OASL RNA binding and ubiquitin-like domains are not required for interaction with ORF20.

<p>(A) Multiple OASL constructs were utilized, including WT OASL and OASL mutants, including ΔUBL lacking the ubiquitin-like domain, the P-loop mutants V67G and N72K, the RNA binding mutants R45E/K66E/R196E/K200E (RKRK), K63E, and K66E, and the catalytic triad mutants, E81A, E83A, and T152A. (B) 293T cells were transfected with myc-tagged ORF20WT or LacZ, V5-tagged OAS1, WT or mutant OASL, and/or empty vector (EV) as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Immunoblots are representative of at least three independent experiments. Similar results were obtained with simultaneous and sequential antibody exposures. (C) The amino acid sequence for ORF20B is shown, with the predicted nuclear localization sequence indicated in red, the predicted nucleolar localization sequence indicated in green, and predicted disordered regions underlined. The predictions formed the basis for three ORF20B C-terminal truncation mutants: ORF20B 1–186, 1–220, and 1–235. (D) 293T cells were transfected with myc-tagged ORF20 isoforms, ORF20B mutants, or LacZ, OASL-V5, and/or EV as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed. Input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Immunoblots are representative of three independent experiments.