Immunofluorescence analysis (IFA).

<p><b> (A)</b> IFA was performed with anti-PML (Green) and anti-K-Rta (Red) antibodies. Arrows indicate cells showing overexpression of K-Rta. <b>(B)</b> K-Rta degrades PML. PML wild type or PML del-SUMO mutant was cotransfected with K-Rta or K-Rta mutants. K-Rta wild type preferentially degrades PML wild type, which can be modified by SUMO in vivo. K-Rta Ring mutant (H145L) or SUMO-binding mutant (<i>Δ</i>SIM) was not able to degrade PML. <b>(C)</b> Endogenous SUMO-modified proteins. K-Rta expression was induced by addition of doxycycline (Dox). Cell lysates were prepared 48 hours post-induction. Indicated proteins were probed with specific antibodies and 25 µg of total protein was loaded in each lane. GAPDH was used as a loading control.</p

STUbL-like function is important for K-Rta transactivation activity.

<p>Reporter assays were performed in 293 cells using the indicated K-Rta target gene reporters. Reporter plasmids were cotransfected with K-Rta Wt or mutants, and luciferase activity was measured at 48 hours post-transfection. Luciferase activity of reporter with empty expression plasmids was normalized to a value of 1. Fold activation over control is shown.</p

K-Rta Wt but not Ring-finger domain mutant can degrade SUMO-modified proteins.

<p>(<b>A</b>) Flag-SUMO and K-Rta wild type or mutant were cotransfected into 293T cells, and probed with indicated antibodies. The K-Rta Ring-like domain (C141, H145) is important for SUMO degradation. (<b>B</b>) MG132 recovered SUMO proteins from degradation. 24 hours after the K-Rta transfection, MG132 or DMSO (vehicle) was added into the culture media and cells were harvested after 12 hours of treatment. SUMO-modified proteins were probed with anti-Flag antibody. (<b>C</b>) SUMO degradation during KSHV reactivation in BCBL-1. After induction of K-Rta expression in TREx-K-Rta-BCBL-1 with doxycycline (Dox), SUMO-modified proteins were probed with anti-Flag antibody. K-Rta induction was confirmed with an anti-K-Rta antibody and GAPDH was served as the loading control. (<b>D</b>) Recovery of SUMO-modified proteins with MG132 in BCBL-1. KSHV reactivation was triggered by induction of K-Rta in BCBL-1 cells in either the presence or absence of MG132. The amount of SUMO-modified proteins was examined by immunoblotting with an anti-Flag antibody. The accumulation of SUMO-modified conjugates was evident by increments of higher molecular weight entities.</p

Degradation of SUMO by K-Rta.

<p>Plasmids expressing E1 (Uba2/Aos), E2 (Ubc9), SUMO-2, K-Rta or empty vector (Vec) were transfected into 293T cells. SUMO, K-Rta or tubulin was probed with respective antibody. <b>(A)</b> K-Rta reduced total SUMO-modified protein levels in a dose-dependent manner. <b>(B)</b> Overexpression of SUMO but not other SUMO enzymes recover the cellular SUMO-modified proteins.</p

K-Rta degrades SUMO(+)K-bZIP but not SUMO(−)K-bZIP.

<p>K-Rta was cotransfected with the indicated K-bZIP plasmid and immunoblotting was performed with anti-K-bZIP antibody. Proteasome (MG132, MG; Epoxomicin, Epox) or lysosome inhibitors (chloroquine, Chlor) were added to the culture media after 24 hours transfection and incubated another 12 hours.</p

SUMO-degradation function is important for KSHV replication.

<p><b>(A)</b> Recombinant KSHV, which harbors a point mutation in the Ring domain of K-Rta or SIM domain was constructed and transfected into 293T cells. LANA expression was examined by RT-qPCR (a). Values are normalized to cellular GAPDH. Recombinant KSHV containing a K-Rta point mutation or revertant wild type virus was reactivated with combination of sodium butyrate (SB, 1 mM) and TPA (20 nM). Expression of K-Rta protein was confirmed by immunoblotting (b), and K-Rta target gene expression was examined by RT-qPCR (c). Values are normalized to cellular β-actin, and fold induction over latent cell is shown. <b>(B)</b> Viral replication. Recombinant KSHV replication was measured after reactivation with TPA, sodium butyrate, or combination of K-Rta expression and TPA treatment. KSHV encapsidated (DNase resistant) DNA copy number in the supernatant was measured by qt-PCR. Absolute viral copy number in 1 mL of supernatant is shown. K-Rta Wt but not mutant expression rescued viral replication.</p

K-Rta physically associates with SUMO.

<p>(<b>A</b>) (a) Recombinant GST and GST-SUMO were purified from an <i>E. coli</i> expression system, and GST-SUMO chains were generated <i>in vitro</i>. Flag-K-Rta protein was purified from recombinant baculovirus infected Sf9 cells (right panel). (b)The GST-Pull down assay was performed by incubating GST, GST-SUMO-1, -SUMO-2, -SUMO-3, -SUMO-2 chain or –SUMO-3 chain with purified Flag-K-Rta. Association was detected by immunoblotting with anti-Flag antibody. (<b>B</b>) Mapping of SUMO-binding domain. The indicated K-Rta deletion protein was incubated with SUMO-2 chains, and the interaction was probed with an anti-SUMO monoclonal antibody (upper panel). The membrane was also stained with Ponceau to show amount of GST-K-Rta deletion protein on the membrane (bottom panel). Asterisks showed non-specific interaction between C-terminal K-Rta and IgG. (<b>C</b>) Generation of K-RtaΔSIM. (a) Multiple alignments between KSHV Rta and RRV Rta. Conserved hydrophobic clusters (putative SIM) are marked in bold letters. Mutations that showed decreased SUMO degradation are underlined. (b) Mutations introduced in this study are shown. (c) SUMO degradation by K-Rta mutants. Immunoblotting was performed after the transfection of indicated K-Rta mutant plasmids along with SUMO expression vector in 293T cells. Mutations at SIM-3, -4, or -5 impaired K-Rta SUMO degradation function. (d) K-Rta Wt but not K-RtaΔSIM degrades SUMO modified proteins. Increasing amounts of K-Rta Wt or K-RtaΔSIM3&5 (0.25, 0.5, or 1.0 ug) were cotransfected with SUMO. The SIM mutation completely abolished the SUMO degradation function of K-Rta. (<b>D</b>) (a) GST pull-down analyses with purified K-Rta Wt or K-Rta ΔSIM mutants are shown. The interaction was probed with anti-Flag antibody. (b) Ponceau staining shows the amount and purity of GST- or GST-SUMO proteins used in the assay.</p

Sumo inhibits KSHV gene expression and reactivation.

<p><b>(A)</b> A dual-inducible cell line was generated with the TREx FRT recombination system. Indicated protein was probed with specific antibodies <b>(B)</b>, and gene expression was examined by northern blotting with probes to the coding regions of K-bZIP or ORF65 <b>(C)</b>. Ubc9 wild type decreased gene expression; however, the Ubc9 mutant increased viral gene expression compared to control cells. <b>(D)</b> SUMO inhibits viral reactivation. Indicated plasmids were cotransfected into recombinant KSHV infected Vero cells. (a) RFP positive cells were counted 72 hours after transfection in five randomly selected fields. (b) Average of RFP positive cells are shown. The total amount of plasmid/well was kept at 2 micrograms by adding control vector DNA. (c) Viral copy number after transfection of K-Rta or mutants into recombinant KSHV infected Vero cells is shown. Supernatants were collected 5 days post-transfection and encapsidated viral DNA was measured by qt-PCR (left panel). K-Rta expression was confirmed by immunoblotting (right panel).</p