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. 2020 Jul 2:283:197961.
doi: 10.1016/j.virusres.2020.197961. Epub 2020 Apr 10.

Porcine deltacoronavirus activates the Raf/MEK/ERK pathway to promote its replication

Ji Hyun Jeon  1 , Yoo Jin Lee  2 , Changhee Lee  3
Affiliations

Porcine deltacoronavirus activates the Raf/MEK/ERK pathway to promote its replication

Ji Hyun Jeon et al. Virus Res. .

Abstract

Porcine deltacoronavirus (PDCoV) is a newly emerged swine coronavirus that causes acute enteritis in neonatal piglets. To date, little is known about the host factors or cellular signaling mechanisms associated with PDCoV replication. Since the Raf/MEK/ERK pathway is involved in modulation of various important cellular functions, numerous DNA and RNA viruses coopt this pathway for efficient propagation. In the present study, we found that PDCoV induces the activation of ERK1/2 and its downstream substrate Elk-1 early in infection irrespective of viral biosynthesis. Chemical inhibition or knockdown of ERK1/2 significantly suppressed viral replication, whereas treatment with an ERK activator increased viral yields. Direct pharmacological inhibition of ERK activation had no effect on the viral entry process but sequentially affected the post-entry steps of the virus life cycle. In addition, pharmacological sequestration of cellular or viral cholesterol downregulated PDCoV-induced ERK signaling, highlighting the significance of the cholesterol contents in ERK activation. However, ERK inhibition had no effect on PDCoV-triggered apoptosis through activation of the cytochrome c-mediated intrinsic mitochondrial pathway, suggesting the irrelevance of ERK activation to the apoptosis pathway during PDCoV infection. Altogether, our findings indicate that the ERK signaling pathway plays a pivotal role in viral biosynthesis to facilitate the optimal replication of PDCoV.

Keywords: Cholesterol; ERK1/2; PDCoV; Signal transduction; Viral replication.

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Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
PDCoV activates the ERK1/2 signaling pathway in cultured cells. ST cells were mock infected or infected with PDCoV at an MOI of 1 or an equal amount of UV-inactivated PDCoV. (A and B) Whole cell lysates were prepared for the indicated time points following infection and subjected to western blot analysis with the antibody specific for phosphorylated ERK1/2 (p-ERK1/2), ERK1/2, or the PDCoV N protein. The blot was also reacted with mouse MAb against β-actin to verify equal protein loading. Fold changes in the p-ERK1/2:total ERK1/2 ratio compared by densitometry of the corresponding bands using a computer densitometer are plotted. (C and D) ERK1/2 activation induced by PDCoV infection was quantitatively determined using a FACE assay. ST cells were fixed at the indicated time points with 4% formaldehyde and incubated with an anti-ERK1/2 or anti-p-ERK1/2 antibody followed by HRP-conjugated IgG antibodies. The absorbance of the solution was determined at 450 nm using a spectrophotometer. Data are the representative of the means from three independent experiments, and error bars denote the mean ± SDM. *P <  0.05; **P <  0.001.
Fig. 2
Fig. 2
PDCoV induces nuclear translocation of active ERK1/2. ST cells were mock infected or infected with PDCoV (A) or UV-inactivated PDCoV (B). At the indicated time points, infected cells were fixed and co-stained with antibodies against p-ERK1/2 (green) and PDCoV N (red). The cells were then counterstained with DAPI, and the intracellular localization of p-ERK1/2 in virus-infected cells was examined under a confocal microscope at 800× magnification. Western blot analysis of cell extracts infected with PDCoV (C) or UV-inactivated PDCoV (D). The cytosolic and nuclear fractions were prepared for the indicated time points and subjected to western blotting with an antibody directed against p-ERK (top panel), PDCoV N (second panel), Sp1 (nuclear protein marker; third panel), α-tubulin (cytosolic protein marker; fourth panel), or β-actin (bottom panel). All subcellular protein markers served as loading controls. The number under each band in the top panel represents fold changes in p-ERK level expressed as the densitometric unit of the band normalized to the corresponding loading control level as compared to the mock-infected control. Note that the presence of the PDCoV N protein in the nucleic fraction is shown as described previously (Lee and Lee, 2015).
Fig. 3
Fig. 3
Phosphorylated ERK1/2 leads to the activation of downstream _target Elk-1. ST cells were mock infected or infected with PDCoV at an MOI of 1 (A) or an equal amount of UV-inactivated PDCoV (B). Whole cell lysates were prepared for the indicated time points following infection and subjected to western blot analysis with the antibody to p-Elk-1 or PDCoV N. The blot was also reacted with mouse MAb against β-actin to verify equal protein loading. Fold changes in the p-Elk-1:β-actin ratio are plotted. Results are the representative of the means from three independent experiments, and error bars denote the mean ± SDM. *P <  0.05; **P <  0.001.
Fig. 4
Fig. 4
Inhibition of ERK1/2 activation impairs PDCoV propagation. (A) ST cells were preincubated with DMSO, PD98059 (50 and 100 μM), or U0126 (50 and 100 μM) for 1 h prior to infection and were mock infected or infected with PDCoV at an MOI of 1. The virus-infected cells were further maintained for 12 h in the presence of DMSO or inhibitors. PDCoV-specific CPEs were monitored and photographed at 12 hpi under an inverted microscope at the magnification of 200× (first panels). For immunostaining, the infected cells were fixed at 12 hpi and incubated with MAb against the PDCoV N protein, followed by incubation with Alexa green-conjugated goat anti-mouse secondary antibody (second panels). The cells were then counterstained with DAPI (third panels) and examined under a fluorescent microscope at 200× magnification. (B) PDCoV production in the presence of each inhibitor was quantified by measuring the percentage of cells expressing N proteins through flow cytometry. (C) Chemical inhibition of ERK1/2 was quantitatively determined using a FACE assay. ST cells were mock infected or PDCoV infected in the presence of DMSO, PD98059, or U0126. The cells were fixed at 6 hpi with 4% formaldehyde and incubated with an anti-ERK1/2 or anti-phospho-ERK1/2 antibody followed by HRP-conjugated IgG antibodies. The absorbance of the solution was determined at 450 nm using a spectrophotometer. (D) At 6 hpi, cellular lysates were prepared and subjected to immunoblotting using an antibody against p-ERK1/2, ERK1/2, p-Elk-1, or PDCoV N. The blot was also reacted with an anti-β-actin antibody to verify equal protein leading. Each protein expression was quantitatively analyzed by densitometry, and fold changes in each p-ERK1/2:total ERK1/2, p-Elk-1:β-actin, and PDCoV N:β-actin ratio are independently plotted (right panel). Data are the representative of the means from three independent experiments, and error bars denote the mean ± SDM. *P <  0.05; **P <  0.001.
Fig. 5
Fig. 5
Chemical inhibitor of ERK1/2 is present at the early stage to inhibit virus production. (A) ST cells were pretreated with DMSO or U0126 (100 μM) and mock or PDCoV (MOI of 1) infected. At the indicated time points post-infection, U0126 was added to achieve the intended final concentration. At 12 hpi, virus-infected cells were trypsinized, and virus infectivity was determined by measuring the percentage of cells expressing N proteins through FACS. (B) Whole cell lysates were prepared for the indicated time points of U0126 treatment and subjected to western blot analysis with the antibody to p-ERK1/2, ERK1/2, p-Elk-1, or PDCoV N. The blot was also reacted with an anti-β-actin antibody to verify equal protein leading. Each protein expression was quantitatively analyzed by densitometry, and fold changes in each p-ERK1/2:total ERK1/2, p-Elk-1:β-actin, and PDCoV N:β-actin ratio are independently plotted. Results are presented as the mean values of three independent experiments, and error bars denote the mean ± SDM. *P <  0.05; **P <  0.001.
Fig. 6
Fig. 6
ERK1/2 knockdown suppresses PDCoV infection. (A) ST cells were transfected with 100 nM ERK-specific siRNA or control siRNA using Lipofectamine 3000 followed by PDCoV infection at 24 h post-transfection. At 12 hpi, cellular lysates were prepared and subjected to immunoblotting using an antibody against ERK1/2, p-ERK1/2, or p-Elk-1. The blot was also reacted with an anti-β-actin antibody to verify equal protein leading. Each protein expression was quantitatively analyzed by densitometry and presented as the relative density value to the β-actin gene, and fold changes in each protein:β-actin ratio are plotted. (B) For immunostaining, transfected and infected cells were fixed at 12 hpi and stained with an anti-PDCoV N antibody followed by Alexa green-conjugated goat anti-mouse secondary antibody (first panels). The cells were then counterstained with DAPI (second panels) and examined under a fluorescent microscope at 200× magnification. Quantification of PDCoV production was assessed exactly as described in the legend of Fig. 4B and presented (left panel) (C) The virus supernatants were collected at 12 hpi, and viral titers were determined. Values are representative of the mean of three independent experiments, and error bars represent the mean ± SDM. **P <  0.001.
Fig. 7
Fig. 7
Pharmacological activation enhances PDCoV multiplication. (A) ST cells were mock or PDCoV infected (MOI of 1) for 1 h and then maintained in fresh medium containing DMSO or various concentrations of PMA. At 6 hpi, the virus supernatants were collected, and virus titers were determined. (B) Whole cell lysates were prepared at 6 hpi and subjected to western blot analysis with the antibody specific for p-ERK1/2, ERK1/2, p-Elk-1, or PDCoV N. The blot was also reacted with an anti β-actin antibody to verify equal protein loading. Fold changes in each p-ERK1/2:total ERK1/2, p-Elk-1:β-actin, and PDCoV N:β-actin ratio are independently plotted. Values shown are representative of the mean of three independent experiments, and error bars denote the mean ± SDM. *P < 0.05; **P < 0.001.
Fig. 8
Fig. 8
Pharmacological inhibition of ERK1/2 activation has no effect on virus entry but interferes with each post-entry step of PDCoV replication. (A) Effect of ERK inhibition on virus binding and internalization. ST cells were infected with PDCoV at an MOI of 1 at 4 °C for 1 h. After washing with cold PBS, infected cells were maintained in the presence or absence of U0126, either at 4 °C (red bars) or 37 °C (blue bars) for an additional hour. 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 ST cells. At 24 h post-incubation, internalized viruses were titrated by IFA. (B) Effect of ERK inhibition on viral RNA synthesis. ST cells pretreated with DMSO or U0126 were mock infected or infected with PDCoV (MOI of 1) for 1 h and then incubated in either DMSO or U0126. Total cellular RNA was extracted at 12 hpi, and strand-specific viral genomic RNAs (red bars) and sg mRNAs (blue bars) of PDCoV were amplified by quantitative real-time RT-PCR. Viral positive-sense genomic RNA and sg mRNA levels were normalized to those of porcine β-actin, and relative quantities (RQ) of accumulated mRNA were determined. The results obtained with U0126-treated samples were compared with those obtained with DMSO-treated samples. (C) Effect of ERK inhibition on viral protein translation. U0126-treated ST cells were mock infected or infected with PDCoV for 1 h and further cultivated in the presence or absence of U0126. At 12 hpi, cellular lysates were prepared, resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted using the antibody against the PDCoV N protein. The blot was also reacted with an anti β-actin antibody to verify equal protein loading. Viral protein expression was quantitatively estimated by densitometry and presented as the density value relative to that of the β-actin gene. U0126-treated sample results were compared to those of the vehicle control. (D) Effect of ERK inhibition on the intensity of viral protein expression. Under the same experimental conditions as in panel C, the PDCoV-infected cells were trypsinized at 12 hpi and subjected to FACS analysis using an anti-PDCoV N antibody to determine the mean fluorescence intensity. The results of U0126-treated samples were compared to those of DMSO-treated control. (E) Effect of ERK inhibition on viral progeny production. ST cells were pretreated with DMSO or U0126 for 1 h and were mock or PDCoV infected (MOI of 1). Vehicle or U0126 was present in the medium throughout the infection. At 24 hpi, virus-containing culture supernatants were collected, and PDCoV titers were determined. (F) Effect of ERK inhibition on viral growth kinetics. PDCoV reproduction after treatment with U0126 was assessed exactly as in panel A. At the indicated time points post-infection, culture supernatants were harvested and virus titers were measured. Results are expressed as the mean values of three independent experiments, and error bars represent standard deviations. *P <  0.05; **P <  0.001.
Fig. 9
Fig. 9
Depletion of cellular or viral cholesterol modulates PDCoV-induced ERK activation. Effects of cholesterol sequestration after treatment of cells (A) or virions (B) with MβCD (1 and 3 mM) on PDCoV-induced ERK activation and viral progeny production were quantitatively determined at 6 hpi using a FACE assay and virus titration under the same experimental conditions as in Figs. 4C and 8E, respectively. Values are representative of the mean from three independent experiments, and error bars denote the mean ± SDM. *P < 0.05; **P < 0.001.
Fig. 10
Fig. 10
Inhibition of ERK1/2 activation does not influence PDCoV-induced apoptosis. (A) ST cells were treated with DMSO or U0126 (100 μM) for 1 h prior to infection and then mock infected or infected with PDCoV in the presence of DMSO or U0126. The cells harvested at the indicated time points were subjected to dual Annexin V and PI labeling and analyzed by FACS. Lower left quadrants represent intact cells (Annexin V negative/PI negative); Lower right quadrants represent early apoptotic cells (Annexin V positive/PI negative); Upper right quadrants indicate late apoptotic or/and necrotic cells (Annexin V positive/PI positive); and Upper left quadrants indicate necrotic cells (Annexin V negative/PI positive). The figure is representative of three independent experiments. (B) The graph represents the percentage of each quadrant, and the non-significant percentages of Annexin V-negative and PI-positive cells were excluded. (C) The U0126-treated and infected cells were labeled with MitoTracker Red CMXRos (red), fixed at 12 hpi, and independently incubated with a primary antibody against Bax or cyt c (green). The cells were then counterstained with DAPI and examined under a confocal microscope at 800× magnification. PDCoV-specific CPEs were also photographed at 12 hpi under an inverted microscope at the magnification of 200× (left panels). Bax mitochondrial translocation is represented as the merger of Bax and mitochondrial marker (yellow), while the residual cytosolic localization is indicated by single staining signal (green). Conversely, cyt c cytosolic translocation is represented by single staining signal (green), and residual mitochondrial accumulation is indicated as the merger of cyt c and MitoTracker (yellow). The inset images are enlarged versions of parts of the picture.
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