An Interaction between KSHV ORF57 and UIF Provides mRNA-Adaptor Redundancy in Herpesvirus Intronless mRNA Export
Figure 6
KSHV ORF57 may preferentially bind Aly over UIF.
(A) Competition assays were performed using recombinant GST-ORF57 bound to glutathione agarose beads and incubated with (i) a constant amount of purified Aly-His (1 µg) and increasing amounts of purified UIF-His (0, 0.5, 1, 2, 3 µg), (ii) a constant amount of purified UIF-His (1 µg) and increasing amounts of purified Aly-His (0, 0.5, 1, 2, 3 µg), Precipitated protein and inputs were analysed by western blotting using a His-specific antibody. (B) Dose-dependent co-immunoprecipitations were performed using 293T cells cotransfected with (i) 0.5 ug of pORF57GFP and 0.5 ug of pAly-myc, in addition to increasing amounts (0, 0.1, 0.5, 0.8, 1.2 ug) of pUIF-Flag or (ii) 0.5 ug of pORF57GFP and 0.5 ug of pUIF-Flag, in addition to increasing amounts (0, 0.1, 0.5, 0.8, 1.2 ug) of pAly-myc, empty vector was also added to ensure a similar amount of DNA was transfected in each sample. After 24 hours, cell lysates were incubated with GFP-TRAP-Affinity agarose beads and the amount of precipitated (i) Aly or (ii) UIF was identified by immunoblotting with Myc- or Flag-specific antibodies, respectively. Western blots for input loading are also shown for ORF57, Aly and UIF constructs.