K160 in the RNA‐binding domain of the orf virus virulence factor OV20.0 is critical for its functions in counteracting host antiviral defense

Abstract

The OV20.0 virulence factor of orf virus antagonizes host antiviral responses. One mechanism through which it functions is by inhibiting activation of the dsRNA-activated protein kinase R (PKR) by sequestering dsRNA and by physically interacting with PKR. Sequence alignment indicated that several key residues critical for dsRNA binding were conserved in OV20.0, and their contribution to OV20.O function was investigated in this study. We found that residues F141, K160, and R164 were responsible for the dsRNA-binding ability of OV20.0. Interestingly, mutation at K160 (K160A) diminished the OV20.0-PKR interaction and further reduced the inhibitory effect of OV20.0 on PKR activation. Nevertheless, OV20.0 homodimerization was not influenced by K160A. The contribution of the dsRNA-binding domain and K160 to the suppression of RNA interference by OV20.0 was further demonstrated in plants. In summary, K160 is essential for the function of OV20.0, particularly its interaction with dsRNA and PKR that ultimately contributes to the suppression of PKR activation.

The orf virus (ORFV) protein OV20.0 interacts with the microprocessor complex subunit DGCR8 to regulate miRNA biogenesis and ORFV infection

“…ΔC, which lacks the C‐terminal RBD, served as a negative control. Of note, K160A is an OV20.0 mutant deficient in binding with dsRNA and other OV20.0‐interacting proteins [20], including DGCR8 (Fig. S3).…”

“…1). Since DGCR8 is not required for shRNA production, this may partially explain why the failure of OV20.0 to suppress RNA interference via shRNA in mammalian cells [20] is likely due to the shRNA system bypassing the step of pri-miRNA processing.…”

“…Our previous work showed that OV20.0 is able to suppress RNA silencing (RNAi) in the plant system, despite having no significant effect on short hairpin (shRNA)-mediated silencing in mammalian cells [20]. Given the differences in the biogenesis of shRNA and miRNA in mammalian cells, particularly in the process of pre-miRNA maturation requiring the formation of microprocessors in the nucleus, we investigated whether OV20.0 participates in the regulation of the miRNA biogenesis pathway by interacting with critical cellular proteins, such as DGCR8, Drosha, TRBP and Dicer.…”

“…OV20.0 has been shown to interact with dsRNA and PACT, which are two components of the RNAi machinery. In our previous study, OV20.0 served as a strong suppressor of the RNA silencing pathway, as evidenced in a plant system [20]. However, whether ORFV OV20.0 can regulate miRNA biogenesis remains unclear.…”

Inhibition of PKR by Viruses

“…PACT can be targeted as a PKR evasion strategy. In addition to binding to dsRNA and PKR itself ( Liao et al, 2021 ), the Orf virus (ORFV)-encoded protein OV20.0 was shown to interact with PACT, thereby blocking PACT-mediated PKR activation ( Tseng et al, 2015 ).…”