The double-stranded RNA-activated protein kinase (PKR) is a Type I interferon (IFN) stimulated gene that has important biological and immunological functions. In viral infections, PKR inhibits or promotes viral replication. In the present study, PKR homologues of orange-spotted grouper (Epinephelus coioides) (EcPKR) were cloned and the involvement of EcPKR during Red-spotted grouper nervous necrosis virus (RGNNV) infection was investigated. EcPKR encodes a 621-amino acid polypeptide that is closely related to the equivalent protein in Larimichthys crocea. EcPKR encoded two dsRNA binding domains and a Serine/Threonine protein kinase domain. Quantitative real-time PCR (qRT-PCR) analysis indicated that EcPKR was present in all examined tissues, with higher expression in spleen, intestine and gill. When stimulated with poly(I:C), the expression of EcPKR in the grouper spleen was increased, with highest expression 12 h post stimulation. EcPKR concentration was significantly increased in RGNNV-infected cells, with highest expression at 36 h post stimulation. EcPKR is mainly present in the cytoplasm. Overexpression of EcPKR in grouper spleen (GS) cells inhibits the transcription of the RGNNV genes. Furthermore, our results show that EcPKR overexpression significantly enhances the immune response of interferon and the activation of interferon-beta (IFN-β), interferon stimulated response element (ISRE) and nuclear factor-kappa B (NF-κB). Taken together, these results are important for better understanding of the function of PKR in fish and reveal its involvement in host response to immune challenges in RGNNV.
Keywords: Cellular localization; Epinephelus coioides; PKR; RGNNV; Replication.
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