LANA Promotes Polyubiquitylation of Tumor Suppressors <i>p53</i> and <i>VHL</i>

<div><p>(A) LANA induces <i>p53</i> and <i>VHL</i> polyubiquitylation. Saos-2 (left panel) or 786-O (right panel) cells were cotransfected with different combinations of myc-LANA (WT and ΔSOCS), myc-<i>p53</i> (or HA-VHL), and HA-Ub as indicated. At 48 h posttransfection, transfected cells were harvested, lysed, and protein normalized. Total protein (40 μg) was subjected to resolve and IB assays against myc (LANA), <i>p53</i> (or <i>VHL</i>), and β-actin. The data showed LANA can induce <i>p53</i> and <i>VHL</i> ubiquitylation in vivo.</p><p>(B) The LANA–Elongin BC–Cul5–Rbx1 complex induces <i>p53</i> and <i>VHL</i> polyubiquitylation in vitro. The cell lysates of WT LANA (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020116#ppat-0020116-g003" target="_blank">Figure 3</a>C, lane 4) or SOCS-box–like motif deletion mutant (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020116#ppat-0020116-g003" target="_blank">Figure 3</a>C, lane 3) as control was subjected to anti-myc immunoaffinity purification. The purified LANA immune complex (IC) was incubated with various combinations of Uba1 (E1), Ubc5a (E2), His-Ub, or GST-VHL (or GST-p53) in the presence of ATP in vitro. The proteins were separated by SDS-PAGE and IB against GST antibody. Left panel, GST-p53; right panel, GST-VHL.</p>