Characterization of a ranavirus inhibitor of the antiviral protein kinase PKR

Background: Ranaviruses (family Iridoviridae) are important pathogens of lower vertebrates. However, little is known about how they circumvent the immune response of their hosts. Many ranaviruses contain a predicted protein, designated vIF2 alpha, which shows homology with the eukaryotic translation initiation factor 2 alpha. In analogy to distantly related proteins found in poxviruses vIF2 alpha might act as an inhibitor of the antiviral protein kinase PKR. Results: We have characterized the function of vIF2a from Rana catesbeiana virus Z (RCV-Z). Multiple sequence alignments and secondary structure prediction revealed homology of vIF2a with eIF2 alpha throughout the S1-, helical- and C-terminal domains. Genetic and biochemical analyses showed that vIF2 alpha blocked the toxic effects of human and zebrafish PKR in a heterologous yeast system. Rather than complementing eIF2 alpha function, vIF2a acted in a manner comparable to the vaccinia virus (VACV) K3L protein (K3), a pseudosubstrate inhibitor of PKR. Both vIF2 alpha and K3 inhibited human PKR-mediated eIF2 alpha phosphorylation, but not PKR autophosphorylation on Thr446. In contrast the E3L protein ( E3), another poxvirus inhibitor of PKR, inhibited both Thr446 and eIF2 alpha Ser51 phosphorylation. Interestingly, phosphorylation of eIF2 alpha by zebrafish PKR was inhibited by vIF2 alpha and E3, but not by K3. Effective inhibition of PKR activity coincided with increased PKR expression levels, indicative of relieved autoinhibition of PKR expression. Experiments with vIF2 alpha deletion constructs, showed that both the N-terminal and helical domains were sufficient for inhibition of PKR, whereas the C-terminal domain was dispensable. Conclusions: Our results show that RCV-Z vIF2 alpha is a functional inhibitor of human and zebrafish PKR, and probably functions in similar fashion as VACV K3. This constitutes an important step in understanding the interaction of ranaviruses and the host innate immune system.