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, 11 (2)

Interferon Regulatory Factor 3-Mediated Signaling Limits Middle-East Respiratory Syndrome (MERS) Coronavirus Propagation in Cells From an Insectivorous Bat

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Interferon Regulatory Factor 3-Mediated Signaling Limits Middle-East Respiratory Syndrome (MERS) Coronavirus Propagation in Cells From an Insectivorous Bat

Arinjay Banerjee et al. Viruses.

Abstract

Insectivorous bats are speculated to be ancestral hosts of Middle-East respiratory syndrome (MERS) coronavirus (CoV). MERS-CoV causes disease in humans with thirty-five percent fatality, and has evolved proteins that counteract human antiviral responses. Since bats experimentally infected with MERS-CoV do not develop signs of disease, we tested the hypothesis that MERS-CoV would replicate less efficiently in bat cells than in human cells because of its inability to subvert antiviral responses in bat cells. We infected human and bat (Eptesicus fuscus) cells with MERS-CoV and observed that the virus grew to higher titers in human cells. MERS-CoV also effectively suppressed the antiviral interferon beta (IFNβ) response in human cells, unlike in bat cells. To determine if IRF3, a critical mediator of the interferon response, also regulated the response in bats, we examined the response of IRF3 to poly(I:C), a synthetic analogue of viral double-stranded RNA. We observed that bat IRF3 responded to poly(I:C) by nuclear translocation and post-translational modifications, hallmarks of IRF3 activation. Suppression of IRF3 by small-interfering RNA (siRNA) demonstrated that IRF3 was critical for poly(I:C) and MERS-CoV induced induction of IFNβ in bat cells. Our study demonstrates that innate antiviral signaling in E. fuscus bat cells is resistant to MERS-CoV-mediated subversion.

Keywords: IRF3; MERS-CoV; bat; interferon.

Conflict of interest statement

The authors declare no competing interest.

Figures

Figure 1
Figure 1
MERS-CoV replication is attenuated in bat cells and does not inhibit IFNβ responses in these cells. To assess if MERS-CoV would replicate at the same rate in human and bat cells, we infected human (MRC5, A549, CaLu3, Huh7) and bat (Efk3, Tb1-Lu) cell lines with MERS-CoV and assessed viral replication at several time-points (by TCID50/mL). Transcript levels for IFNβ and TNFα were quantified by qRT-PCR at the indicated time-points. (A) MERS-CoV replication in human (MRC5) and bat (Efk3) cells that were infected with a low multiplicity of infection (MOI) of 0.01 infectious unit/cell (mean ± SD, n = 3). (B) MERS-CoV replication in human (MRC5) and bat (Efk3) cells infected with a high MOI of 10 infectious units/cell (mean ± SD, n = 3). (C) MERS-CoV replication in human lung (A549, CaLu3 and MRC5) and liver (Huh7) cells that were infected with an MOI of 0.01 infectious units/cell (mean ± SD, n = 2). (D) MERS-CoV replication in insectivorous bat kidney (Efk3) and lung (Tb1-Lu) cells that were infected with an MOI of 0.01 infectious unit/cell (mean ± SD, n = 2). (E) Putative MERS-CoV receptor, dipeptidyl peptidase 4 (DPP4) transcript levels in MRC5 and Efk3 cells (mean ± SD, n = 2). (F) IFNβ transcript levels at different times after MERS-CoV infection in Efk3 and MRC5 cells (mean ± SD, n = 4). (G) TNFα transcript levels at several time points in MERS-CoV infected MRC5 and Efk3 cells (mean ± SD, n = 4). (H) Cytopathic effects (CPE) observed in MRC5 and Efk3 cells twenty-four hours after MERS-CoV infection (MOI = 10). qRT-PCR results are represented as fold increases over mock-infected cells, normalized to GAPDH values (see Methods). Statistical significance was calculated using the Mann Whitney U test for two independent samples. SD = standard deviation. n = number of biological replicates.
Figure 2
Figure 2
MERS-CoV causes visible cytopathic effects in human cells but not in bat cells. Cytopathic effects in human (A549, Huh7, MRC5 and CaLu3) and bat (Efk3 and Tb1-Lu) cells that were infected with MERS-CoV with an MOI of 0.01 infectious unit/cell. Arrows indicate visible cytopathic effects. hpi = hours post infection.
Figure 3
Figure 3
Human and bat IRF3 localize to the nucleus of the cell following poly(I:C) treatment. To determine if bat IRF3, like human IRF3, responded to poly(I:C)-mediated activation by post-translational modification and nuclear translocation, we performed immunofluorescent microscopy and immunoblots on poly(I:C)-treated and mock-treated cells. (A) The cellular location of endogenous IRF3 in mock and poly(I:C) treated human (MRC5) and bat (Efk3) cells. IRF3 is stained red. GAPDH is stained green to highlight the cellular cytoplasm and the nucleus is stained blue. (B) Mean IRF3 fluorescence ratio (nucleus:cytoplasm) in MRC5 and Efk3 cells (mean ± SD, n = 5). (C) Immune blots of nuclear and cytoplasmic fractions of mock and poly(I:C) treated MRC5 and Efk3 cells. C = cytoplasmic fraction, N = nuclear fraction, arrow = higher molecular weight IRF3, calnexin = cytoplasmic marker, and lamin = nuclear marker. Statistical significance was calculated using the Mann Whitney U test for two independent samples. * p < 0.05. SD = standard deviation. n = number of fields. For the original, full size blots see Figure S1.
Figure 4
Figure 4
IRF3 is required for IFNβ signaling in response to poly(I:C) in human and bat cells. To determine the role of IRF3 in antiviral interferon signaling, we partially knocked-down IRF3 in both bat (Efk3) and human (MRC5) cells and quantified the increase in IFNβ transcripts in these cells in response to poly(I:C) by quantitative real-time PCR (qRT-PCR). To further demonstrate the dependency of poly(I:C)-mediated expression of IFNβ on IRF3 in Efk3 cells, we generated IRF3 knockout bat cells and quantified the increase in IFNβ transcripts in these cells in response to poly(I:C) by quantitative real-time PCR (qRT-PCR). (A) Fold increase in IFNβ transcript levels in IRF3 knocked-down Efk3 cells (siIRF3) and negative control siRNA (ncsiRNA) treated cells on stimulation with poly(I:C) (mean ± SD, n = 4). (B) Fold increase in IFNβ transcript levels in IRF3 knocked-down MRC5 cells (siIRF3) and negative control siRNA (ncsiRNA) treated cells on stimulation with poly(I:C) (mean ± SD, n = 4). (C) To generate IRF3 knockout Efk3 cells, we deleted a portion of the first exon in the genomic DNA using CRISPR-Cas9. This schematic represents the deletion in the first exon of IRF3 in Efk3-c3-8 cells, one of the IRF3 deleted cell lines. “ATG” in the wildtype cells (Efk3-WT) marks the start codon of IRF3. Similar nucleotides are indicated by “*”. (D) Fold increase in IFNβ transcript levels in IRF3 knocked-out bat cells (IRF3-KO cells) and wildtype bat cells (Efk3-WT). IRF3 knockdown and knockout were confirmed by immune blots (protein panel; Figures A,B,D). Results are represented as fold increases over mock poly(I:C) transfected cells, normalized to GAPDH values (see Methods). Statistical significance was calculated using the Mann Whitney U test for two independent samples. * p < 0.05. SD = standard deviation. n = number of biological replicates. For full size blots, see Figures S2 and S3.
Figure 5
Figure 5
Reduction (knock-down) or deletion (knock-out) of IRF3 increases levels of MERS-CoV and decreases IFNβ transcripts in big brown bat cells. To determine if IRF3 mediated signaling is crucial for limiting MERS-CoV replication in big brown bat cells, we knocked-down IRF3 in bat (Efk3) and human (MRC5) cells and infected them with MERS-CoV. A multiplicity of infection (MOI) of 0.01 or 0.1 infectious unit/cell was used for virus propagation studies and an MOI of 10 infectious units/cell was used for cytokine studies. We quantified virus replication in these cells by the TCID50 assay 48 h post infection (hpi) and transcripts for IFNβ and OAS1 by qRT-PCR 24 hpi. (A) MERS-CoV titers in MRC5 and Efk3 cells that were either treated with human or bat siIRF3 (IRF3 knockdown), respectively, or ncsiRNA (mock/no IRF3 knockdown) 48 hpi (mean ± SD, n = 4). IRF3 knockdown in MRC5 and Efk3 cells by siRNA was confirmed by immune blots (protein panel). An MOI of 0.01 infectious unit/cell was used for MERS-CoV infections. (B) Fold change in IFNβ transcript levels in siIRF3 (IRF3 knockdown) or ncsiRNA (mock/no IRF3 knockdown) treated Efk3 and MRC5 cells infected with MERS-CoV (mean ± SD, n = 4). An MOI of 10 infectious units/cell was used for the infections. (C) Fold change in interferon stimulated gene (OAS1) transcript levels in siIRF3 (IRF3 knockdown) or ncsiRNA (mock/no IRF3 knockdown) treated Efk3 and MRC5 cells infected with MERS-CoV (mean ± SD, n = 4). An MOI of 10 infectious units/cell was used for the infections. (D) Cytopathic effect (CPE) observed in siIRF3 (IRF3 knockdown) or ncsiRNA (mock/no IRF3 knockdown) treated Efk3 and MRC5 cells 48 hpi with MERS-CoV. (E) MERS-CoV titer in bat (Efk3), human (MRC5) and IRF3 knockout bat (cr3-8) cells 48 hpi with an MOI of 0.1 infectious unit/cell (mean ± SD, n = 4). (F) MERS-CoV titer in wildtype bat (Efk3) and IRF3 deleted bat (cr3-8) cells infected with an MOI of 0.01 infectious unit/cell over a period of 72 h. qRT-PCR results are represented as fold increases over mock infected cells, normalized to GAPDH values (see Methods). Statistical significance was calculated using the Mann Whitney U test for two independent samples. * p < 0.05. SD = standard deviation. n = number of biological replicates. For full size blots, see Figure S4.
Figure 6
Figure 6
Bat IRF3 nucleotide sequences are divergent from their human counterpart. IRF3 nucleotide sequences for several other mammals and big brown bat IRF3 sequence (bold) were aligned to identify similarities. The maximum likelihood tree for IRF3 nucleotide sequences (1000 Bootstrap) is represented here. Bar represents nucleotide substitution per site. For IRF3 nucleotide sequence information, see Table 2.

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