Studies on the inhibition of RIG-I mediated signaling by FAT10 protein

Abstract

As the first line of host defense against virus infection, antiviral innate immune system utilizes pattern recognition receptors to detect specific pathogens derived molecules. Cytosolic viral RNAs are recognized by RIG-I-like receptors, as consequence, RIG-I activation triggers production of IFNs and proinflammatory cytokines. Although RIG-I mediated immune-surveillance is critical for clearing pathogens, aberrant RIG-I activity leads to severe pathological states such as autosomal dominant multi-system disorder, inflammatory myophathies and dermatomyositis. Therefore, identification of regulators that ensure the double-edged function of RIG-I inflammatory response is particularly important to elucidate approaches for preventing and treatment of RIG-I-associated diseases. FAT10 is a UBL protein uniquely found in mammal, is well-known as a signal for proteasomal degradation. FAT10 is involved in several cellular processes that occur via either conjugation to target proteins or non-covalent association with other proteins. However, FAT10 contribution in host-virus interaction, especially in the innate immunity against infection has not been explored. Here, we presented the inflammatory inducible FAT10 as a novel negative regulator of RIG-I-mediated inflammatory response. In various cell lines, FAT10 protein level is undetectable unless it is induced by pro-inflammatory cytokines. We showed that FAT10 overexpression inhibited viral RNA-induced IRF3 and NF-kB activation in conjugation independent manner. Cellular fractionation and immunohistochemistry studies revealed that FAT10 inhibited RIG-I containing antiviral stress granules formation and restricted activated RIG-I targeting to mitochondria. Upon viral infection, overexpressed FAT10 elicited changes in RIG-I solubility, results in RIG-I translocation from cytosol to a detergent-insoluble fraction. FAT10 associated non-covalently with the RIG-I, but not with the natural splice variant RIG-I SV. The 2CARD domain of RIG-I protein was required for the interaction with FAT10. More specifically, RIG-I mutation at T55 residue, a known interaction site for E3 ligase TRIM25, blocked the interaction with FAT10 as well as translocation into the insoluble fraction. TRIM25 stabilized FAT10, an extremely unstable protein, thereby, strengthens inhibitory effect of FAT10 on RIG-I activity. As TRIM25 has a dual function, coordinated interplay of RIG-I, TRIM25 and FAT10 adds another layer of regulation to maintain antiviral innate inflammatory response in check. Our study presented a novel mechanism to dampen RIG-I activity, thus, create requisite checkpoint of RIG-I mediated inflammatory response. Highly accumulated FAT10 is observed in various cancers with pro-inflammatory environment, therefore, our finding which uncovered the role of accumulated FAT10 in antiviral response may also provide the molecular clue to understand the carcinogenesis related with infection and inflammation. Suppressive effect of the accumulated FAT10 during inflammation might also contribute to establishment of persistent infection that initiates chronic infection-triggered pathology.