The C-Terminal Domain of Salmonid Alphavirus Nonstructural Protein 2 (nsP2) Is Essential and Sufficient To Block RIG-I Pathway Induction and Interferon-Mediated Antiviral Response.

Salmonid alphavirus (SAV) is an atypical alphavirus that has a considerable impact on salmon and trout farms. Unlike other alphaviruses, such as the chikungunya virus, SAV is transmitted without an arthropod vector, and it does not cause cell shutoff during infection. The mechanisms by which SAV escapes the host immune system remain unknown. By studying the role of SAV proteins on the RIG-I signaling cascade, the first line of defense of the immune system during infection, we demonstrated that nonstructural protein 2 (nsP2) effectively blocks the induction of type I interferon (IFN). This inhibition, independent of the protease activity carried by nsP2, occurs downstream of IRF3, which is the transcription factor allowing the activation of the IFN promoter and its expression. The inhibitory effect of nsP2 on the RIG-I pathway depends on the localization of nsP2 in the host cell nucleus, which is linked to two nuclear localization sequences (NLS) located in its C-terminal part. The C-terminal domain of nsP2 by itself is sufficient and necessary to block IFN induction. Mutation of the NLS of nsP2 is deleterious to the virus. Finally, nsP2 does not interact with IRF3, indicating that its action is possible through a targeted interaction within discrete areas of chromatin, as suggested by its punctate distribution observed in the nucleus. These results therefore demonstrate a major role for nsP2 in the control by SAV of the host cell's innate immune response. IMPORTANCE The global consumption of fish continues to rise, and the future demand cannot be met by capture fisheries alone due to limited stocks of wild fish. Aquaculture is currently the world's fastest-growing food production sector, with an annual growth rate of 6 to 8%. Recurrent outbreaks of SAV result in significant economic losses with serious environmental consequences for wild stocks. While the clinical and pathological signs of SAV infection are fairly well known, the molecular mechanisms involved are poorly described. In the present study, we focus on the nonstructural protein nsP2 and characterize a specific domain containing nuclear localization sequences that are critical for the inhibition of the host innate immune response mediated by the RIG-I pathway.

Deciphering the Fine-Tuning of the Retinoic Acid-Inducible Gene-I Pathway in Teleost Fish and Beyond.

Interferons are the first lines of defense against viral pathogen invasion during the early stages of infection. Their synthesis is tightly regulated to prevent excessive immune responses and possible deleterious effects on the host organism itself. The RIG-I-like receptor signaling cascade is one of the major pathways leading to the production of interferons. This pathway amplifies danger signals and mounts an appropriate innate response but also needs to be finely regulated to allow a rapid return to immune homeostasis. Recent advances have characterized different cellular factors involved in the control of the RIG-I pathway. This has been most extensively studied in mammalian species; however, some inconsistencies remain to be resolved. The IFN system is remarkably well conserved in vertebrates and teleost fish possess all functional orthologs of mammalian RIG-I-like receptors as well as most downstream signaling molecules. Orthologs of almost all mammalian regulatory components described to date exist in teleost fish, such as the widely used zebrafish, making fish attractive and powerful models to study in detail the regulation and evolution of the RIG-I pathway.

A20 (tnfaip3) is a negative feedback regulator of RIG-I-Mediated IFN induction in teleost.

Interferon production is tightly regulated in order to prevent excessive immune responses. The RIG-I signaling pathway, which is one of the major pathways inducing the production of interferon, is therefore finely regulated through the participation of different molecules such as A20 (TNFAIP3). A20 is a negative key regulatory factor of the immune response. Although A20 has been identified and actively studied in mammals, nothing is known about its putative function in lower vertebrates. In this study, we sought to define the involvement of fish A20 orthologs in the regulation of RIG-I signaling. We showed that A20 completely blocked the activation of IFN and ISG promoters mediated by RIG-I. Furthermore, A20 expression in fish cells was sufficient to reverse the antiviral state induced by the expression of a constitutively active form of RIG-I, thus allowing the efficient replication of a fish rhabdovirus, the viral hemorrhagic septicemia virus (VHSV). We brought evidence that A20 interrupted RIG-I signaling at the level of TBK1 kinase, a critical point of convergence for many different pathways that activates important transcription factors involved in the expression of many cytokines. Finally, we showed that A20 expression was directly induced by the RIG-I pathway demonstrating that fish A20 acts as a negative feedback regulator of this key pathway for the establishment of an antiviral state.