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Interferon-γ-Directed Inhibition of a Novel High-Pathogenic Phlebovirus and Viral Antagonism of the Antiviral Signaling by Targeting STAT1

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Interferon-γ-Directed Inhibition of a Novel High-Pathogenic Phlebovirus and Viral Antagonism of the Antiviral Signaling by Targeting STAT1

Yun-Jia Ning et al. Front Immunol.

Abstract

Severe fever with thrombocytopenia syndrome (SFTS) is a life-threatening infectious disease caused by a novel phlebovirus, SFTS virus (SFTSV). Currently, there is no vaccine or antiviral available and the viral pathogenesis remains largely unknown. In this study, we demonstrated that SFTSV infection results in substantial production of serum interferon-γ (IFN-γ) in patients and then that IFN-γ in turn exhibits a robust anti-SFTSV activity in cultured cells, indicating the potential role of IFN-γ in anti-SFTSV immune responses. However, the IFN-γ anti-SFTSV efficacy was compromised once viral infection had been established. Consistently, we found that viral nonstructural protein (NSs) expression counteracts IFN-γ signaling. By protein interaction analyses combined with mass spectrometry, we identified the transcription factor of IFN-γ signaling pathway, STAT1, as the cellular target of SFTSV for IFN-γ antagonism. Mechanistically, SFTSV blocks IFN-γ-triggered STAT1 action through (1) NSs-STAT1 interaction-mediated sequestration of STAT1 into viral inclusion bodies and (2) viral infection-induced downregulation of STAT1 protein level. Finally, the efficacy of IFN-γ as an anti-SFTSV drug in vivo was evaluated in a mouse infection model: IFN-γ pretreatment but not posttreatment conferred significant protection to mice against lethal SFTSV infection, confirming IFN-γ's anti-SFTSV effect and viral antagonism against IFN-γ after the infection establishment. These findings present a picture of virus-host arm race and may promote not only the understanding of virus-host interactions and viral pathogenesis but also the development of antiviral therapeutics.

Keywords: IFN-γ; NSs; STAT1; antiviral immunity; immune evasion; inclusion body; severe fever with thrombocytopenia syndrome virus (SFTSV); virus-host interaction.

Figures

Figure 1
Figure 1
Levels of IFN-γ in SFTS patients and healthy individuals. IFN-γ in serum samples from SFTS cases (n = 33) and healthy donors (n = 17) was detected by ELISA. Each dot represents the IFN concentration in an individual. Horizon bars indicate the respective group mean. ****P < 0.0001.
Figure 2
Figure 2
IFN-γ suppresses SFTSV infection in vitro. (A) HepG2 cells were treated with IFN-γ (200 ng/ml) for 6 h prior to the infection with SFTSV (MOI = 3). Twenty-four hours post infection (p.i.), cells were fixed for immunofluorescence assay (IFA) using the antibody against NP. Nuclei were stained with Hoechst 33258 as shown in blue. (B) Cells were pretreated with the indicated doses of IFN- γ and then infected with SFTSV (MOI = 0.1). At 24 h p.i., cells were fixed for IFA as in (A). Percentages of infected cells in the IFN-γ-treated groups were normalized to the infection percentage of the untreated group. (C) Cells were pretreated with IFN-γ prior to the infection of SFTSV. At 24 h p.i., virus titers in the culture media were measured by the TCID50 method. (D) Cells were infected with SFTSV and then treated with IFN-γ at 4 h p.i. Twenty-four hours post infection, cells were fixed and treated as in (A). (E) Cells were treated with the indicated doses of IFN-γ at 4 h following SFTSV infection. Relative infection ratios were calculated as in (B). (F) Cells were treated with IFN-γ at 4 h following SFTSV infection. At 24 h p.i., virus titers in the culture media were measured as in (C). Graphs show means ± standard deviation (SD), n = 3. *P < 0.05; ***P < 0.001.
Figure 3
Figure 3
SFTSV NSs antagonizes IFN-γ signaling. (A,B) HEK293 cells were cotransfected with the reporter plasmid of IFN-γ-responsive promoter and the Renilla luciferase control plasmid (pRL-TK), along with an empty control plasmid (vector) or the NSs expression plasmid. Twenty-four hours after transfection, cells were treated with IFN-γ or left untreated for 16 h, followed by the measurement of luciferase activities. Relative luciferase activity (Rel. Luc. Act.) (A) and the fold activation (over the untreated controls) (B) were presented, respectively. (C,D) HepG2 cells were transfected with the NSs expression plasmid or the vector and at 24 h post transfection, cells were treated with IFN-γ for 10 h or left untreated, followed by the analyses of OAS2 and IP-10 mRNA expression with real-time quantitative PCR. Graphs show means ± SD, n = 3. **P < 0.01.
Figure 4
Figure 4
Identification of STAT1 as the NSs target for IFN-γ signaling suppression. (A) Results of mass spectrum analysis. The purified NSs-associated proteins or the agarose bead-binding products (control) were subjected to LC-MS/MS analysis. STAT1, the key transcription factor in IFN-γ signaling pathway, was specifically identified in the NSs coprecipitates. The tandem spectra of two representative peptides (identified with >99% confidence) of STAT1 (accession, NP_009330) were shown. (B) Validation of the NSs-STAT1 interaction. HEK293 cells were transfected with the control plasmid (vector) or plasmids encoding NSs-S or NP-S. At 24 h post-transfection, protein interactions were examined by pulldown assay. Subsequently, pulldown products and cell lysates (lysate input) were subjected to WB analyses using the indicated antibodies.
Figure 5
Figure 5
NSs blocks IFN-γ-induced nuclear translocation of STAT1 by trapping STAT1 into IBs. (A,B) HEK293 cells transfected with the NSs expression plasmid were left untreated or treated with IFN-γ (as indicated) for 30 min at 48 h posttransfection and then fixed to visualize the expression and localization of NSs (green) and endogenous STAT1 (red) by IFA and confocal microscopy. Nuclei stained with Hoechst were shown in blue. Asterisks indicate the blockade of STAT1 nuclear accumulation in NSs-expressing cells. Arrows in the enlarged images of the dotted box areas show the sequestration of STAT1 into NSs IBs. An intensity line graph in the lower right corner of each panel shows the signal intensity of the green and red channels along the line in the enlarged merged images. To better visualize the NSs-positive cells, over-exposed images of the green/NSs channel were also presented. (C) Cells with or without NSs expression from the experiments of (A,B) were respectively scored for STAT1 nuclear translocation. Approximately 100 cells were counted for each group. Percentages of cells with noticeable STAT1 nuclear accumulation were shown, respectively.
Figure 6
Figure 6
SFTSV antagonizes IFN-STAT1 signaling via sequestration of STAT1 into NSs IBs and down-regulation of STAT1 abundance. (A–C) HEK293 cells infected with SFTSV were left untreated (A) or treated with IFN-γ (B) or IFN-α (C) for 30 min at 48 hpi and fixed for visualizing the expression and localization of NSs (green) and endogenous STAT1 (red) by IFA. Nuclei were stained with Hoechst. Asterisks indicate the deprivation of STAT1 nuclear accumulation in SFTSV-infected cells. Arrows in the enlarged panels show the sequestration of STAT1 into SFTSV NSs IBs. The intensity line graphs show the green and red signal intensity along the white lines in enlarged merged images. Over-exposed images of the green/NSs channel were also presented to visualize all the infected cells. (D) Cells with or without NSs expression from the experiments of (A–C) were respectively scored for STAT1 nuclear translocation. Percentages of cells with noticeable STAT1 nuclear import were shown.
Figure 7
Figure 7
Down-regulation of STAT1 abundance at the protein level by SFTSV infection but not expression of NSs or the other viral proteins. (A–D) HEK293 cells were mock infected or infected with SFTSV (A), or transfected with the vector plasmid (pCAGGS) or NSs expression plasmid (pCAGGS-NSs) (C), and collected at the indicated time points post infection or transfection for WB analyses. Using the ImageJ software, protein band intensities of STAT1 and STAT2 from (A,C) were measured and then normalized to those of β-actin, as shown in (B,D), respectively. Note that the relative band intensities at 0 h post infection or transfection were set to 1 as indicated by the dotted reference lines. (E) HEK293 cells transfected with the S-tagged STAT1 (STAT1-S) expression plasmid or empty vector were infected with SFTSV or mock infected. At 24 h p.i., cells were lysed for S-pulldown assays, followed by WB analysis with the indicated antibodies. (F) Cells were transfected with plasmids encoding the indicated viral proteins or the control vector. At 72 h posttransfection, cells were harvested and subjected to WB analysis using antibodies against the indicated proteins. (G) Relative band intensities of STAT1 from (F) were analyzed using ImageJ. Dotted reference line indicates the ordinate value 1. (H) HEK293 cells were mock infected or infected with SFTSV for the indicated times. Relative mRNA levels of the cellular proteins in the infected cells (normalized to mock-infected groups at the corresponding time points) were analyzed by real-time quantitative PCR. B-cell CLL/lymphoma factor (BCL), a virus-induced gene control. Dotted line indicates the ordinate value 1 for reference. Data are shown as means ± SD, n = 3.
Figure 8
Figure 8
IFN-γ pretreatment confers protection to mice against lethal SFTSV infection in vivo. (A,B) Indicated doses of IFN-γ were administered to suckling mice (n ≥ 9/treatment) 24 h prior to infection of SFTSV (1.5×103 TCID50). (C,D) Indicated doses of IFN-γ were administered 24 h following SFTSV inoculation (n ≥ 8/treatment). Control animals were injected with culture medium instead of virus. The survival rates and body weights of survived mice were monitored and recorded at the indicated times following virus challenge. Dotted line represents the average weight of the neonatal mice for virus challenge. Statistical conclusions obtained from Mantel-Cox log-rank and Gehan-Breslow-Wilcoxon tests were unanimous compared to the virus-infected group without IFN-γ treatment, *P < 0.05.
Figure 9
Figure 9
Model for the interplays between SFTSV infection and IFN-γ-STAT1 signaling. SFTSV infection leads to the substantial production of IFN-γ which can direct anti-SFTSV action through induction of antiviral ISGs and other immune or inflammatory factors by IFN-γ-STAT1 signaling. In turn, the host anti-SFTSV response mounted by IFN-γ can be counteracted by the virus through the following two mechanisms: (1) NSs IB sequestration of STAT1 by the NSs-STAT1 interaction and (2) virus infection-induced downregulation of STAT1 protein abundance. Together, these viral and cellular actions reflect a complex arm race between SFTSV and its host, shedding lights on the virus-host interactions and viral pathogenesis.

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