STAT2 negatively regulates RIG-I in the antiviral innate immunity of black carp.

The signal transducer and activator of transcription 2 (STAT2), a downstream factor of type I interferons (IFNs), is a key component of the cellular antiviral immunity response. However, the role of STAT2 in the upstream of IFN signaling, such as the regulation of pattern recognition receptors (PRRs), remains unknown. In this study, STAT2 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized. The open reading frame (ORF) of bcSTAT2 comprises 2523 nucleotides and encodes 841 amino acids, which presents the conserved structure to that of mammalian STAT2. The dual-luciferase reporter assay and the plaque assay showed that bcSTAT2 possessed certain IFN-inducing ability and antiviral ability against both spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). Interestingly, we detected the association between bcSTAT2 and bcRIG-I through co-immunoprecipitation (co-IP) assay. Moreover, when bcSTAT2 was co-expressed with bcRIG-I, bcSTAT2 obviously suppressed bcRIG-I-induced IFN expression and antiviral activity. The subsequent co-IP assay and immunoblotting (IB) assay further demonstrated that bcSTAT2 inhibited K63-linked polyubiquitination but not K48-linked polyubiquitination of bcRIG-I, however, did not affect the oligomerization of bcRIG-I. Thus, our data conclude that black carp STAT2 negatively regulates RIG-I through attenuates its K63-linked ubiquitination, which sheds a new light on the regulation of the antiviral innate immunity cascade in vertebrates.

The phenylalanine-28 is crucial for black carp RIG-I mediated antiviral signaling.

Retinoic acid-inducible gene I (RIG-I) functions as a cytosolic sensor to recognize RNA products of the invading microorganisms and induce the production of type I interferons（IFNs）. In this study, two RIG-I variants, named as bcRIG-Ia and bcRIG-Ib, were characterized in black carp (Mylopharyngodon piceus) respectively. RNA pull-down assay revealed that both bcRIG-Ia and bcRIG-Ib could bind to synthetic poly(I:C) and the RD domain was crucial for RNA binding of these two molecules. However, over-expression of bcRIG-Ib, but not bcRIG-Ia, induced the transcription of IFN promoter, and led to the improved antiviral activity against both spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). And knockdown of bcRIG-I dampened the transcription of bcViperin and bcIFNb in host cells. Truncation mutation and site mutation analysis identified that phenylalanine (F)- 28 was crucial for bcRIG-Ib oligomerization and its mediated IFN signaling. Interestingly, F28 was conserved among teleost RIG-Is and site mutation analysis revealed that F28 was essential for RIG-I mediated IFN signaling in the cyprinid fish. Thus, our study concludes that F28 is crucial for black carp RIG-I mediated antiviral signaling and suggests F28 is also essential for the activation of IFN signaling by RIG-Is from other teleost fish.