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. 2016 Aug 26;90(18):8281-92.
doi: 10.1128/JVI.01091-16. Print 2016 Sep 15.

Porcine Epidemic Diarrhea Virus Infection Inhibits Interferon Signaling by Targeted Degradation of STAT1

Longjun Guo  1 , Xiaolei Luo  1 , Ren Li  1 , Yunfei Xu  1 , Jian Zhang  1 , Jinying Ge  1 , Zhigao Bu  1 , Li Feng  1 , Yue Wang  2
Affiliations

Porcine Epidemic Diarrhea Virus Infection Inhibits Interferon Signaling by Targeted Degradation of STAT1

Longjun Guo et al. J Virol. .

Abstract

Porcine epidemic diarrhea virus (PEDV) is a worldwide-distributed alphacoronavirus, but the pathogenesis of PEDV infection is not fully characterized. During virus infection, type I interferon (IFN) is a key mediator of innate antiviral responses. Most coronaviruses develop some strategy for at least partially circumventing the IFN response by limiting the production of IFN and by delaying the activation of the IFN response. However, the molecular mechanisms by which PEDV antagonizes the antiviral effects of interferon have not been fully characterized. Especially, how PEDV impacts IFN signaling components has yet to be elucidated. In this study, we observed that PEDV was relatively resistant to treatment with type I IFN. Western blot analysis showed that STAT1 expression was markedly reduced in PEDV-infected cells and that this reduction was not due to inhibition of STAT1 transcription. STAT1 downregulation was blocked by a proteasome inhibitor but not by an autophagy inhibitor, strongly implicating the ubiquitin-proteasome targeting degradation system. Since PEDV infection-induced STAT1 degradation was evident in cells pretreated with the general tyrosine kinase inhibitor, we conclude that STAT1 degradation is independent of the IFN signaling pathway. Furthermore, we report that PEDV-induced STAT1 degradation inhibits IFN-α signal transduction pathways. Pharmacological inhibition of STAT1 degradation rescued the ability of the host to suppress virus replication. Collectively, these data show that PEDV is capable of subverting the type I interferon response by inducing STAT1 degradation.

Importance: In this study, we show that PEDV is resistant to the antiviral effect of IFN. The molecular mechanism is the degradation of STAT1 by PEDV infection in a proteasome-dependent manner. This PEDV infection-induced STAT1 degradation contributes to PEDV replication. Our findings reveal a new mechanism evolved by PEDV to circumvent the host antiviral response.

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Figures

FIG 1
FIG 1
PEDV infection is resistant to IFN-α. (A) Vero E6 cells were infected either with NDV-GFP at an MOI of 0.01, as a control, or with PEDV at an MOI of 0.1 for 24 h. Subsequently, 1,000 IU/ml or 10,000 IU/ml of IFN-α was added to the medium. At 24 h post-IFN-α treatment, PEDV-infected cells were stained with a MAb to PEDV spike protein as described in Materials and Methods. NDV-GFP-infected cells were observed directly under a fluorescence microscope. (B) PEDV-infected Vero E6 cells were grown for 24 h in the presence of various concentrations of IFN-α as indicated. Supernatant samples were collected, and titers were determined. Data represent the means of duplicate measurements (±SD) of virus titers from three independent experiments. *, P < 0.05. The P value was calculated using Student's t test.
FIG 2
FIG 2
STAT1, but not STAT2, is downregulated in PEDV-infected cells. (A and B) PEDV infection reduces the expression levels of STAT1, but not STAT2, in Vero E6 and IPEC-J2 cells. Cells were infected with PEDV at an MOI of 0.1 for 24 h or 48 h. Detergent lysates collected from Vero E6 (A) or IPEC-J2 (B) cells were directly subjected to reducing SDS-PAGE and immunoblotting with antibodies to STAT1, STAT2, or β-actin (loading control). Densitometric data for STAT1/actin, STAT2/actin, and STAT1/N ratios from three independent experiments are expressed as means ± SD. (C) An IFA and a Western blot assay verified that UV-inactivated PEDV was replication defective. PEDV S protein was stained with MAb 3F12, followed by FITC-conjugated goat anti-mouse IgG. Vero E6 cells were inoculated with a mock infection control, PEDV, or UV-inactivated PEDV at an MOI of 1 for 24 h. Cells were lysed and were analyzed by Western blotting with a MAb against PEDV N protein. (D) The levels of STAT1 protein were not reduced by UV-inactivated PEDV in cells. Vero E6 and IPEC-J2 cells were inoculated with UV-inactivated PEDV at an MOI of 0.1 for 24 h or 48 h. Western blotting was used to detect the levels of STAT1 protein. Densitometric data for STAT1/actin ratios from three independent experiments are expressed as means ± SD. *, P < 0.05. The P value was calculated using Student's t test.
FIG 3
FIG 3
PEDV infection activates the transcription of STAT1. Vero E6 and IPEC-J2 cells were infected with PEDV at an MOI of 0.1 for 24 h or 48 h. Cells were treated with IFN-α at 10 IU/ml as a positive control. Total RNA was extracted from cells, and STAT1 mRNA levels were assessed by quantitative RT-PCR using the primers listed in Table 1. Three independent experiments were performed in triplicate, and values are means ± SD for all three experiments. *, P < 0.05. The P value was calculated using Student's t test.
FIG 4
FIG 4
PEDV-induced STAT1 downregulation occurs through proteasome-mediated degradation but not through an autophagy mechanism. (A) Cells were first treated with the proteasome inhibitor MG132 or the carrier control DMSO for 1 h and were then either infected with PEDV at an MOI of 0.1 or left uninfected. Cells were further cultured in the absence or presence of MG132 for various times as indicated. Detergent lysates were collected from cells and were subjected to reducing SDS-PAGE and immunoblotting with a STAT1 antibody. Densitometric data for STAT1/actin ratios from three independent experiments are expressed as means ± SD. (B) PEDV infection induced the ubiquitination (Ub) of STAT1. Vero E6 cells were infected with PEDV at an MOI of 0.1 for 24 h. Detergent lysates were first immunoprecipitated with a STAT1 antibody as described in Materials and Methods and then subjected to reducing SDS-PAGE and immunoblotting with a rabbit polyclonal anti-Ub antibody. Detergent lysates blotted with the actin antibody were used as an input protein control. Densitometric data for Ub STAT1/actin ratios from three independent experiments are expressed as means ± SD. (C) Vero E6 and IPEC-J2 cells were treated either with 3-MA (5 mM) or with the carrier control DMSO for 4 h prior to PEDV infection. At 24 h or 48 h postinfection, cell lysates were subjected to blotting with a STAT1 antibody. Densitometric data for STAT1/actin ratios from three independent experiments are expressed as means ± SD. *, P < 0.05. The P value was calculated using Student's t test.
FIG 5
FIG 5
Established PEDV infection inhibits STAT1 phosphorylation. (A) Vero E6 and IPEC-J2 cells were infected with PEDV at an MOI of 0.1 for 12, 24, 30, 36, 48, or 55 h. Cell lysates were collected and were subjected to blotting with a phospho-STAT1 (Tyr701) antibody. Densitometric data for p-STAT1/actin ratios from three independent experiments are expressed as means ± SD. (B) Vero E6 and IPEC-J2 cells were first infected with PEDV at an MOI of 0.1 for 36 h and then treated with IFN-α (10 U/ml). At 30 min post-IFN-α treatment, cells were lysed and were subjected to Western blotting with the antibody against phospho-STAT1 (Tyr701). Densitometric data for p-STAT1/actin ratios from three independent experiments are expressed as means ± SD. *, P < 0.05. The P value was calculated using Student's t test.
FIG 6
FIG 6
Both phosphorylated and nonphosphorylated forms of STAT1 are degraded by PEDV. Vero E6 and IPEC-J2 cells were pretreated with the kinase inhibitor genistein (100 μM) or the carrier control DMSO for 1 h and were then incubated either with PEDV at an MOI of 0.1 or with a mock infection control. Cells were cultured in the presence or absence of genistein for a further 36 h. Detergent lysates collected from Vero E6 (A) and IPEC-J2 (B) cells were directly subjected to reducing SDS-PAGE and immunoblotting with a STAT1 antibody or a phospho-STAT1 (Tyr701) antibody. Densitometric data for p-STAT1/actin or STAT1/actin ratios from three independent experiments are expressed as means ± SD. *, P < 0.05. The P value was calculated using Student's t test.
FIG 7
FIG 7
PEDV inhibits the type I interferon pathway. Vero E6 and IPEC-J2 cells were either mock infected or infected with PEDV at an MOI of 0.1 for 36 h; they were then treated with IFN-α (10 U/ml) or IFN-γ (10 U/ml) for different times as indicated. Vero E6 (A) and IPEC-J2 (B) cells were collected and lysed and were then subjected to Western blot analysis with an anti-STAT1 antibody or an anti-phospho-STAT1 (Tyr701) antibody. Densitometric data for p-STAT1/actin ratios from three independent experiments are expressed as means ± SD. *, P < 0.05. The P value was calculated using Student's t test.
FIG 8
FIG 8
STAT1 degradation promotes PEDV replication. (A) Immunofluorescent assay to detect PEDV infection. Vero E6 cells were treated with MG132 at 5 μM for 1 h before inoculation with PEDV CV777. Cells were further cultured in the absence or presence of MG132 as described above. At 24 h or 48 h after inoculation, the cell monolayers were fixed and were examined for PEDV infection by an IFA with a MAb (3F12) against PEDV spike protein. (B) Blocking STAT1 degradation with MG132 inhibits PEDV replication. Vero E6 cells were treated with MG132 as mentioned above. At 24 h or 48 h postinfection, the progeny viruses were recovered from culture fluids. The virus titer was determined by the TCID50 assay. Results represent means ± SD for three independent experiments. (C and D) Blocking STAT1 degradation with bortezomib (C) or lactacystin (D) inhibits PEDV replication. Vero E6 cells were treated with bortezomib at 80 nM or lactacystin at 20 μM during PEDV infection. The virus titer was determined by the TCID50 assay. (E) Overexpression of STAT1 decreases PEDV replication. Vero E6 cells were transfected with the pcDNA3.1/STAT1 or pcDNA3.1 vector prior to PEDV inoculation. At 24 h posttransfection, Vero E6 cells were infected with PEDV for 24 h or 48 h. Results represent means ± SD for three independent experiments. *, P < 0.05. The P value was calculated using Student's t test.
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