The 3C Protein of Enterovirus 71 Inhibits Retinoid Acid-Inducible Gene I-Mediated Interferon Regulatory Factor 3 Activation and Type I Interferon Responses

Abstract

Enterovirus 71 (EV71) is a human pathogen that induces hand, foot, and mouth disease and fatal neurological diseases. Immature or impaired immunity is thought to associate with increased morbidity and mortality. In a murine model, EV71 does not facilitate the production of type I interferon (IFN) that plays a critical role in the first-line defense against viral infection. Administration of a neutralizing antibody to IFN-␣/␤ exacerbates the virus-induced disease. However, the molecular events governing this process remain elusive. Here, we report that EV71 suppresses the induction of antiviral immunity by targeting the cytosolic receptor retinoid acid-inducible gene I (RIG-I). In infected cells, EV71 inhibits the expression of IFN-␤, IFN-stimulated gene 54 (ISG54), ISG56, and tumor necrosis factor alpha. Among structural and nonstructural proteins encoded by EV71, the 3C protein is capable of inhibiting IFN-␤ activation by virus and RIG-I. Nevertheless, EV71 3C exhibits no inhibitory activity on MDA5. Remarkably, when expressed in mammalian cells, EV71 3C associates with RIG-I via the caspase recruitment domain. This precludes the recruitment of an adaptor IPS-1 by RIG-I and subsequent nuclear translocation of interferon regulatory factor 3. An R84Q or V154S substitution in the RNA binding motifs has no effect. An H40D substitution is detrimental, but the protease activity associated with 3C is dispensable. Together, these results suggest that inhibition of RIG-I-mediated type I IFN responses by the 3C protein may contribute to the pathogenesis of EV71 infection.

Enterovirus 71 Inhibits Pyroptosis through Cleavage of Gasdermin D

“…3C, lanes 5 and 6). These two mutants are abolished for the RNA binding activity but not the protease activity of 3C (15). All of these mutants were expressed at levels comparable to the level wild-type 3C, as determined by Western blotting (Fig.…”

“…Several studies have suggested that EV71 suppresses antiviral immunity (9,10,(13)(14)(15)(16)(17). In addition to inhibition of type I IFN induction, EV71 has evolved strategies to counter inflammasome activation through cleavage of NLRP3 by viral proteases 2A and 3C (18).…”

“…In addition, the 3C protease induces apoptosis by inducing cleavage of PinX1, an intrinsic telomerase inhibitor, to facilitate EV71 release (12). Other important roles of 3C are to dampen immune responses by impeding the RIG-I-MAVS interaction or by cleaving a series of host factors, including TIR domain-containing adaptor inducing IFN-␤ (TRIF), interferon regulatory factor 7/9, and TAK1/TAB1/TAB2/TAB3 complex (13)(14)(15)(16)(17).…”

TRIM14 is a mitochondrial adaptor that facilitates retinoic acid-inducible gene-I–like receptor-mediated innate immune response

“…Immunofluorescence assays were done as described (36). For mitochondria staining, cells were incubated for 10 min at 37°C in growth medium containing 200 nM MitoTracker Red CMXRos (Invitrogen), followed by washes with prewarmed PBS buffer.…”

“…Lysates were incubated overnight at 4°C with the indicated antibodies, followed by incubation with protein A/G agarose beads (Santa Cruz Biotechnology) for 2 h at 4°C. After four washes with RIPA buffer, immunoprecipitates were subjected to Western blot analysis as described (36).…”