Species-specific cleavage of cGAS by picornavirus protease 3C disrupts mitochondria DNA-mediated immune sensing

Abstract

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 approaches against picornaviruses.

The antiviral response triggered by the cGAS/STING pathway is subverted by the foot-and-mouth disease virus proteases

“…5a, b). It is worth mentioning that Yan et al [ 30 ] also failed to detect the swine cGAS C-terminal cleavage fragment generated by 3Cpro during SVV infection in HEK293T cells stably expressing cGAS/STING. In our case, the combined cleavage by 3C and Lpro may contribute to the difficulty in detecting the cGAS fragments derived from the activity of the FMDV-encoded proteases.…”

“…However, taking into account that autophagy triggered by RE stress during FMDV infection has been associated with STING degradation [ 52 ], the contribution of this pathway to the decay of STING observed at late times after infection cannot be ruled out. Though detection of endogenous swine cGAS is known to be difficult [ 30 ], a rapid decrease in the protein levels at early times after infection (3–6 h pi) could be detected in SK6 and WSL cells. When WSL cells were infected with an Lpro-deficient FMDV mutant, the stability of the protein was significantly increased, confirming the role of the Leader protease in cGAS degradation.…”

“…By co-expression assays, we were able to precisely identify the cleavage sites for each protease. Though the autophagy-dependent degradation of swine cGAS during SVV infection had been suggested [ 31 ], while this work was in preparation, the cleavage of cGAS by the 3C protease of SVV between K138 and L139 was reported [ 30 ]. Interestingly, we identified the FMDV 3Cpro cleavage site on swine cGAS between Q140 and T141, partly overlapping with the specific motif W 137 KLQ 140 defined as SVV 3C target site, and consistent with the specificity differences documented for the 3C proteases encoded by different picornaviruses [ 53 ].…”

“…Using recombinant proteins and in vitro cleavage assays, Bhattacharya et al showed that the DENV NS2B3 protease is capable of cleaving human cGAS in the N-terminus potentially disrupting the C-terminal enzymatic activity of the protein [ 29 ]. Also, a recent work showed that the 3C protease encoded by the Seneca valley virus (SVV) can cleave porcine cGAS overexpressed in human cells [ 30 ]. Moreover, triggering of IFN-I responses has been associated to mitochondrial damage and DNA release during picornaviral infection and overexpression of the FMDV non-structural 2B protein induced mtDNA release to the cytosol in swine PK-15 cells [ 31 ].…”

Natural-Target-Mimicking Translocation-Based Fluorescent Sensor for Detection of SARS-CoV-2 PLpro Protease Activity and Virus Infection in Living Cells

“…This sensor could potentially be used for a plethora of viruses as MAVS was shown to be digested by proteases of cytomegalovirus [ 13 ], MERS [ 14 ], hepatitis A virus [ 15 , 16 ] and other infections [ 17 ]. Notably, similar sensors with other proteins of the antiviral response of a host cell targeted by infections were also described [ 18 ].…”