FIG. 6.

The vaccinia virus I1 protein binds telomeres and is responsible for the upper doublet of shifted complexes. (A and B) Identification of I1 by affinity purification. The 200-bp viral hairpins were biotinylated and conjugated to streptavidin-coated magnetic beads. Hairpin beads were then incubated with an infected cell extract using conditions similar to those used in EMSA reactions, including the presence of poly(dI-dC) and 150 mM KCl. Beads were collected using a magnet and developed with buffer containing increasing concentrations of KCl. Washes were assayed for telomere binding activity by EMSA using the 65-bp+tet hairpin probe (A) and analyzed in parallel by SDS-PAGE and silver staining (B). Lanes 1, cytoplasmic extract before incubation with beads; lanes 2, cytoplasmic extract after incubation with beads (flow through); lanes 3 and 4, 150 mM KCl washes; lanes 5, 250 mM KCl wash; lanes 6, 500 mM KCl wash; lanes 7, 1,000 mM KCl wash. The 35-kDa band in the 500 mM KCl wash (panel B, lane 6, lower gray arrow) was excised and identified as the vaccinia virus I1 protein by mass spectroscopy (see the text). Protein standards are shown at the right with their molecular masses indicated in kilodaltons. (C) The vaccinia virus I1 protein is necessary and sufficient for complex formation. Cytoplasmic extracts from uninfected cells (lane 1) or infected cells harvested at 24 hpi (lane 2 to 4) were analyzed by EMSA using the 65-bp+tet hairpin probe (upper panel) and by immunoblot analysis using a polyclonal anti-I1 serum (lower panel). Cells were infected with the following: lane 2, wt virus (wtVV); lane 3, vLacI (a virus expressing the lac repressor protein); lane 4, vindI1 in the absence of IPTG. In lane 5, 320 ng of His-tagged recombinant I1 protein (HisI1) was used in the EMSA reaction (upper panel) and immunoblot analysis (lower panel). Dots and black arrows in panels A and C indicate the upper and lower doublets of shifted complexes, respectively; gray arrows in panel B indicate the 35- and 40-kDa proteins discussed in the text. Protein standards are shown at the right with their molecular masses indicated in kilodaltons.