ยฉ 2016, American Society for Microbiology. Coronavirus (CoV) nonstructural protein 14 (nsp14) is a 60-kDa protein encoded by the replicase gene that is part of the replication- transcription complex. It is a bifunctional enzyme bearing 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) activities. ExoN hydrolyzes single-stranded RNAs and double-stranded RNAs (dsRNAs) and is part of a proofreading system responsible for the high fidelity of CoV replication. nsp14 N7-MTase activity is required for viral mRNA cap synthesis and prevents the recognition of viral mRNAs as "non-self" by the host cell. In this work, a set of point mutants affecting different motifs within the ExoN domain of nsp14 was generated, using transmissible gastroenteritis virus as a model of Alphacoronavirus. Mutants lacking ExoN activity were nonviable despite being competent in both viral RNA and protein synthesis. A specific mutation within zinc finger 1 (ZF-C) led to production of a viable virus with growth and viral RNA synthesis kinetics similar to that of the parental virus. Mutant recombinant transmissible gastroenteritis virus (TGEV) ZF-C (rTGEV-ZF-C) caused decreased cytopathic effect and apoptosis compared with the wild-type virus and reduced levels of dsRNA accumulation at late times postinfection. Consequently, the mutant triggered a reduced antiviral response, which was confirmed by evaluating different stages of the dsRNA-induced antiviral pathway. The expression of beta interferon (IFN-ฮฒ), tumor necrosis factor (TNF), and interferon-stimulated genes in cells infected with mutant rTGEV-ZF-C was reduced compared to the levels seen with the parental virus. Overall, our data revealed a potential role for CoV nsp14 in modulation of the innate immune response.