PACT Facilitates RNA-Induced Activation of MDA5 by Promoting MDA5 Oligomerization

Abstract

MDA5 is a RIG-I-like cytoplasmic sensor of dsRNA and certain RNA viruses, such as encephalomyocarditis virus, for the initiation of the IFN signaling cascade in the innate antiviral response. The affinity of MDA5 toward dsRNA is low, and its activity becomes optimal in the presence of unknown cellular coactivators. In this article, we report an essential coactivator function of dsRNA-binding protein PACT in mediating the MDA5-dependent type I IFN response. Virus-induced and polyinosinic-polycytidylic acid-induced activation of MDA5 were severely impaired in PACT-knockout cells and attenuated in PACT-knockdown cells, but they were potentiated when PACT was overexpressed. PACT augmented IRF3-dependent type I IFN production subsequent to dsRNA-induced activation of MDA5. In contrast, PACT had no influence on MDA5-mediated activation of NF-κB. PACT required dsRNA interaction for its action on MDA5 and promoted dsRNA-induced oligomerization of MDA5. PACT had little stimulatory effect on MDA5 mutants deficient for oligomerization and filament assembly. PACT colocalized with MDA5 in the cytoplasm and potentiated MDA5 recruitment to the dsRNA ligand. Taken together, these findings suggest that PACT functions as an essential cellular coactivator of RIG-I, as well as MDA5, and it facilitates RNA-induced formation of MDA5 oligomers.

Middle East respiratory syndrome coronavirus-encoded ORF8b strongly antagonizes IFN-β promoter activation: its implication for vaccine design

“…PACT binds to dsRNA and recruit it to MDA5 or RIG-I. The authors claimed that ORF4a interrupt dsRNA-PACT interaction with the RLR's (Kok et al, 2011;Ho et al, 2016;Lui et al, 2017). Furthermore, when a recombinant MERS-CoV with ORF4a deletion infects cells, IFN induction was only marginally reduced (Comar et al, 2019).…”

Double-Stranded RNA Sensors and Modulators in Innate Immunity

“…Despite having a helicase domain (including Hel2i) closely related to that of Dicer, PACT was proposed to bind RIG-I CTD, not Hel2i, and to stimulate the ATPase activity of RIG-I (190). However, in an apparent contradiction to these findings, more recent studies have shown that PACT is specifically involved in MDA5-mediated antiviral signaling, not RIG-I-mediated signaling (196,197). It was also proposed that PACT interacts with LGP2 in a dsRNA-dependent manner and that this interaction is required for PACT-mediated MDA5 stimulation (197).…”

Antiviral activity of double‐stranded RNA‐binding protein PACT against influenza A virus mediated via suppression of viral RNA polymerase

“…Expression plasmid for FLAG-RIG-I was provided by Dr. Takashi Fujita (Kyoto University, Kyoto, Japan) (5). Expression plasmids for FLAG-PACT, Myc-PACT, and FLAG-PACT-DM have been previously described (6,11,27). PACT-DM is an RNA binding-defective mutant of PACT in which both RNA-binding domains are disrupted (27).…”

“…Expression plasmids for FLAG-PACT, Myc-PACT, and FLAG-PACT-DM have been previously described (6,11,27). PACT-DM is an RNA binding-defective mutant of PACT in which both RNA-binding domains are disrupted (27). V5-PACT expression plasmid was based on the pcDNA3.1/V5-his vector.…”

“…In addition, TLR and another RIG-I-like receptor, MDA5, also contribute to host defense against IAV (24)(25)(26). In this context, PACT functions as a cellular partner and activator of RIG-I and MDA5 to facilitate the viral induction of type I IFNs (6,27). To survive the innate antiviral response, IAV has developed various countermeasures to subvert IFN production and signaling (28,29).…”