal cells were determined by Western blotting. Full-length gels are presented in Supplementary Fig. 4. (d) The internalized viral titers were determined in plaque assays. The internal cells were collected at 10 hpi, and proceeded plaque assay after frozen and thaw. Cells treated with DMSO were set as 100%. The data represent 3 independent experiments, and error bars represent SD (**P < 0.01, t test).
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f5: Amphotericin B impedes the binding and internalization of EV71 virions to host cells.EV71 was incubated with Amphotericin B pretreated RD (a) and 293 cells (b). Then background, bound, internal cells (described in materials and methods) were lysed for RNA extraction. Viral RNA was quantified by semi-RT PCR (upper panel) and viral RNA copy number was quantified by quantitative RT-PCR (lower panel). The results are plotted relative to virus background in DMSO treated cells. The data represent 3 independent experiments, and error bars represent SD (*P < 0.05, **P < 0.01, t test). (c) Levels of SCARB2 expression in background, bound, internal cells were determined by Western blotting. Full-length gels are presented in Supplementary Fig. 4. (d) The internalized viral titers were determined in plaque assays. The internal cells were collected at 10 hpi, and proceeded plaque assay after frozen and thaw. Cells treated with DMSO were set as 100%. The data represent 3 independent experiments, and error bars represent SD (**P < 0.01, t test).

Mentions: To further identify which step of EV71 infection was inhibited by Amphotericin B, we examined binding of EV71 particles to target cells and viral internalization. Virus binding assay was performed by incubating EV71 viruses with RD or 293 cells with or without Amphotericin B at 4 °C as previously described4445. After extensive washing to remove unbound viruses, amounts of viruses that were associated with target cells under Amphotericin B treatment decreased to less than 60% of that measured in the absence of Amphotericin B (Fig. 5, Bound). Protease treatment of the target cells decreased the PCR signal, supporting cell-bound nature of these viruses (Fig. 5, Background). To examine virus uptake, cells were first incubated with EV71 at 4 °C, then transferred to 37 °C for one hour to allow virus internalization. Cells were then treated with protease to remove viruses that bound to cell surface (Fig. 5, Internal). The results showed significant reduction of EV71 internalization by Amphotericin B. We also examined the expression of EV71 receptor SCARB2 by western blotting and found that SCARB2 expression was not affected by Amphotericin B (Fig. 5c). In addition, the internalized viruses were quantitated in plaque assay. As shown in Fig. 5d, the internalized viruses were reduced as a result of Amphotericin B treatment. Together, these data suggest that Amphotericin B impairs the binding and internalization of EV71 virus to host cells without affecting viral receptor expression.


Amphotericin B Inhibits Enterovirus 71 Replication by Impeding Viral Entry
Amphotericin B impedes the binding and internalization of EV71 virions to host cells.EV71 was incubated with Amphotericin B pretreated RD (a) and 293 cells (b). Then background, bound, internal cells (described in materials and methods) were lysed for RNA extraction. Viral RNA was quantified by semi-RT PCR (upper panel) and viral RNA copy number was quantified by quantitative RT-PCR (lower panel). The results are plotted relative to virus background in DMSO treated cells. The data represent 3 independent experiments, and error bars represent SD (*P < 0.05, **P < 0.01, t test). (c) Levels of SCARB2 expression in background, bound, internal cells were determined by Western blotting. Full-length gels are presented in Supplementary Fig. 4. (d) The internalized viral titers were determined in plaque assays. The internal cells were collected at 10 hpi, and proceeded plaque assay after frozen and thaw. Cells treated with DMSO were set as 100%. The data represent 3 independent experiments, and error bars represent SD (**P < 0.01, t test).
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Related In: Results  -  Collection

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f5: Amphotericin B impedes the binding and internalization of EV71 virions to host cells.EV71 was incubated with Amphotericin B pretreated RD (a) and 293 cells (b). Then background, bound, internal cells (described in materials and methods) were lysed for RNA extraction. Viral RNA was quantified by semi-RT PCR (upper panel) and viral RNA copy number was quantified by quantitative RT-PCR (lower panel). The results are plotted relative to virus background in DMSO treated cells. The data represent 3 independent experiments, and error bars represent SD (*P < 0.05, **P < 0.01, t test). (c) Levels of SCARB2 expression in background, bound, internal cells were determined by Western blotting. Full-length gels are presented in Supplementary Fig. 4. (d) The internalized viral titers were determined in plaque assays. The internal cells were collected at 10 hpi, and proceeded plaque assay after frozen and thaw. Cells treated with DMSO were set as 100%. The data represent 3 independent experiments, and error bars represent SD (**P < 0.01, t test).
Mentions: To further identify which step of EV71 infection was inhibited by Amphotericin B, we examined binding of EV71 particles to target cells and viral internalization. Virus binding assay was performed by incubating EV71 viruses with RD or 293 cells with or without Amphotericin B at 4 °C as previously described4445. After extensive washing to remove unbound viruses, amounts of viruses that were associated with target cells under Amphotericin B treatment decreased to less than 60% of that measured in the absence of Amphotericin B (Fig. 5, Bound). Protease treatment of the target cells decreased the PCR signal, supporting cell-bound nature of these viruses (Fig. 5, Background). To examine virus uptake, cells were first incubated with EV71 at 4 °C, then transferred to 37 °C for one hour to allow virus internalization. Cells were then treated with protease to remove viruses that bound to cell surface (Fig. 5, Internal). The results showed significant reduction of EV71 internalization by Amphotericin B. We also examined the expression of EV71 receptor SCARB2 by western blotting and found that SCARB2 expression was not affected by Amphotericin B (Fig. 5c). In addition, the internalized viruses were quantitated in plaque assay. As shown in Fig. 5d, the internalized viruses were reduced as a result of Amphotericin B treatment. Together, these data suggest that Amphotericin B impairs the binding and internalization of EV71 virus to host cells without affecting viral receptor expression.

View Article: PubMed Central - PubMed

ABSTRACT

Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease that leads to cardiopulmonary complications and death in young children. There is thus an urgent need to find new treatments to control EV71 infection. In this study, we report potent inhibition of EV71 by a polyene antibiotic Amphotericin B. Amphotericin B profoundly diminished the expression of EV71 RNA and viral proteins in the RD cells and the HEK293 cells. As a result, EV71 production was inhibited by Amphotericin B with an EC50 (50% effective concentration) of 1.75&thinsp;&mu;M in RD cells and 0.32&thinsp;&mu;M in 293 cells. In addition to EV71, EV68 was also strongly inhibited by Amphotericin B. Results of mechanistic studies revealed that Amphotericin B targeted the early stage of EV71 infection through impairing the attachment and internalization of EV71 by host cells. As an effective anti-fungi drug, Amphotericin B thus holds the promise of formulating a novel therapeutic to treat EV71 infection.

No MeSH data available.


Related in: MedlinePlus