title: 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 eIF2α 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.
  
  
  OBJECTIVE
  The 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.
authors.affiliation: Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Yeu-Yang
authors.initials: YY
authors.lastname: Tseng
authors.affiliation: Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Fong-Yuan
authors.initials: FY
authors.lastname: Lin
authors.affiliation: Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Sun-Fang
authors.initials: SF
authors.lastname: Cheng
authors.affiliation: Research School of Biology, Australian National University, Canberra, Australian Capital Territory, Australia.
authors.forename: David
authors.identifier: http://orcid.org/0000-0001-6825-9172
authors.initials: D
authors.lastname: Tscharke
authors.affiliation: Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Songkhla
authors.initials: S
authors.lastname: Chulakasian
authors.affiliation: Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Chia-Chi
authors.initials: CC
authors.lastname: Chou
authors.affiliation: Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Ya-Fen
authors.initials: YF
authors.lastname: Liu
authors.affiliation: Department and Graduate Institute of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Wei-Shan
authors.initials: WS
authors.lastname: Chang
authors.affiliation: Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Min-Liang
authors.initials: ML
authors.lastname: Wong
authors.affiliation: Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan wlhsu@dragon.nchu.edu.tw.
authors.forename: Wei-Li
authors.initials: WL
authors.lastname: Hsu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004195:Disease Models, Animal; D004474:Ecthyma, Contagious / Q000473:pathology / Q000821:virology; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D006801:Humans; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009923:Orf virus / Q000502:physiology*; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D016601:RNA-Binding Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: C081374:E3L protein, Vaccinia virus; D015852:Eukaryotic Initiation Factor-2; C112834:OV20.0L protein, Orf virus; D020033:Protein Isoforms; D016601:RNA-Binding Proteins; D014764:Viral Proteins; D004247:DNA; D019892:eIF-2 Kinase