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.

The sequencing of ASFV WT and ASFV E120R Δ72-73aa recombinant virus genomes was performed using next-generation sequencing (NGS), as previously described. Briefly, purified DNA was used to construct Illumina standard shotgun library followed the NEBNext Ultra DNA Library Prep Kit (NEB, Catalog no. E7370), and then was sequenced using the Illumina NovaSeq 6000 platform with PE150 model.

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