Innate immune response and autophagy flux in MERS-CoV-MA-WT and MERS-CoV-MA-Δ4b infected MRC-5 cells after the autophagy pathway was chemically inhibited.

mRNA expression levels of genes related to autophagy pathway and pro-inflammatory response were quantified by RT-qPCR in MRC-5 cells either mock-infected or infected with MERS-CoV-MA-WT or MERS-CoV-MA-Δ4b at a m.o.i of 1 for 24 h p.i after the treatment (+ 3-MA) or not (- 3-MA) with 5mM 3-methyladenine for 16 h. Bars represent means and error bars represent standard deviations (n = 3). Mock, Mock-infected cells. Differences were analyzed by Student’s t test: *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001. Asterisks on the columns represent significant differences between mock-treated and cells.</p

Analysis of autophagy pathway during infection with MERS-CoV-MA-WT or MERS-CoV-MA-Δ4b viruses in MRC-5 cells.

MRC-5 cells were mock-infected or infected with the WT or Δ4b viruses at a m.o.i. of 1. The amount of p62 (A) and ULK1 (B) at 24 h p.i. was quantified from Western blots. Actin or GAPDH were used as loading controls. (C) Analysis of rRNA degradation pattern at 24 h p.i. or post-transfection with poly I:C in MRC-5 cells using a Bioanalyzer. Representative results from one experiment out of three (m.o.i. of 1 PFU; n = 3) are shown.</p

Innate immune response to MERS-CoV-MA-Δ4b in mice.

hDPP4-KI mice were infected with 5x104 PFU of MERS-CoV-MA-WT, MERS-CoV-MA-Δ4b or MERS-CoV-MA-mNLS virus. Mice were sacrificed at 4 and 6 dpi and the RNA of the lungs was extracted for gene expression assays. mRNA expression levels of genes related to the IFN response (A), the pro-inflammatory response (B), and autophagy (C) were quantified by RT-qPCR. Error bars represent standard deviations of the mean (n = 3). Differences between groups were analyzed by Student’s t test: *, p-value < 0.1; **, p-value < 0.01.</p

Taqman assays.

Taqman assays.</p

Characterization of MERS-CoV-MA-WT and MERS-CoV-MA-Δ4b in MRC-5 cells.

(A) Subconfluent monolayers of MRC-5 were infected with wild-type or Δ4b at a m.o.i. of 0.1 or 0.001. Culture supernatants were collected at 24, 48 and 72 h p.i. and titrated by plaque assay. The average of two independent experiments is represented. (B) The mRNA expression levels of genes related to the IFN or the pro-inflammatory responses were quantified by RT-qPCR in MRC-5 cells either mock-infected or infected with WT or Δ4b viruses at m.o.i. 1 and 24 h p.i. Error bars represent standard deviations of the mean (n = 3). Differences were analyzed by Student’s t test: *, p-value < 0.05; **, p-value < 0.01.</p

Virulence of MERS-CoV-MA-Δ4b and MERS-CoV-MA-mNLS in mice.

30-week-old hDPP4-KI mice were intranasally inoculated with 5x104 pfu of MERS-CoV-MA-WT (WT), MERS-CoV-MA-Δ4b (Δ4b) or MERS-CoV-MA-mNLS (mNLS) viruses. Weight loss (A) and survival (B) were monitored for 12 days. Error bars represent standard deviations of the mean (n = 5 mice per group). (C) Representative images of pulmonary histopathological lesions (H&E staining; magnification 10x) observed in mice euthanized at 4 and 6 dpi. Alveolar septal thickening, perivascular and peribronchiolar cuffs populated by lymphocytes (black arrows) and alveolar edema (red arrowheads) were among the most characteristic inflammatory lesions observed. (D) Scores associated with edema and cell infiltrates at 4 and 6 dpi. (E) Viral titers in the lungs were determined by plaque assay at 4 and 6 dpi (n = 3).</p

Proposed mechanism of action of 4b protein in MERS-CoV virulence.

During MERS-CoV-WT infection, 4b protein accumulates in the nucleus, while in the absence of a functional nuclear localization signal (mNLS), 4b protein is retained in the cytoplasm. The presence of either nuclear or cytoplasmatic 4b is associated with activation of inflammation and inhibition of autophagy, which contribute to virulence in vivo, as shown for MERS-CoV- WT and MERS-CoV-mNLS in a mouse model of infection. According to this work, autophagy contributes to limit inflammation. In the absence of 4b protein, autophagy was activated and a lower inflammatory response was induced, which contributes to attenuation. Chemokine mRNAs increased both in MERS-CoV-WT and MERS-CoV-mNLS infection are indicated in orange. Cytokines mRNA differentially induced in MERS-CoV-WT infection are shown in red. Changes in protein levels are indicated in green.</p

Characterization of MERS-CoV-MA-Δ4b and MERS-CoV-MA-mNLS mutants.

(A) Huh-7 cells were infected with MERS-CoV-MA, MERS-CoV-MA-Δ4b or MERS-CoV-MA-mNLS at a m.o.i. of 0.1 for 24 h and the localization of 4b (in green) was analyzed by confocal microscopy. (B) Subconfluent monolayers of Huh-7 were infected with wild-type (black), Δ4b (red) or mNLS (purple) at a m.o.i. of 0.1 or 0.001. Culture supernatants were collected at 24, 48, 72 and 96 h p.i. and titrated by plaque assay. The average of two independent experiments is represented. (C) Pro-inflammatory response induced in Huh-7 by the infection with WT, Δ4b or mNLS. RNA was collected at 24 h p.i and quantified by RT-qPCR. Error bars represent standard deviations of the mean (n = 3). Differences with WT group were analyzed by Student’s t test: *, p-value < 0.05; **, p-value < 0.01.</p

Subcellular localization of NF-κB during infection with MERS-CoV 4b-NLS mutants.

<p>Huh-7 (A) or Calu-3 (B) cells were mock-infected or infected (MOI = 0.1 PFU/cell) with WT, Δ4b or 4b-NLS mutants. At 18 hpi, cells were fractionated into cytoplasmic (C) and nuclear (N) fractions and analyzed by Western-blot for 4b and p65 detection. GAPDH and histone H3 were used as cytoplasmic and nuclear markers, respectively. Huh-7 (C) or Calu-3 (D) cells were infected with WT, Δ4b or 4b-NLS mutants (MOI 0.1 PFU/cell). At 24 hpi, cells were fixed and stained with antibodies against 4b (green) and p65 (red). Cell nuclei were stained with DAPI (blue).</p

Interference of MERS-CoV 4b protein in NF-κB-karyopherin 4 binding during infection.

<p>Huh-7 cells were transfected with a plasmid encoding karyopherin 4 (KPNA4)-FLAG. At 24 hpt, cells were infected (MOI 0.1 PFU/cell) with WT, Δ4b or 4b-NLS mutants. At 20 hpi, cells lysates were immunoprecipitated with anti-FLAG antibodies. Cell lysates (CL) and eluted proteins were analyzed by immunoblotting with indicated antibodies.</p