Regulation of PACT-Mediated Protein Kinase Activation by the OV20.0 Protein of Orf Virus

Abstract

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...

ADAR1 and PACT contribute to efficient translation of transcripts containing HIV-1 trans-activating response (TAR) element

“…Several viruses have been shown to inactivate PACT function in the infected cells as PACT is involved both in activating PKR to suppress viral protein synthesis and in IFN production via RIG-I like receptors (RLRs) [88]. Middle East Respiratory Syndrome Coronavirus 4a protein [89], Herpes Simplex Virus US 11 protein [90,91], Ebola virus VP35 protein [92], Influenza virus NS1 protein [93] and orf virus ov20.2 protein [94] have been shown to inactivate PACT. Overall, our results show that the suppression of PACT activity to effectively inactivate PKR in HIV-1-producing cells is the result of the combined activity of the recruited ADAR1 that mediates PKR kinase inhibition, and Tat most probably stabilizes the complex formed by PKR, PACT and ADAR1.…”

Orf virus: A promising new therapeutic agent

“…Inactivation of eIF2‐α suppresses the processes of cellular and virus protein translation, effectively blocking the replication of the virus. Moreover, ORFV020 is able to curb PKR activation by preventing PKR from being bound by PKR Activator (PACT), the first identified protein activator of PKR …”

“…Moreover, ORFV020 is able to curb PKR activation by preventing PKR from being bound by PKR Activator (PACT), the first identified protein activator of PKR. 44 CBP, functionally similar to CBP-II of other poxviruses in its ability to bind with high affinity and inhibit many CC-chemokines, is critical in ORFV pathogenesis and virulence during the early stage of infection.…”

Adenosine Deaminase Acting on RNA 1 Associates with Orf Virus OV20.0 and Enhances Viral Replication

“…Multiple strategies that are used by OV20.0 to suppress PKR activation were revealed recently (5,6). OV20.0 is able to associate with PKR and compete with two cellular activators of PKR, i.e., dsRNA and PACT.…”

“…This physical association also occupies the binding site for PACT in the RBD2 and kinase domains of PKR. Another possible mechanism is that OV20.0 forms heterodimers with PACT via their RBDs, which may further reduce PACT-dependent PKR activation (6).…”