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Cyclic GMP-AMP synthase (cGAS) is a key sensor of double-stranded DNA (dsDNA), initiating oligomerization and phase separation to drive immune responses against pathogens and endogenous damage. Porcine circovirus (PCV) induces immunosuppression, heightening susceptibility to secondary infections, but the underlying mechanisms remain unclear. Here, we report PCV type 2d (PCV2d) infection fails to induce type I interferons (IFN-I) and significantly suppresses IFN-I production upon poly (dA:dT) stimulation in a dose-dependent manner. Mechanistically, the replication-related protein (Rep) proteins of PCV2, PCV3 and PCV4 inhibit cGAS-mediated IFN-I induction by competitively binding dsDNA, thereby disrupting cGAS oligomerization and phase separation. Interestingly, Rep also suppresses mitochondria DNA-induced cGAS activation. We further identify Rep residues Q12 and R199-W202 as key regions facilitating dsDNA binding. Our findings reveal a previously unrecognized mechanism by which circovirus Rep antagonizes cGAS activation, providing new insights into PCV-induced immunosuppression.

RNA viruses cause numerous infectious diseases in humans and animals. The crosstalk between RNA viruses and the innate DNA sensing pathways attracts increasing attention. Recent studies showed that the cGAS-STING pathway plays an important role in restricting RNA viruses via mitochondria DNA (mtDNA) mediated activation. However, the mechanisms of cGAS mediated innate immune evasion by RNA viruses remain unknown. Here, we report that seneca valley virus (SVV) protease 3C disrupts mtDNA mediated innate immune sensing by cleaving porcine cGAS (pcGAS) in a species-specific manner. Mechanistically, a W/Q motif within the N-terminal domain of pcGAS is a unique cleavage site recognized by SVV 3C. Three conserved catalytic residues of SVV 3C cooperatively contribute to the cleavage of pcGAS, but not human cGAS (hcGAS) or mouse cGAS (mcGAS). Additionally, upon SVV infection and poly(dA:dT) transfection, pcGAS and SVV 3C colocalizes in the cells. Furthermore, SVV 3C disrupts pcGAS-mediated DNA binding, cGAMP synthesis and interferon induction by specifically cleaving pcGAS. This work uncovers a novel mechanism by which the viral protease cleaves the DNA sensor cGAS to evade innate immune response, suggesting a new antiviral approach against picornaviruses.

Alpha-herpesviruses, including pseudorabies virus (PRV) and herpes simplex virus type 1 (HSV-1), cause severe diseases in a wide range of hosts. However, the precise mechanisms of immune evasion by alpha-herpesviruses remains elusive, hindering the development of broad-spectrum antiviral vaccines and drugs. Here, we demonstrate that the immediate early protein US1, encoded by alpha-herpesviruses, directly interacts with cGAS, suppressing its dsDNA binding and enzymatic activity. Structural analysis using AlphaFold reveals a conserved overlapping region within PRV and HSV-1 US1 proteins. Deletion of these peptides leads to increased cGAS-mediated IFN-β production. Meanwhile, both synthetic and purified SUMO-fused US1 peptides significantly inhibit cGAS activity across species, with the SUMO-fused US1 peptides directly binding to the catalytic domain of cGAS. Both US1-deficient viruses (PRV-ΔUS1 and HSV-1-ΔUS1) exhibit higher IFN-β production and enhanced signaling through the cGAS-STING pathway. Importantly, mice infected with PRV-ΔUS1 or HSV-1-ΔUS1 show increased IFN-β secretion and reduced viral loads. In conclusion, overlapping peptides from US1 protein of alpha-herpesviruses antagonize cGAS-mediated innate immune responses, highlighting a promising target for the development of broad-spectrum inhibitors to counteract herpesvirus infections.

Inflammasomes play pivotal roles in inflammation by processing and promoting the secretion of IL-1β. Caspase-1 is involved in the maturation of IL-1β and IL-18, while human caspase-4 specifically processes IL-18. Recent structural studies of caspase-4 bound to Pro-IL-18 reveal the molecular basis of Pro-IL-18 activation by caspase-4. However, the mechanism of caspase-1 processing of pro-IL-1β and other IL-1β-converting enzymes remains elusive. Here, we observed that swine Pro-IL-1β (sPro-IL-1β) exists as an oligomeric precursor unlike monomeric human Pro-IL-1β (hPro-IL-1β). Interestingly, Seneca Valley Virus (SVV) 3C protease cleaves sPro-IL-1β to produce mature IL-1β, while it cleaves hPro-IL-1β but does not produce mature IL-1β in a specific manner. When the inflammasome is blocked, SVV 3C continues to activate IL-1β through direct cleavage in porcine alveolar macrophages (PAMs). Through molecular modeling and mutagenesis studies, we discovered that the pro-domain of sPro-IL-1β serves as an ’exosite’ with its hydrophobic residues docking into a positively charged 3C protease pocket, thereby directing the substrate to the active site. The cleavage of sPro-IL-1β generates a monomeric and active form of IL-1β, initiating the downstream signaling. Thus, these studies provide IL-1β is an inflammatory sensor that directly detects viral protease through an independent pathway operating in parallel with host inflammasomes.

Inflammasomes play pivotal roles in inflammation by processing and promoting the secretion of IL-1[beta]. Caspase-1 is involved in the maturation of IL-1[beta] and IL-18, while human caspase-4 specifically processes IL-18. Recent structural studies of caspase-4 bound to Pro-IL-18 reveal the molecular basis of Pro-IL-18 activation by caspase-4. However, the mechanism of caspase-1 processing of pro-IL-1[beta] and other IL-1[beta]-converting enzymes remains elusive. Here, we observed that swine Pro-IL-1[beta] (sPro-IL-1[beta]) exists as an oligomeric precursor unlike monomeric human Pro-IL-1[beta] (hPro-IL-1[beta]). Interestingly, Seneca Valley Virus (SVV) 3C protease cleaves sPro-IL-1[beta] to produce mature IL-1[beta], while it cleaves hPro-IL-1[beta] but does not produce mature IL-1[beta] in a specific manner. When the inflammasome is blocked, SVV 3C continues to activate IL-1[beta] through direct cleavage in porcine alveolar macrophages (PAMs). Through molecular modeling and mutagenesis studies, we discovered that the pro-domain of sPro-IL-1[beta] serves as an 'exosite' with its hydrophobic residues docking into a positively charged 3C protease pocket, thereby directing the substrate to the active site. The cleavage of sPro-IL-1[beta] generates a monomeric and active form of IL-1[beta], initiating the downstream signaling. Thus, these studies provide IL-1[beta] is an inflammatory sensor that directly detects viral protease through an independent pathway operating in parallel with host inflammasomes.