LANA Associates with the Elongin BC Complex and Cul5 through the SOCS-Box–Like Motif

<div><p>(A) KSHV LANA contains a putative SOCS-box motif which includes a consensus of BC-box and Cul-box motifs. Upper panels show hydrophobic sequence alignment of the BC-box and Cul-box motifs from viral LANA and cellular SOCS-box–containing proteins that bind Elongin BC and Cullin proteins individually. Conserved residues with similarity are shaded in the upper panel. Lower panels show ORF73 proteins from other gammaherpesvirus contain an SOCS-box–like motif which is similar to KSHV LANA. HVS, herpesvirus saimiri; RRV, rhesus monkey rhadinovirus. The site mutations of LANA in leucine (L) and/or cystine (C) are indicated with underlining.</p><p>(B) LANA associates with the Elongin BC complex through the BC-box motif. Saos-2 cells were transfected with expression vector encoding the indicated proteins. The WT LANA and its mutant with BC-box (ΔBC) deletion and point mutation (LPCF, L213G or C217G) within BC box were compared. Since LANA lost its normally top bands (approximately 230 kDa) in the L213G mutant, another two lower bands of LANA (between 190 kDa and 230 kDa) were specifically present. The lysates underwent IP and IB assays as described previously. RD, relative density.</p><p>(C) LANA associates with Cullin 5 through the Cul-box motif. Saos-2 cells were transfected with expression vector encoding the indicated proteins. The WT LANA with Cul-box (ΔCul) or SOCS-box (ΔSCOS) deletion was compared. The lysates underwent IP and IB assays as described previously. Crude extracts (left panels) and immune complexes (right panels) are shown.</p><p>(D) LANA assembles with Elongin BC complex and Cul5/Rbx1 module to reconstitute a multiprotein complex with E3 ubiquitin ligase activity. Left panel shows the LANA–Elongin BC–Cul5–Rbx1 complex had ubiquitin ligase activity. 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), and GST-ubiquitin (GST-Ub) in the absence or presence of ATP, to assess its ability to stimulate ubiquitylation in vitro. The proteins were separated by SDS-PAGE and IB using anti-GST antibodies. Right panel shows SOCS-box–like motif deletion reduced the capacity of LANA to induce polyubiquitylation. Saos-2 cells were transfected with WT LANA or its SOCS mutant. After 24-h transfection, cells were treated with MG132 (0.5 μM) for 2 h. Lysates were subjected to IB against Ub or myc antibodies.</p>