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. 2024 Sep 13:15:1382675.
doi: 10.3389/fimmu.2024.1382675. eCollection 2024.

African swine fever virus MGF360-4L protein attenuates type I interferon response by suppressing the phosphorylation of IRF3

Zhen Wang  1   2 , Yuheng He  2 , Ying Huang  2 , Wenzhu Zhai  2 , Chunhao Tao  2 , Yuanyuan Chu  2 , Zhongbao Pang  2 , Hongfei Zhu  2 , Peng Zhao  1 , Hong Jia  2
Affiliations

African swine fever virus MGF360-4L protein attenuates type I interferon response by suppressing the phosphorylation of IRF3

Zhen Wang et al. Front Immunol. .

Abstract

African swine fever (ASF) is a highly contagious and lethal disease of swine caused by African swine fever virus (ASFV), and the mortality rate caused by virulent stains can approach 100%. Many ASFV viral proteins suppress the interferon production to evade the host's innate immune responses. However, whether ASFV MGF360-4L could inhibit type I interferon (IFN-I) signaling pathway and the underlying molecular mechanisms remain unknown. Our study, indicated that ASFV MGF360-4L could negatively regulates the cGAS-STING mediated IFN-I signaling pathway. Overexpressing ASFV MGF360-4L could inhibit the cGAS/STING signaling pathway by inhibiting the interferon-β promoter activity, which was induced by cGAS/STING, TBK1, and IRF3-5D, and further reduced the transcriptional levels of ISG15, ISG54, ISG56, STAT1, STAT2, and TYK2. Confocal microscopy and immunoprecipitation revealed that MGF360-4L co-localized and interacted with IRF3, and WB revealed that ASFV MGF360-4L suppressed the phosphorylation of IRF3. 4L-F2 (75-162 aa) and 4L-F3 (146-387 aa) were the crucial immunosuppressive domains and sites. Altogether, our study reveals ASFV MGF360-4L inhibited cGAS-STING mediated IFN-I signaling pathways, which provides insights into an evasion strategy of ASFV involving in host's innate immune responses.

Keywords: African swine fever virus; MGF360-4L; phosphorylation; suppress; type I interferon.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
ASFV MGF360-4L suppressed IFN-β transcription mediated by the cGAS/STING pathway. (A) HEK293T cells were transfected with ASFV MGF360-4L-Flag, treated with 1 µg/ml poly (dA:dT), and then harvested. The IFN-β mRNA levels were then detected. (B, C) HEK293T cells were co-transfected with IFN-β-luc (20 ng) or NF-κB-luc (20 ng), pRL-TK (2 ng), cGAS-HA (10 ng), STING-HA (40 ng), and MGF360-4L-Flag (100 ng). After 24 h of transfection, promoter activation was detected using DLR assay kits, and the expression of cGAS-HA, STING-HA, and MGF360-4L-Flag was examined using western blot assay. **, p<0.01; and ***, p<0.001.
Figure 2
Figure 2
ASFV MGF360-4L inhibited IFN-β promoter activity and the mRNA levels of IFN-β in a dose-dependent manner. HEK293T cells were co-transfected with cGAS-HA, STING-HA and p3×Flag-CMV-7.1 empty vector were the control comparison group. (A) HEK293T cells were co-transfected with IFN-β-luc (20 ng), pRL-TK (2 ng), cGAS-HA (40 ng), and STING-HA (160 ng), along with increased amounts of ASFV MGF360-4L-flag (0 ng, 50 ng, 100 ng, 200 ng). At 24 h post-transfection, the expression of cGAS-HA, STING-HA, and MGF360-4L-Flag was detected using western blotting. Promoter activity was measured using a DLR kit. (B) HEK293T cells were co-transfected with cGAS-HA (40 ng) and STING-HA (160 ng), along with increased amounts of ASFV MGF360-4L-flag (0 ng, 50 ng, 100 ng, 200 ng) or the p3×Flag-CMV-7.1 empty vector, and the mRNA levels of IFN-β were then detected using qPCR. **, p<0.01; ***, p<0.001; and ****, p<0.0001.
Figure 3
Figure 3
ASFV MGF360-4L reduced the mRNA levels of ISG15, ISG54 and ISG56. (A–C) HEK293T cells were transfected with MGF360-4L-Flag (200 ng) expression plasmid or the p3×Flag-CMV-7.1 empty vector (200 ng). At 16 h post-transfection, cells were treated with 2′3′-cGAMP (2 µg/ml) for another 12 h, then harvested and lysed, followed by RT-qPCR detection of ISG15 (A), ISG54 (B), and ISG56 (C) mRNA. (D–F) PK-15 cells were transfected with MGF360-4L-Flag (200 ng) expression plasmid or the p3×Flag-CMV-7.1 empty vector (200 ng). At 16 h post-transfection, cells were treated with 2′3′-cGAMP (2 µg/ml) for another 12 h, then harvested and lysed, followed by RT-qPCR detection of ISG15 (D), ISG54 (E), and ISG56 (F) mRNA. *, p<0.05; and ****, p<0.0001.
Figure 4
Figure 4
ASFV MGF360-4L suppressed the transcription levels of STAT1, STAT2, and TYK2. (A–E) HEK293T cells were transfected with MGF360-4L-Flag (200 ng) expression plasmid or the p3×Flag-CMV-7.1 empty vector (200 ng). At 16 h post-transfection, cells were treated with 2′3′-cGAMP (2 µg/ml) for another 12 h. Total RNA was then extracted from cell lysates, followed by RT-qPCR detection of JAK1 (A), STAT1 (B), STAT2 (C), STAT3 (D), and TYK2 (E) mRNA. (F–J) PK-15 cells were transfected with MGF360-4L-Flag (200 ng) expression plasmid or the p3×Flag-CMV-7.1 empty vector (200 ng). At 16 h post-transfection, cells were treated with 2′3′-cGAMP (2 µg/ml) for another 12 h. Total RNA was then extracted from cell lysates, followed by RT-qPCR detection of JAK1 (F), STAT1 (G), STAT2 (H), STAT3 (I), and TYK2 (J) mRNA. *, p<0.05; **, p<0.01; and ****, p<0.0001.
Figure 5
Figure 5
Effects of MGF360-4L on the cGAS/STING signaling mediated anti-VSV activity. (A–E) HEK293T cells were cultured overnight to 80% confluence. They were then co-transfected with cGAS-HA (40 ng), STING-HA (160 ng), and MGF360-4L-Flag (200 ng) for 16 h. The cells were infected with VSV-GFP for 8 h. (A–C) The mRNA levels of VSV, INF-β, and ISG56 were detected using RT-qPCR. The GFP signals from the VSV replicates were observed using fluorescence microscopy (E) and analyzed using western blotting (D). *, p<0.05; and ***, p<0.001.
Figure 6
Figure 6
ASFV MGF360-4L inhibited the IFN- β promoter activity induced by TBK1 and IRF3-5D. (A, B) HEK 293T cells were co-transfected with IFN-β-luc (20 ng), pRL-TK (2 ng), MGF360-4L-Flag (200 ng) and TBK1-Flag (200 ng) or IRF3-5D-HA (200 ng), then harvested and lysed. Promoter activation was detected using DLR assay kits, and the expression of cGAS-HA, STING-HA, and MGF360-4L was examined using western blot assay. ***, p<0.001, and ****, p<0.0001.
Figure 7
Figure 7
ASFV MGF360-4L inhibited the cGAS/STING pathway by suppressing the phosphorylation of IRF3. (A) HEK293T cells were co-transfected with cGAS-HA (40 ng), STING-HA (160 ng) and ASFV MGF360-4L-Flag (200 ng). After 24 h of transfection, the expression of cGAS-HA, STING-HA, and MGF360-4L-Flag was examined using western blot assay. (B) HEK293T cells were co-transfected with cGAS-HA (40 ng), and STING-HA (160 ng), along with increased amounts of ASFV MGF360-4L-flag (0 ng, 50 ng, 100 ng, 200 ng). At 24 h post-transfection, the expression of cGAS-HA, STING-HA, and MGF360-4L-Flag was detected using western blotting. (C) PK-15 cells were co-transfected with cGAS-HA (40 ng), STING-HA (160 ng) and ASFV MGF360-4L-Flag (200 ng). After 24 h of transfection, the expression of cGAS-HA, STING-HA, and MGF360-4L-Flag was examined using western blot assay.
Figure 8
Figure 8
MGF360-4L interacted and co-localized with IRF3. (A) MGF360-4L interacted with IRF3 in the overexpression system. HEK 293T cells were transfected with the indicated plasmids (5 µg each). IgG was used for control immunoprecipitation. Co-immunoprecipitation and immunoblotting analyses were performed with the indicated Abs. (B, C) Co-localization of MGF360-4L with IRF3. HEK 293T (B) cells and PK-15 cells (C) were transfected with MGF360-4L-Flag and IRF3-HA expression plasmids. After 24 h of transfection, MGF360-4L-Flag and IRF3-HA were stained with mouse-anti-Flag (green) and rabbit-anti-HA (red), and the nuclei were stained with DAPI (blue). Confocal assays were performed using Leica TCS SP8.
Figure 9
Figure 9
Regions of ASFV MGF360-4L responsible for inhibitory activity. (A–C) Luciferase assays in HEK293T cells were performed to detect the activation of the IFN-β promoter following the expression of empty vector, cGAS-HA and STING-HA, TBK1-Flag, or IRF3-5D-HA in the presence of empty vector, and full-length MGF360-4L-Flag, 4L-F1-Flag (1-88 aa), 4L-F2-Flag (75-162 aa), or 4L-F3-Flag (146-387 aa) plasmids. (D) HEK293T cells were co-transfected with cGAS-HA, STING-HA, and full-length MGF360-4L-Flag, 4L-F1-Flag (1-88 aa), 4L-F2-Flag (75-162 aa), or 4L-F3-Flag (146-387 aa) plasmids. At 24 h post-transfection, the mRNA levels of IFN-β were detected using RT-qPCR. *, p<0.05; **, p<0.01; and ***, p<0.001.
Figure 10
Figure 10
Schematic diagram of the mechanism of MGF360-4L-mediated IFN-β signaling inhibition. ASFV MGF360-4L interacted with IRF3 and suppressed the phosphorylation of IRF3 to help the virus escape the host immune response.
See this image and copyright information in PMC

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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the National Key Research and Development Program of China (2021YFD1801200) and the Yunnan Key Research and Development Project (202103AC100001), the Earmarked Fund for CARS (CARS36), and the Agricultural Science and Technology Innovation Program (no. ASTIP‐IAS‐11).

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