Functional Analysis of the Short Isoform of Orf Virus Protein OV20.0

Abstract

Orf virus (ORFV) OV20.0L is an ortholog of vaccinia virus (VACV) gene E3L. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACV E3L, OV20.0L encodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminally truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localization, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. Nonetheless, both OV20.0 isoforms were able to bind double-stranded RNA (dsRNA)-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2␣ subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of function in vitro, a recombinant ORFV that was able to express only the sh20 isoform was attenuated in a mouse model. IMPORTANCEThe OV20.0 protein of orf virus (ORFV) has two isoforms and contributes to virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localization from full-length OV20.0. Similar to the full-length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR, and thus act as an antagonist of the interferon response in vitro. In vivo, however, wild-type OV20.0 could not be replaced with sh20 alone without a loss of virulence, suggesting that the functions of the isoforms are not simply redundant. Orf virus (ORFV), a member of the Parapoxvirus genus and the Poxviridae family, is the causative agent of contagious ecthyma in sheep, goats, and other ruminants. The disease is characterized by the development of pustular lesions around the nostrils and mouth with a high incidence rate and a low mortality rate in healthy adult animals. In contrast, infection in immunosuppressed animals or in lambs may be fatal (1). ORFV is also of concern as a source of zoonotic infection because it can cause cutaneous lesions in humans in contact with infected animals. Persistent infection with ORFV can be observed in goats and sheep, and while the severity of lesions is reduced compared with that seen in primary infection, this persistence suggests that the virus is able to evade host immunity (2-4). In line with this observation, ORFV has been shown to encode several proteins that modulate the host response to infection. These include viral homologues of ovine cytokines, such as vascular endothelial growth factor, interleukin-10 (IL-10), and a granulocyte-macrophage colony-stimulating factor (GM-CSF)-inhibiting protein, as well as an apoptosis inhibitor (5-7). ORFV also antagonizes interferon (IFN) signaling, and this is done by the product...

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

“…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.…”

“…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).…”

“…Both events lead to an inhibition of PKR activation (8). However, the functional domain of OV20.0 is not known.…”

“…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).…”

Contagious Pustular Dermatitis (Orf Disease) - Epidemiology, Diagnosis, Control and Public Health Concerns

“…Peralta et al (2015) described the sequencing and analysis of five molecular markers of orf virus: a partial B2L gene (ORF011), VIR (ORF020), an envelope mature protein (ORF109), vIL10 (ORF127), and GIF (ORF117) from two outbreaks in Argentina. Tseng et al (2015) showed that the shorter isoform (sh20) of OV20.0 protein of orf virus (ORFV) arises due to use of a downstream initiation codon and is amino-terminally truncated. This form also differs in expression kinetics and cellular localization from full-length OV20.0.…”