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. 2018 Nov;163(11):3119-3124.
doi: 10.1007/s00705-018-3967-7. Epub 2018 Jul 26.

Cholesterol Is Important for the Entry Process of Porcine Deltacoronavirus

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Free PMC article

Cholesterol Is Important for the Entry Process of Porcine Deltacoronavirus

Ji Hyun Jeon et al. Arch Virol. .
Free PMC article

Abstract

The present study was conducted to examine whether cellular and/or viral cholesterol levels play a role in porcine deltacoronavirus (PDCoV) replication. Our results showed that depletion of cholesterol from cells or virions by treating them with methyl-β-cyclodextrin (MβCD) diminished PDCoV infection in a dose-dependent manner. The addition of exogenous cholesterol to MβCD-treated cells or virions moderately restored PDCoV infectivity. Furthermore, the pharmacological sequestration of cellular or viral cholesterol efficiently blocked both virus attachment and internalization. Taken together, the current data indicate that the cholesterol present in the cell membrane and viral envelope contributes to PDCoV replication by acting as a key component in viral entry.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Effects of cellular cholesterol depletion and replenishment on the replication of PDCoV. (A) PDCoV infection efficiency after cholesterol depletion from the cell membrane. ST cells were preincubated with MβCD at the indicated concentrations for 1 h and were mock-infected or infected with PDCoV. Viral production in the presence of MβCD was calculated by measuring the percentage of cells expressing PDCoV N proteins using flow cytometry. (B) Viral progeny production by cellular cholesterol depletion. ST cells pretreated with MβCD were infected with PDCoV and maintained in the presence of MβCD. At 24 hpi, virus culture supernatants were collected and the PDCoV titer was determined. (C and D) PDCoV infection efficiency after cholesterol depletion and replenishment from the cell membrane. ST cells were preincubated with MβCD with (+) or without (–) exogenous cholesterol and infected with PDCoV in the presence or absence of MβCD and/or exogenous cholesterol as indicated. Viral infectivity was determined by measuring the percentage of cells expressing PDCoV N proteins using FACS analysis (C) and by virus titration (D). (E) Cholesterol content determination after cholesterol depletion and replenishment from the cell membrane. ST cells were preincubated with MβCD with (+) or without (–) exogenous cholesterol and infected with PDCoV in the presence or absence of MβCD and/or exogenous cholesterol as indicated. Virus-specific CPE were observed daily and photographed at 24 hpi using a fluorescent/bright-field microscope at a magnification of 200× (first row of panels). For immunostaining, infected cells were fixed at 24 hpi and incubated with a cholesterol-binding, fluorescent antibiotic, Filipin III (second row of panels). The cells were examined using a fluorescent microscope at 200× magnification. The values shown are the means of three independent experiments, and error bars represent standard deviations. *, P = 0.001–0.05; **, P < 0.001
Fig. 2
Fig. 2
Effects of viral cholesterol depletion and replenishment on the replication of PDCoV. (A) PDCoV infection efficiency after cholesterol depletion from the virus envelope. PDCoV suspensions were treated with MβCD to remove cholesterol in the viral envelope, followed by ultracentrifugation, and the purified PDCoV was used to infect fresh ST cells. Virus infectivity was determined by measuring the percentage of cells expressing N proteins of PDCoV using FACS analysis. (B) Viral progeny production after viral cholesterol depletion. Virus culture supernatants were collected at the same time-point, and PDCoV titers were determined. (C and D) PDCoV infection efficiency after cholesterol depletion and replenishment from the viral envelope. PDCoV suspensions were treated with MβCD with (+) or without (–) exogenous cholesterol, followed by ultracentrifugation and infection. Virus infectivity was determined by measuring the percentage of cells expressing PDCoV N proteins using FACS (C) and by virus titration (D). (E) Cholesterol content determination after cholesterol depletion and replenishment from the viral envelope. PDCoV suspensions were incubated with MβCD with (+) or without (–) exogenous cholesterol, followed by ultracentrifugation. Virion cholesterol contents were determined using filipin III, and fluorescence intensity was measured with a fluorescence microplate reader. The values shown are the means of three independent experiments, and error bars represent standard deviations. *, P = 0.001–0.05; **, P < 0.001
Fig. 3
Fig. 3
Effects of cellular (A) or viral (B) cholesterol depletion on virus entry. (A) ST cells were pretreated MβCD infected with PDCoV at 4 °C for 1 h. After washing with cold PBS, infected cells were maintained in the presence or absence of MβCD, either at 4 °C (binding) or 37 °C (internalization), for 1 h. The virus-infected cells maintained at 37 °C were further treated with proteinase K at 37 °C. The infected cells were then serially diluted and plated onto fresh target cells. At 2 days post-incubation, bound or internalized viruses were titrated. (B) PDCoV was treated with MβCD and ultracentrifuged, and the purified virus was used to infect ST cells in the absence of MβCD to measure bound or internalized viruses exactly as described above. The results are expressed as the mean values from three independent experiments performed in triplicate, and error bars represent standard deviations. *, P = 0.001–0.05; **, P < 0.001

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