African swine fever virus MGF505-11R inhibits type I interferon production by negatively regulating the cGAS-STING-mediated signaling pathway

African swine fever (ASF) is a devastating infectious disease caused by African swine fever virus (ASFV). The ASFV genome encodes multiple structural and non-structural proteins that contribute to evasion of host immunity. In this study, we determined that the viral non-structural protein MGF360-14L inhibits interferon-β (IFN-β) promoter activity induced by cGAS-STING signaling. MGF360-14L was also found to downregulate expression of the IRF3 protein and promote its degradation through ubiquitin-meditated proteolysis. Moreover, MGF360-14L was shown to interact with and destabilize IRF3 by facilitating E3 ligase TRIM21-mediated K63-linked ubiquitination of IRF3. Overall, our study revealed that MGF360-14L promotes degradation of IRF3 through TRIM21, thereby inhibiting type I interferon production. These findings provide new insights into the mechanisms underlying ASFV immune evasion.

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African swine fever virus E120R protein inhibits interferon-β production by interacting with IRF3 to block its activation

Journal of Virology ◽

10.1128/jvi.00824-21 ◽

2021 ◽

Author(s):

Huisheng Liu ◽

Zixiang Zhu ◽

Tao Feng ◽

Zhao Ma ◽

Qiao Xue ◽

...

Keyword(s):

Type I Interferon ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Interferon Regulatory Factor ◽

Fever Virus ◽

Type I ◽

Interferon Production ◽

Structural Protein ◽

Regulatory Factor ◽

Host Antiviral Response

African swine fever is a devastating disease of swine caused by African swine fever virus (ASFV). The pathogenesis of the disease remains largely unknown, leaving the uncontrolled spreading of the disease in many countries and regions. Here, we identified the E120R, a structural protein of ASFV, as a key virulent factor and late phase expression protein of the virus. E120R revealed an activity to suppress host antiviral response through blocking IFN-β production, and the 72-73 amino acid sites in the C-terminal domain were essential for this function. E120R interacted with the interferon regulatory factor 3 (IRF3) and interfered with the recruitment of IRF3 to TBK1, which in turn suppressed IRF3 phosphorylation, decreasing interferon production. The recombinant mutant ASFV was further constructed to confirm the claimed mechanism. The ASFV lacking the complete E120R region could not be rescued, whereas the virus could tolerate the deletion of the 72nd and 73rd residuals in the E120R (ASFV E120R-Δ72-73aa). ASFV E120R with the two amino acids deletion failed to interact with IRF3 during ASFV E120R-Δ72-73aa infection, and the viral infection highly activated IRF3 phosphorylation and induced more robust type I interferon production in comparison with its parental ASFV. An unbiased transcriptome-wide analysis of gene expression also confirmed that a considerably higher level of ISGs was detected in ASFV E120R-Δ72-73aa-infected porcine alveolar macrophages (PAMs) than that in the wildtype ASFV-infected PAMs. Together, our findings found a novel mechanism evolved by ASFV to inhibit host antiviral response and provide a new target for guiding the development of ASFV live-attenuated vaccine. IMPORTANCE African swine fever is a highly contagious animal disease affecting pig industry worldwide, which has brought enormous economic losses. The causative agent African swine fever virus (ASFV) infection causes severe immunosuppression during viral infection, attributing to serious clinical manifestation. Therefore, identification of the viral proteins involved in immunosuppression is critical for ASFV vaccine design and development. Here, for the first time, we demonstrated that E120R protein, a structural protein of ASFV, played an important role in suppression of interferon regulatory factor 3 (IRF3) phosphorylation and type I interferon production by binding to IRF3 and blocking the the recruitment of IRF3 to TBK1. Deletion of the crucial binding sites in E120R critically increased interferon response during ASFV infection. This study explored a novel antagonistic mechanism of ASFV, which is critical for guiding the development of ASFV live-attenuated vaccines.

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Modulation of Type I Interferon System by African Swine Fever Virus

Pathogens ◽

10.3390/pathogens9050361 ◽

2020 ◽

Vol 9 (5) ◽

pp. 361 ◽

Cited By ~ 2

Author(s):

Elisabetta Razzuoli ◽

Giulia Franzoni ◽

Tania Carta ◽

Susanna Zinellu ◽

Massimo Amadori ◽

...

Keyword(s):

Type I Interferon ◽

African Swine Fever Virus ◽

Statistical Significance ◽

Expression Patterns ◽

African Swine Fever ◽

Fever Virus ◽

Type I ◽

Viral Dissemination ◽

Type I Ifns ◽

Interferon System

African Swine Fever Virus (ASFV) has tropism for macrophages, which seems to play a crucial role in disease pathogenesis and viral dissemination. Previous studies showed that ASFV developed mechanisms to evade type I interferon (IFN) responses. Hence, we analyzed the ability of ASFV strains of diverse virulence to modulate IFN-β and IFN-α responses. Porcine monocyte-derived macrophages un-activated (moMΦ) or activated with IFN-α (moMΦ + FN-α) were infected with virulent (22653/14) or attenuated (NH/P68) ASFV strains, and expressions of IFN-β and of 17 IFN-α subtypes genes were monitored over time. ASFV strains of diverse virulence induced different panels of IFN genes: infection of moMΦ with either strains caused statistically significant up-regulation of IFN-α3, -α7/11, whereas only attenuated NH/P68 determined statistically significant up-regulation of IFN-α10, -α12, -α13, -α15, -α17, and IFN-β. Infection of activated moMΦ with either strains resulted in up-regulation of IFN-β and many IFN-α subtypes, but statistical significance was found only for IFN-α1, -α10, -α15, -α16, -α17 in response to NH/P68-infection only. These data revealed differences in type I IFNs expression patterns, with differences between strains of diverse virulence. In addition, virulent 22653/14 ASFV seems to have developed mechanisms to suppress the induction of several type I IFN genes.

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Modulation of type I interferon signaling by African swine fever virus (ASFV) of different virulence L60 and NHV in macrophage host cells

Veterinary Microbiology ◽

10.1016/j.vetmic.2018.02.008 ◽

2018 ◽

Vol 216 ◽

pp. 132-141 ◽

Cited By ~ 6

Author(s):

Raquel Portugal ◽

Alexandre Leitão ◽

Carlos Martins

Keyword(s):

Type I Interferon ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Host Cells ◽

Type I ◽

Interferon Signaling

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Sensitivity of African swine fever virus to type I interferon is linked to genes within multigene families 360 and 505

Virology ◽

10.1016/j.virol.2016.03.019 ◽

2016 ◽

Vol 493 ◽

pp. 154-161 ◽

Cited By ~ 40

Author(s):

Josephine P. Golding ◽

Lynnette Goatley ◽

Steve Goodbourn ◽

Linda K. Dixon ◽

Geraldine Taylor ◽

...

Keyword(s):

Type I Interferon ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Multigene Families ◽

Type I

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African Swine Fever Virus Multigene Family Genes Inhibit the Type-I Interferon Response by Acting on the NFκB and IRF3 Signalling Pathways at the Transcription Factor Level or below

Proceedings ◽

10.3390/proceedings2020050057 ◽

2020 ◽

Vol 50 (1) ◽

pp. 57

Author(s):

Samuel Connell ◽

Ana Reis ◽

Anusyah Rathakrishnan ◽

Sarah Gilbert ◽

Linda Dixon

Keyword(s):

Transcription Factor ◽

Multigene Family ◽

Type I Interferon ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Interferon Response ◽

Signalling Pathways ◽

Type I ◽

Factor Level

African Swine Fever Virus (ASFV) is a haemorrhagic infection of swine, which routinelydisplays 100% lethality. [...]

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Deletion of the Gene for the Type I Interferon Inhibitor I329L from the Attenuated African Swine Fever Virus OURT88/3 Strain Reduces Protection Induced in Pigs

Vaccines ◽

10.3390/vaccines8020262 ◽

2020 ◽

Vol 8 (2) ◽

pp. 262 ◽

Cited By ~ 1

Author(s):

Ana Luisa Reis ◽

Lynnette C. Goatley ◽

Tamara Jabbar ◽

Elisabeth Lopez ◽

Anusyah Rathakrishnan ◽

...

Keyword(s):

Type I Interferon ◽

African Swine Fever Virus ◽

Clinical Signs ◽

African Swine Fever ◽

Type I ◽

Lethal Challenge ◽

Host Innate Immune Response ◽

Antibody Levels

Live attenuated vaccines are considered to be the fastest route to the development of a safe and efficacious African swine fever (ASF) vaccine. Infection with the naturally attenuated OURT88/3 strain induces protection against challenge with virulent isolates from the same or closely related genotypes. However, adverse clinical signs following immunisation have been observed. Here, we attempted to increase the OURT88/3 safety profile by deleting I329L, a gene previously shown to inhibit the host innate immune response. The resulting virus, OURT88/3ΔI329L, was tested in vitro to evaluate the replication and expression of type I interferon (IFN) and in vivo by immunisation and lethal challenge experiments in pigs. No differences were observed regarding replication; however, increased amounts of both IFN-β and IFN-α were observed in macrophages infected with the deletion mutant virus. Unexpectedly, the deletion of I329L markedly reduced protection against challenge with the virulent OURT88/1 isolate. This was associated with a decrease in both antibody levels against VP72 and the number of IFN-γ-producing cells in the blood of non-protected animals. Furthermore, a significant increase in IL-10 levels in serum was observed in pigs immunised with OURT88/3ΔI329L following challenge. Interestingly, the deletion of the I329L gene failed to attenuate the virulent Georgia/2007 isolate.

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African Swine Fever Virus Multigene Family 360 and 530 Genes Affect Host Interferon Response

Journal of Virology ◽

10.1128/jvi.78.4.1858-1864.2004 ◽

2004 ◽

Vol 78 (4) ◽

pp. 1858-1864 ◽

Cited By ~ 81

Author(s):

C. L. Afonso ◽

M. E. Piccone ◽

K. M. Zaffuto ◽

J. Neilan ◽

G. F. Kutish ◽

...

Keyword(s):

Viral Infection ◽

Multigene Family ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Cdna Clones ◽

Type I ◽

Mrna Levels ◽

Type I Ifn ◽

Transcriptional Responses

ABSTRACT African swine fever virus (ASFV) multigene family 360 and 530 (MGF360/530) genes affect viral growth in macrophage cell cultures and virulence in pigs (L. Zsak, Z. Lu, T. G. Burrage, J. G. Neilan, G. F. Kutish, D. M. Moore, and D. L. Rock, J. Virol. 75:3066-3076, 2001). The mechanism by which these novel genes affect virus-host interactions is unknown. To define MGF360/530 gene function, we compared macrophage transcriptional responses following infection with parental ASFV (Pr4) and an MGF360/530 deletion mutant (Pr4Δ35). A swine cDNA microarray containing 7,712 macrophage cDNA clones was used to compare the transcriptional profiles of swine macrophages infected with Pr4 and Pr4Δ35 at 3 and 6 h postinfection (hpi). While at 3 hpi most (7,564) of the genes had similar expression levels in cells infected with either virus, 38 genes had significantly increased (>2.0-fold, P < 0.05) mRNA levels in Pr4Δ35-infected macrophages. Similar up-regulation of these genes was observed at 6 hpi. Viral infection was required for this induced transcriptional response. Most Pr4Δ35 up-regulated genes were part of a type I interferon (IFN) response or were genes that are normally induced by double-stranded RNA and/or viral infection. These included monocyte chemoattractant protein, transmembrane protein 3, tetratricopeptide repeat protein 1, a ubiquitin-like 17-kDa protein, ubiquitin-specific protease ISG43, an RNA helicase DEAD box protein, GTP-binding MX protein, the cytokine IP-10, and the PKR activator PACT. Differential expression of IFN early-response genes in Pr4Δ35 relative to Pr4 was confirmed by Northern blot analysis and real-time PCR. Analysis of IFN-α mRNA and secreted IFN-α levels at 3, 8, and 24 hpi revealed undetectable IFN-α in mock- and Pr4-infected macrophages but significant IFN-α levels at 24 hpi in Pr4Δ35-infected macrophages. The absence of IFN-α in Pr4-infected macrophages suggests that MGF360/530 genes either directly or indirectly suppress a type I IFN response. An inability to suppress host type I IFN responses may account for the growth defect of Pr4Δ35 in macrophages and its attenuation in swine.

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pMGF505-7R determines pathogenicity of African swine fever virus infection by inhibiting IL-1β and type I IFN production

PLoS Pathogens ◽

10.1371/journal.ppat.1009733 ◽

2021 ◽

Vol 17 (7) ◽

pp. e1009733

Author(s):

Jiangnan Li ◽

Jie Song ◽

Li Kang ◽

Li Huang ◽

Shijun Zhou ◽

...

Keyword(s):

Immune Responses ◽

Inflammatory Responses ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Innate Immune ◽

Fever Virus ◽

Type I ◽

Type I Ifn ◽

Innate Immune Responses

Inflammatory factors and type I interferons (IFNs) are key components of host antiviral innate immune responses, which can be released from the pathogen-infected macrophages. African swine fever virus (ASFV) has developed various strategies to evade host antiviral innate immune responses, including alteration of inflammatory responses and IFNs production. However, the molecular mechanism underlying inhibition of inflammatory responses and IFNs production by ASFV-encoded proteins has not been fully understood. Here we report that ASFV infection only induced low levels of IL-1β and type I IFNs in porcine alveolar macrophages (PAMs), even in the presence of strong inducers such as LPS and poly(dA:dT). Through further exploration, we found that several members of the multigene family 360 (MGF360) and MGF505 strongly inhibited IL-1β maturation and IFN-β promoter activation. Among them, pMGF505-7R had the strongest inhibitory effect. To verify the function of pMGF505-7R in vivo, a recombinant ASFV with deletion of the MGF505-7R gene (ASFV-Δ7R) was constructed and assessed. As we expected, ASFV-Δ7R infection induced higher levels of IL-1β and IFN-β compared with its parental ASFV HLJ/18 strain. ASFV infection-induced IL-1β production was then found to be dependent on TLRs/NF-κB signaling pathway and NLRP3 inflammasome. Furthermore, we demonstrated that pMGF505-7R interacted with IKKα in the IKK complex to inhibit NF-κB activation and bound to NLRP3 to inhibit inflammasome formation, leading to decreased IL-1β production. Moreover, we found that pMGF505-7R interacted with and inhibited the nuclear translocation of IRF3 to block type I IFN production. Importantly, the virulence of ASFV-Δ7R is reduced in piglets compared with its parental ASFV HLJ/18 strain, which may due to induction of higher IL-1β and type I IFN production in vivo. Our findings provide a new clue to understand the functions of ASFV-encoded pMGF505-7R and its role in viral infection-induced pathogenesis, which might help design antiviral agents or live attenuated vaccines to control ASF.

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