Functional domains and the antiviral effect of the dsRNA-dependent protein kinase PKR from Paralichthys olivaceus

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Wuhan 430072, China

^* To whom correspondence should be addressed. Email: jfgui{at}ihb.ac.cn.

	Abstract

The double-stranded RNA (dsRNA)-dependent protein kinase PKR is thought to mediate a conserved antiviral pathway by inhibiting viral protein synthesis via phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2). However, little is known about the data related to low vertebrate including fish. Recently, identification of PKR-like or PKZ has addressed the question of whether there is orthologous PKR in fish. Herein, we identify the first fish PKR gene from Japanese flounder Paralichthys olivaceus (PoPKR). PoPKR encodes a protein that shows conserved structure characteristic of mammalian PKRs, with the N-terminal region for dsRNA binding and the C-terminal region for inhibition of protein translation. The catalytic activity of PoPKR is further validated to require for protein translation inhibition in vitro. PoPKR is constitutively transcribed at low levels and is highly induced after virus infection. Strikingly, PoPKR overexpression increases eIF2 phosphorylation and inhibits replication of SMRV (Scophthalmus maximus rhabdovirus) in FEC cells, whereas the phosphorylation and antiviral effect are impaired in transfected cells expressing the catalytically inactive PKR-K421R variant, indicating that PoPKR inhibits virus replication by phosphorylating substrate eIF2. The interaction between PoPKR and eIF2 is demonstrated by coimmunoprecipitation assays, and transfection of PoPKR-specific siRNA further reveals that the enhanced eIF2 phosphorylation is catalyzed by PoPKR during SMRV infection. The current data provide significant evidence for the existence of PKR-mediated antiviral pathway in fish, and reveal considerable conservation in the functional domains and the antiviral effect of PKR proteins between fish and mammals.