Hsp105 is essential for polyomavirus infection.

<p><b>A</b>. CV-1 cells were transfected with a ctrl siRNA, or <i>Hsp105</i> siRNA #1 or #2 for 24 h, and the resulting WCE were immunoblotted with the indicated antibodies (top panel) or RT-PCR analysis was performed to observe the <i>XBP1</i> splicing (bottom panel). Cells treated with DTT were used as a positive control. <b>B</b>. Cells in (A) were infected with SV40 (MOI ~0.5) for 24 h, fixed, and immunostained against SV40 large T antigen (TAg). Infection was scored using immunofluorescence microscopy (counting >1000 cells for each condition). Data are normalized to the ctrl siRNA. Values represent the mean ± SD (n≥3). <b>C</b>. As in (B), except cells were infected with BKV for 40 h before immunostaining for BKV TAg. <b>D</b>. Multiple sequence alignment of Hsp70 and Hsp110 family proteins from yeast and humans. Only the relevant sequences are shown. The highlighted regions indicate the amino acid(s) that were altered to generate the Hsp105 mutants (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005086#sec009" target="_blank">Methods</a>). <b>E</b>. The indicated F-tagged proteins were purified from 293T cells, and their purity analyzed by SDS-PAGE followed by staining with Brilliant Blue R250. Hsc70 was obtained from commercial source (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005086#sec009" target="_blank">Methods</a>). The asterisk indicates an antibody heavy chain band. <b>F</b>. Purified proteins in (E) were incubated with ATP conjugated-agarose beads. Unbound and bound proteins were analyzed by immunoblotting using a FLAG antibody. <b>G</b>. CV-1 cells expressing the indicated F-tagged proteins were immunoprecipitated, and the eluted samples were analyzed by immunoblotting. <b>H</b>. Thin layer chromatography was used to determine the level of radiolabeled ADP that remain bound to Hsc70 after the indicated purified protein was incubated with radiolabeled ADP-Hsc70. The black line indicates that an intervening lane has been spliced out of the same film. <b>I</b>. CV-1 cells were reverse transfected with ctrl or <i>Hsp105</i> siRNA #1 for 24 h prior to transfection with the indicated tagged constructs for 24 h. Cells were then infected with SV40 (MOI ~0.5) for 24 h, fixed, and stained with anti-FLAG/S and anti-TAg antibodies. The percentage of TAg positive cells were counted only in cells expressing the indicated tagged protein by immunofluorescence microscopy (right graph). Values represent mean ± SD (n≥3). The protein expression levels of endogenous Hsp105, as well as transfected Hsp105 WT-F and GFP-F, in cell extracts derived from control and Hsp105-depleted cells (transfected with either <i>GFP</i>-F or <i>Hsp105</i> WT-F) are shown (left panels). The three lanes in the immunoblot correspond to the first three bars in the right graph.