Biochemical and functional characterization of orf virus decapping protein OV71

Nudix enzymes constitute a family of hydrolases that share a conserved Nudix motif, which catalyzes the hydrolysis of nucleoside diphosphates linked to another moiety X. Some members are cellular and viral decapping enzymes that hydrolyze the 5´ cap structure on an mRNA molecule. Unlike vaccinia virus, which encodes two Nudix enzymes, orf virus (ORFV) encodes only a single Nudix-containing gene, ORFV071 (OV71). This study investigates the biochemical properties of recombinant OV71 protein and its role in viral replication.

In vitro decapping assays using radiolabeled capped RNA substrates were performed to assess OV71 activity in the presence or absence of competitors or metal cations. Electrophoretic mobility shift assays and pulldown assays evaluated the RNA-binding ability of OV71. Decapping-deficient mutant viruses were generated by homologous recombination, and their replication was analyzed using one-step growth curve experiments. Reverse transcription-qPCR quantified host and viral mRNA levels.

OV71 exhibited intrinsic decapping activity, hydrolyzing long capped RNAs to release m⁷GDP, with optimal activity in the presence of Mn²⁺. It bound both single- and double-stranded RNA and was expressed early during viral replication. Decapping-deficient mutant viruses replicated poorly in cells. Unlike the vaccinia virus decapping-deficient mutant, which triggers host antiviral responses leading to degradation of viral and host mRNAs as well as rRNAs, an orf virus mutant caused accumulation of host-capped RNAs and a severe reduction in viral mRNAs. Notably, host rRNA remained relatively intact compared to wild-type virus infection.

OV71 is a decapping enzyme that hydrolyzes the cap structure on long capped mRNAs. It binds both single- and double-stranded RNA, suggesting that it may target both RNA species in infected cells. Its decapping activity is critical for efficient orf virus replication. Loss of this activity leads to the accumulation of host-capped mRNAs, a drastic reduction of viral mRNAs, and minimal impact on host rRNAs, indicating a role distinct from that of the vaccinia virus decapping enzymes.