SGTA-Hsc70 dissociates from the B14-B12 complex and engages SV40 during entry.

<p>(A) Endogenous SGTA is released from B14 during entry. CV-1 cells were uninfected or infected with SV40 at a high MOI (≈100) for the indicated time. Cells were harvested, cross-linked, and lysed in buffer containing 1% Triton X-100 followed by immunoprecipitation with SGTA antibodies and immunoblotting. (B) Endogenous Hsc70 is released from B14 during entry. CV-1 cells were infected or uninfected for 12 h and immunoprecipitated as in (A) except Hsc70 antibodies were used. (C) Transfected SGTA is released from B14-B12 during entry. CV-1 cells transfected to express FLAG-tagged GFP or SGTA were infected with SV40 for 16 h. Immunoprecipitation was performed without cross-linking and the precipitated material analyzed by immunoblotting. (D) SV40 lacking VP2 fails to trigger dissociation of SGTA from B14-B12. As in (C) except cells were also infected with ΔVP2 SV40. (E) p97-Derlin-1 interaction is unaffected by SV40. Cells infected with virus as in (A) were lysed without cross-linking and immunoprecipitated with the indicated antibodies with subsequent immunoblotting of the precipitated material. (F) SGTA interacts with SV40 in cells during entry. CV-1 cells transfected to express FLAG-tagged WT SGTA were infected with SV40 at high MOI. Where indicated, cells were treated with BFA. Cells were cross-linked, lysed, and immunoprecipitated with anti-FLAG agarose beads. Precipitated proteins were analyzed by immunoblotting with the indicated antibodies. (G) SGTA requires localization to B14-B12 for interaction with SV40. As in (F) except cells were transfected to express either FLAG-tagged GFP, WT SGTA or mutant SGTA. (H) Coomassie staining of purified proteins. (I) Direct binding of SGTA and SV40 <i>in vitro</i>. Purified proteins were incubated with DTT/EGTA-treated SV40. Reactions were cross-linked, and the sampled precipitated using anti-FLAG agarose beads. Bound material was analyzed by immunoblotting.