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. 2011 May 13;286(28):24702–24713. doi: 10.1074/jbc.M111.222216

FIGURE 1.

FIGURE 1.

Characterization of a temperature-sensitive virus with a lesion in the A20 ORF. A, extracts from cells infected with Dts48 under non-permissive conditions lack processive polymerase activity. BSC40 cells were infected with WT virus (IHD-W strain) or Dts48 (multiplicity of infection of 2) and incubated at non-permissive (39.7 °C) or permissive (31.5 °C) temperature for 24 h. Post-nuclear supernatants were prepared and assayed for processive polymerase activity using a single-primed M13 DNA template in the presence of [32P]dATP. Reactions were resolved on a 0.8% agarose gel and visualized by autoradiography. RFII products, representing synthesis of the complete 7.2-kb daughter strand in a single binding event, are marked. The bottom panels represent immunoblot analyses of the same lysates using α-Pol and α-A20 antibodies. B, the Dts48-A20 protein retains the ability to interact with fUDG in vivo. BSC40 cells were infected and transfected so as to express 3XFLAG-UDG (fUDG) and either WT A20 or Dts48 A20 and incubated at 39.7 or 31.5 °C. At 24 h post-infection, cells were harvested, and clarified lysates were subjected to affinity purification using α-FLAG beads. FUDG and any associated proteins were visualized by silver staining (note the different electrophoretic mobilities of the two A20 proteins). The numbers below the lanes indicate the relative amount of WT or Dts48 A20 that was retrieved with fUDG; the amount of WT A20 retrieved was set at 100%. The bottom two panels represent immunoblot analyses of the lysates using α-A20 or α-FLAG antibodies. C, the Dts48-A20-fUDG association is conserved in vitro. IVTT reactions were programmed to synthesize fUDG and either WT or Dts48 A20; fUDG and any associated proteins were retrieved on α-FLAG beads. The input and eluate fractions were analyzed by SDS-PAGE and visualized by autoradiography. The bracket indicates A20 fragments produced from internal initiation events. The numbers below the eluate lanes indicate the relative amount of WT or Dts48 A20 that was retrieved with fUDG; the level of WT A20 retrieved was set at 100%.