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. 2007 Apr 25;81(13):7077–7085. doi: 10.1128/JVI.00058-07

FIG. 4.

FIG. 4.

Increasing concentrations of purified UL84 produce a downward-staircase pattern of binding with SL-RNA. EMSA were performed as described in Materials and Methods except increasing amounts of purified protein were used in the binding reactions. (A) Autoradiograph of dried agarose gel showing EMSA using SL-RNA with increasing concentrations of purified UL84. Lanes: 1, UL84 SL-RNA alone; 2, SL-RNA plus 0.5 μl of UL84; 3, SL-RNA plus 1 μl of purified UL84; 4, SL-RNA plus 2 μl of purified UL84; 5, SL-RNA plus 3 μl of purified UL84; 6, SL-RNA plus 5 μl of purified UL84; 7, SL-RNA plus 7 μl of purified UL84; 8, SL-RNA plus 10 μl of purified UL84; 9, SL-RNA plus 13 μl of purified UL84. (B) The downward-staircase pattern of binding to SL-RNA is not due to a shortening or degradation of the oligonucleotide. SL-RNA protein bands corresponding to lanes 5, 6, and 7 from the EMSA shown in panel A were extracted from the gel, deproteinized, and resolved through a denaturing urea gel. Shown is the autoradiograph of the dried urea gel. Lanes: 1, SL-RNA alone; 2, SL-RNA extracted from lane 5 from panel A; 3, SL-RNA extracted from lane 6 from panel A; 4, SL-RNA extracted from lane 6 from panel A. (C) The phosphorothioate-capped SL-RNA displays the same downward-staircase pattern as the unmodified version. EMSA experiments were performed using the same conditions as described for panel A except that SL-RNA PS was used instead of the unmodified SL-RNA. (D) Control experiments demonstrating that the UL84-SL-RNA interaction is genuine. Lanes: 1, SL-RNA alone; 2, SL-RNA plus UL84; 3, SL-RNA plus UL84 plus Mab84; 4, SL-RNA plus UL84 plus the K8 MAb; 5, SL-RNA plus heat-treated UL84; 6, SL-RNA plus IE2 (1 μl); 7, SL-RNA plus IE2 (3 μl); 8, SL-RNA plus IE2 (5 μl); 9, SL-RNA plus IE2 (10 μl). (E) UV cross-linking gives rise to a slower-migrating species. Lanes are the same as in panel A except samples were exposed to 30 min of UV light in a Stratalinker 2400. SS, supershifted band upon incubation with anti-UL84 antibody; S, shifted UL84-SL-RNA species; CL, shift resulting from cross-linking.