Double-stranded RNA (dsRNA)-activated protein kinase (PKR), a major component of the cellular antiviral system, is activated by the binding of either dsRNA or the cellular PKR activator, the PACT protein. The suppression of PKR activation is one of the main strategies that viruses employ to circumvent interferon signaling. Orf virus (ORFV), a parapoxvirus from the Poxviridae family, causes contagious pustular dermatitis in small ruminants. Previous studies have demonstrated that various OV20.0 isoforms, encoded by the OV20.0L gene, are able to inhibit PKR activation both by sequestering dsRNA and by physically interacting with PKR in vitro. Thus, this gene acts as a virulence factor of ORFV when tested using a mouse infection model. In the present study, the regions within OV20.0 that interact with dsRNA and with PKR have been mapped. Furthermore, this study demonstrates for the first time that OV20.0 is also able to interact with the dsRNA binding domain of PACT and that the presence of dsRNA strengthened the interaction of these two molecules. The presence of OV20.0 diminishes PKR phosphorylation when this is stimulated by PACT. Nevertheless, the association of OV20.0 with PKR, rather than with PACT, was found to be essential for reducing PACT-mediated PKR phosphorylation. These observations elucidate a new strategy whereby innate immunity can be evaded by ORFV. O RFV OV20.0 is the ortholog of the vaccinia virus (VACV) E3(1). Among all known E3 orthologs, VACV E3 is the most well characterized with respect to innate immune evasion (2). The dsRNA binding domain (RBD) is located at the C terminus (residues 117 to 182) of the VACV E3 protein, and this region also is involved in the protein's association with IFN-stimulating gene 15 (ISG15), which is known to lead to a suppression of the cellular antiviral response (3-5). In addition, the N terminus of VACV E3 interacts with the kinase domain of PKR, which then results in an inhibition of PKR phosphorylation, with the presence of Lys-167 and Arg-168 within the RBD increasing the binding efficiency and inhibition effect (6). Compared with VACV E3, little is known about the function of ORFV OV20.0. Up to the present, only the phenomena of ORFV OV20.0-mediated interferon (IFN) resistance and an inhibition of PKR activation have been reported; however, the underlying mechanism of action of this protein still requires further investigation (7,8). Based on the sequence alignment, four important amino acid residues responsible for the interaction of VACV E3 with dsRNA are known to be conserved in ORFV OV20.0 (1). Indeed, the dsRNA binding activity of OV20.0 protein has been confirmed in several different experimental systems (1,8). Furthermore, recent studies have indicated that both the full-length OV20.0 protein and its N-terminal truncated isoform (sh20.0 or a deleted protein described as ⌬1-79 here) possess similar dsRNA binding abilities (8). In addition, direct association between PKR and both OV20.0 and the various sh20.0 isoforms has been observed. These...
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