Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jan 28;18(1):e1010270.
doi: 10.1371/journal.ppat.1010270. eCollection 2022 Jan.

African swine fever virus I267L acts as an important virulence factor by inhibiting RNA polymerase III-RIG-I-mediated innate immunity

Yong Ran  1   2 , Dan Li  3   4 , Mei-Guang Xiong  1   2   5 , Hua-Nan Liu  3   4 , Tao Feng  3   4 , Zheng-Wang Shi  3   4 , Yu-Hui Li  1   2   5 , Huang-Ning Wu  1   2   5 , Su-Yun Wang  1   2 , Hai-Xue Zheng  3   4 , Yan-Yi Wang  1   2
Affiliations

African swine fever virus I267L acts as an important virulence factor by inhibiting RNA polymerase III-RIG-I-mediated innate immunity

Yong Ran et al. PLoS Pathog. .

Abstract

ASFV is a large DNA virus that is highly pathogenic in domestic pigs. How this virus is sensed by the innate immune system as well as why it is so virulent remains enigmatic. In this study, we show that the ASFV genome contains AT-rich regions that are recognized by the DNA-directed RNA polymerase III (Pol-III), leading to viral RNA sensor RIG-I-mediated innate immune responses. We further show that ASFV protein I267L inhibits RNA Pol-III-RIG-I-mediated innate antiviral responses. I267L interacts with the E3 ubiquitin ligase Riplet, disrupts Riplet-RIG-I interaction and impairs Riplet-mediated K63-polyubiquitination and activation of RIG-I. I267L-deficient ASFV induces higher levels of interferon-β, and displays compromised replication both in primary macrophages and pigs compared with wild-type ASFV. Furthermore, I267L-deficiency attenuates the virulence and pathogenesis of ASFV in pigs. These findings suggest that ASFV I267L is an important virulence factor by impairing innate immune responses mediated by the RNA Pol-III-RIG-I axis.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. AT-rich regions of the ASFV genome induce IFN-β.
(A) Comparison of the total AT-content of the ASFV genome with genomes of HSV-1, EBV and VZV. (B) The content and distribution of AT-rich regions in the genomes of ASFV were compared with those in the genomes of HSV-1 and VZV using the EMBOSS Isochore algorithm. Regions with AT content more than 70% are shown. (C) Effects of knockdown of cGAS or RIG-I on IFNB1 transcription induced by ASFV or HSV-1 infection. PAMs (1×106) were transfected with the indicated siRNA (final concentration, 25 nM each) for 48 hours, and then the cells were infected with ASFV or HSV-1 (MOI = 0.1) for 4 hours. The mRNA levels of IFNB1, cGAS, and RIG-I were analyzed by qPCR. (D) The AT-rich DNA of the ASFV genome activates the IFN-β promoter. HEK293T (1×105) were transfected with the IFN-β promoter luciferase plasmid. Twenty-four hours later, these cells were stimulated with transfected dsDNA as indicated (1 μg/ml) for 10 hours before luciferase assays were performed. ASFV-AT and ASFV-GC are short for ASFV/DNA-AT and ASFV/DNA-GC respectively. (E) qPCR analysis of mRNA levels of the indicated genes stimulated by transfected dsDNA (1 μg/ml) for 6 hours in HEK293T and PK-15 cells.
Fig 2
Fig 2. AT-rich regions of the ASFV genome activate RNA Pol-III-RIG-I axis.
(A) RNAs extracted from poly(dA:dT)-, ASFV-AT-, ASFV-GC (1 μg/ml) or mock-transfected PK-15 cells (2×106) were treated with RNase A or DNase I at 37°C for 1 hour. The RNAs were then transfected into HEK293T (2 μg/ml) that transfected with IFN-β-luciferase reporter. Six-hours later, luciferase assays were performed. The flow diagram of experiment was shown on the left. ASFV-AT indicates ASFV-AT-#1 hereafter. (B) PAMs (2×106) were mock infected or infected with ASFV (MOI = 0.1) for the indicated times. RNAs were extracted and treated with DNase (200 U/ml/mg RNA) at 37°C for 1 hour. The RNAs were then transfected into HEK293T (2 μg/ml) that transfected with IFN-β-luciferase reporter. Six-hours later, luciferase assays were performed. (C) PK-15 cells or PAMs (1×106) were pre-treated with the indicated concentrations of the RNA Pol-III inhibitor ML-60218 for 6 hours before transfection with poly(dA:dT), ASFV-AT, ASFV-GC or poly(I:C) (1 μg/ml) for 4 hours. The mRNA levels of IFNB1 were analyzed by RT-qPCR. (D) PAMs (2×106) were pre-treated with ML-60218 (50 μM) for 6 hours before infection with ASFV (MOI = 0.1) for the indicated times. The mRNA levels of IFNB1, ISG15, ISG54, and MX1 were analyzed by RT-qPCR. (E) The effects of RIG-I-deficiency on poly(dA:dT)-, ASFV-AT- or poly(I:C)-stimulated IFN-β induction. Two sgRNA were used to knockdown RIG-I in PK-15 cells using CRISPR/Cas9 method (sgNT, non-targeting sgRNA). The mRNA levels of IFNB1 were analyzed by RT-qPCR after the cells were stimulated with transfected DNA or RNA analogs poly(I:C) for 6 hours. The knockdown efficiency of RIG-I was confirmed by western blot. (F) PK-15 cells (2×107) prepared as in (E) were transfected with ASFV-AT-#2 for 3 hours. RIP experiments were performed using the RIG-I antibody, and specific primers were used to detect the enrichment of ASFV-AT by RIG-I in cells. The expression of RIG-I in lysates and immunoprecipitations was detected by western blot analysis.
Fig 3
Fig 3. ASFV antagonizes RNA Pol-III-RIG-I axis.
(A) ASFV infection inhibits poly(dA:dT)-induced transcription of IFNB1, IL6 and TNFA. PAMs (2×106) were uninfected or infected with ASFV (MOI = 0.1) for the indicated times, and then were stimulated with transfected poly(dA:dT) (1 μg/ml) for 4 hours before RT-qPCR were performed. (B) Screening of ASFV proteins that inhibits poly(dA:dT)-induced activation of the IFN-β promoter. HEK293T cells were transfected with plasmids of individual ASFV cDNA (0.1 μg), the IFN-β luciferase reporter (0.05 μg) and TK reporter (0.01 μg/ml). Twenty-four hours later, cells were left untreated or stimulated with transfected poly(dA:dT) (1 μg/ml) for 10 hours before luciferase assays were performed. The proteins that showed more than two-fold inhibition are labeled in the figure. (C) The effects of I267L on activation of the IFN-β promoter stimulated by transfected ASFV-AT, poly(dA:dT), and poly(I:C) (1 μg/ml). The experiments were similarly performed as in (B). (D) PK-15 and THP-1 cells (1×106) stably transduced with vector or I267L were stimulated with transfected ASFV-AT, poly(dA:dT), and poly(I:C) (1 μg/ml) for 6 hours before RT-qPCR were performed. (E) THP-1(1×106) stably transduced with vector or I267L were infected with SeV or HSV-1 (MOI = 0.1) for 6 hours before qPCR was performed. (F) The effects of I267L encoded by ASFV genotype I (Ba71V) and genotype II (CN/GS/2018) on activation of the IFN-β promoter stimulated by transfected ASFV-AT, poly(dA:dT), and poly(I:C) (1 μg/ml). The experiments were similarly performed as in (B). (G) HEK293T cells (1×105) were co-transfected with the indicated reporters, expression plasmids and empty vectors or I267L expression plasmids (0.05 μg). Luciferase assays were performed 24 hours after transfection. pNifty-luciferase reporter: a reporter to indicate NF-κB activation, which contains five NF-κB binding sites. (H) HEK293T cells (1×105) were co-transfected with the indicated reporters, empty vectors or I267L expression plasmids (0.05 μg). Twenty-four hours later, cells were infected with SeV (MOI = 0.1) or EMCV (MOI = 1.0) for 12 hours before luciferase assays were performed.
Fig 4
Fig 4. ASFV I267L targets Riplet.
(A) HEK293T cells (5×106) were co-transfected with Flag-tagged I267L (5 μg) and HA-tagged RIG-I (5 μg) or HA-tagged Riplet (5 μg). Coimmunoprecipitation and immunoblotting were performed with the indicated antibodies. (B) HEK293T cells (5×106) were transfected with the indicated plasmids (5 μg). Twenty-four hours later, cell lysates were subjected to pull-down assay with in vitro purified GST or GST-I267L. (C) HEK293T cells (5×106) were co-transfected with plasmids of Flag-RIG-I and HA-Riplet (5 μg), and increasing dose plasmids of HA-I267L (0, 1.0, 5.0 μg). Coimmunoprecipitation and immunoblotting were performed with the indicated antibodies. (D) HEK293T cells (5×106) were transfected with plasmids as indicated. Twenty-four hours later, coimmunoprecipitation and immunoblotting were performed with the indicated antibodies. (E) HEK293T cells (5×106) were transfected with plasmids of Flag-RIG-I and HA-VISA, and increasing dose plasmids of HA-I267L. Coimmunoprecipitation and immunoblotting were performed with the indicated antibodies. (F) HEK293T cells (1×105) were co-transfected with the IFN-β promoter reporter, expression plasmids and empty vectors or I267L expression plasmids (0.05 μg) as indicated. (G) PK-15 cells (1×105) were transfected with control (NC) or Riplet-siRNA (50 nM). Forty-eight hours later, cells were stimulated with transfected poly(dA:dT), ASFV-AT, and poly(I:C) (1 μg/ml) for 6 hours before RT-qPCR were performed.
Fig 5
Fig 5. Generation of I267L-deficient virus.
(A) Schematic presentation of generation of I267L-deletion virus by homologous recombination. (B) Confirmation of the successfully recombination by fluorescence microscopy. (C) Absence of parental ASFV CN/GS/2018 in ASFVΔI267L virus stock was confirmed by PCR.
Fig 6
Fig 6. Deficiency of I267L attenuates the ability of ASFV to antagonize innate immune responses.
(A) Determining the expression level of I267L during ASFV infection by RT-qPCR. The p30 and p72 are shown as indicators of early gene and late gene respectively. (B) PAMs (1×106) were infected by ASFV-WT or ASFVΔI267L (MOI = 0.1) for 6 hours or 12 hours before RT-qPCR were performed. (C) PAMs (1×106) were infected by ASFV-WT or ASFVΔI267L (MOI = 0.1) for 24 hours or 36 hours. Virus DNA was extracted from the cells to determine the viral DNA copies of p72 by qPCR. (D) PAMs (2×106) were uninfected or infected with ASFV-WT or ASFVΔI267L (MOI = 0.1) for 6 hours, and then were stimulated with transfected poly(dA:dT) (1 μg/ml) for 4 hours before RT-qPCR were performed. (E) PAMs (2×107) were uninfected or infected with ASFV-WT or ASFVΔI267L (MOI = 0.1) for the indicated times. Immunoprecipitation and immunoblotting were performed with the indicated antibodies (2 μg anti-RIG-I antibody was used for each immunoprecipitation). (F) PAMs (2x107) were uninfected or infected with ASFV-WT or ASFVΔI267L (MOI = 0.1) for the indicated times. Immunoprecipitation and immunoblotting were performed with the indicated antibodies (2 μg IgG control or anti-RIG-I antibody was used for each immunoprecipitation). IgG-H indicates the heavy chain of IgG. (G) PAMs (5×106) were uninfected or infected with ASFV-WT or ASFVΔI267L (MOI = 0.1) for the indicated times. Cells were lysed and subjected to Western blot analysis for examination of the phosphorylation and expression of IRF3. The β-actin were used as loading control.
Fig 7
Fig 7. Deficiency of I267L attenuates virulence and pathogenesis of ASFV.
(A, B, C, D and E) Pigs were inoculated intramuscularly (i.m.) with either 10 HAD50 of ASFVΔI267L (n = 6) or ASFV-WT (n = 6). The pigs were monitored daily for 21 days for temperature and mortality. The expression level of IFN-β in serum at 5 days post challenge were determined by ELISA (A), the daily temperature changes of pigs were shown in panel (B), the survival rates were shown in panel (C), viral DNA copies in blood, oral swab, fecal swab, and nasal swab collected at 5, 10, 15, and 20 days post challenge were determined by qPCR and shown in panel (D), and the antibodies angaist p30 were determined using an in-house blocking ELISA (E).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Dixon LK, Stahl K, Jori F, Vial L, Pfeiffer DU. African Swine Fever Epidemiology and Control. Annu Rev Anim Biosci. 2020;8:221–46. doi: 10.1146/annurev-animal-021419-083741 - DOI - PubMed
    1. Blome S, Franzke K, Beer M. African swine fever–A review of current knowledge. Virus Research. 2020;287:198099. doi: 10.1016/j.virusres.2020.198099 - DOI - PubMed
    1. Wang F, Zhang H, Hou L, Yang C, Wen Y. Advance of African swine fever virus in recent years. Research in Veterinary Science. 2021;136:535–9. doi: 10.1016/j.rvsc.2021.04.004 - DOI - PubMed
    1. Dixon LK, Islam M, Nash R, Reis AL. African swine fever virus evasion of host defences. Virus Res. 2019;266:25–33. doi: 10.1016/j.virusres.2019.04.002 - DOI - PMC - PubMed
    1. Roberts NJ, Douglas RG, Simons RM, Diamond ME. Virus-Induced Interferon Production by Human Macrophages. The Journal of Immunology. 1979;123:365–9. - PubMed

Publication types

MeSH terms

Grants and funding

This work was supported by the African Swine Fever Research Emergency Program of the Chinese Academy of Sciences (KJZD-SW-L06) to Y.Y.W., the National Natural Science Foundation of China grants (U20A2059) to Y.R. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
-