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. 2021 Oct 6;17(10):e1009971. doi: 10.1371/journal.ppat.1009971

Fig 1. ORFV113 expression kinetics, presence in virions and supernatants, and subcellular localization.

Fig 1

(A) Kinetics of ORFV113 expression. OFTu cells mock infected (M) or infected with revertant virus OV-IA82-RV113Flag (MOI, 10) were harvested at indicated times p.i. Total cell protein extracts were resolved by SDS-PAGE, blotted and incubated with antibodies against Flag and GAPDH. Result is representative of two independent experiments. (B) Detection of ORFV113 in virions. OFTu cells were infected with OV-IA82-Δ113 (Δ113) or OV-IA82-RV113Flag (RV113Flag), and intracellular mature virions (IMV) were purified through double sucrose gradient centrifugation. Whole cell lysate (CL) (50 μg) from OFTu cells mock infected or infected with OV-IA82-RV113Flag, and purified virions (10 μg) were resolved by SDS-PAGE and analyzed by Western blot using antibodies against Flag, structural protein ORFV086 or host GAPDH. Result is representative of three independent experiments. (C and D) Presence of ORFV113 in supernatant of ORFV infected cells and ORFV113 transiently expressing cells. (C) OFTu cells mock infected (M) or infected with OV-IA82-Δ113 or OV-IA82-RV113HA (MOI, 10) were harvested at 24 h p.i. Immunoprecipitation was performed on clarified culture supernatant and immunoprecipitation products were resolved by SDS-PAGE, blotted and incubated with antibodies against HA. Result is representative of two independent experiments. (D) Culture supernatants were harvested from OFTu cells transfected with pCMV-HA (Empty HA plasmid), pCMV-ORFV113HA (ORFV113HA plasmid) or pCMV-ORFV113 (Untagged ORFV113 plasmid) at 24h post transfection. 50 μl of the culture supernatants were resolved by SDS-PAGE, blotted and incubated with antibodies against HA. Result is representative of three independent experiments. # indicates non-specific reactivity with serum protein routinely observed in supernatants. (E) ORFV113 subcellular localization. OFTu cells were mock infected or infected with OV-IA82-RV113HA (MOI, 10), fixed at 1, 3, 6, 9, 12 and 24 h p.i. sequentially incubated with primary antibody against HA and Alexa fluor 488 labeled secondary antibody, counterstained with DAPI and examined by confocal microscopy.