Discussion Ranaviruses are important pathogens of fish, amphibians and reptiles (reviewed in [2]). However, little is known about how they interact with the immune system of their hosts. Herein we show that RCV-Z vIF2α, a homolog of eIF2α, is an effective inhibitor of PKR in a heterologous yeast
assay system. PKR is an important antiviral protein kinase that has been primarily studied in mammals (reviewed in [15]). PKR-related genes have recently been identified in a variety of fish and amphibian species. Fish PKR genes are expressed at low levels constitutively, but they are highly induced after viral infection and stimulation with the dsRNA analog poly(I:C), which mimics viral infection [27, 28]. It was recently shown that PKR of the Japanese flounder (Paralichthys olivaceus) was able to inhibit replication of Scophthalmus Crenigacestat solubility dmso maximus rhabdovirus [28]. To date, only PKR learn more inhibitors from mammalian viruses have been functionally characterized (reviewed in [32]). Moreover, the only well-characterized viral PKR inhibitors that directly target the PKR kinase domain are the pseudosubstrates found in many poxviruses and represented by VACV K3L, which is homologous to the S1 domain of the PKR
target eIF2α [33, 40, 46, 47]. It was speculated that the ranavirus Selleckchem Compound Library vIF2α protein, another eIF2α homolog, might inhibit PKR of infected hosts [38, 39]. A notable difference between K3 and eIF2α is the presence of an extended C-terminal domain in eIF2α. In addition to the C-terminal α/β domain, eIF2α consists of an N-terminal Quinapyramine S1 domain and a central α-helical domain. The K3 protein is homologous to the N-terminal domain in eIF2α. Like K3, vIF2α shows moderate sequence identity to
eIF2α in the S1 domain. In this study we used PSI-BLAST analyses, multiple sequence alignment and secondary structure prediction to show that the C-terminal parts of vIF2α are likewise homologous to the helical and C-terminal domains of eIF2α. Functional analyses using deletion constructs of vIF2α revealed that both the S1 and helical domains are sufficient for inhibition of PKR in yeast (Figure 5). Since the presence of both domains was necessary for detectable vIF2α expression, it appears possible that the domains are important to stabilize each other. The crystal structure of human eIF2α showed that the S1 and helical domains are connected by an intramolecular disulfide bridge formed by cysteine residues 69 and 97 [48]. Interestingly, a cysteine corresponding to position 69 is found in many Metazoa, including Chordata, Echinodermata, Cnidaria and Mollusca, but is missing in most Arthropoda (except Ioxedes scapularis), in all fungi and plants sequences currently found in Genbank, and in all poxviral K3L orthologs (Figure 1 and data not shown).