Effects of HIF-1α inhibitor PX-478 on PELs.

<p>(<b>A</b>) HIF-1α mRNA levels in BCBL-1 cells 24 hours after treatment with various concentrations of PX-478, normalized to 18S internal control and expressed as fold changes compared to no PX-478 control cells. (<b>B)</b> Proliferation rate of PEL cell lines BCBL-1, BC-3, JSC-1, BC-1, and BC-2 and Burkitt’s lymphoma (BL) cell lines BJAB and CA46 measured using the MTS assay 72 hours after treatment with indicated concentrations of PX-478, expressed as fold changes compared to no PX-478 control cells. (<b>C)</b> Growth rates of the PEL and BL cells treated with 0 or 10 μM PX-478. Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between untreated and inhibitor-treated cells are indicated. *<i>P</i> ≤0.05, **<i>P</i> ≤ 0.01.</p

Effect of HIF-1α knockdown on the expression of KSHV miRNAs.

<p><b>(A)</b> Level of primary miRNA transcript as measured by RT-qPCR and normalized to 18S mRNA. <b>(B)</b> Levels of mature miRNAs measured using taqman assays and normalized to that of RNU43 miRNA. Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between shScr and shHIF-1 cells are indicated. *<i>P</i> ≤0.05 (2-tailed t-test).</p

Generation of BCBL-1 and BC-3 cells with stable HIF-1α knockdown.

<p><b>(A)</b> Protein levels of HIF-1α in nuclear extracts of BJAB, BCBL-1, and BC-3 cells measured by Western blot analysis after 24 hours in normoxia. Tata-binding protein (TBP) is used as a loading control. BCBL-1 and BC-3 cells were transduced with lentivirus encoding shRNA to HIF-1α or Scrambled (Scr) RNA and stable cell lines were generated with puromycin selection. Total RNA and nuclear protein extracts were extracted from the cells to confirm the status of the knockdown. (<b>B</b>) mRNA levels of HIF-1α measured by RT-qPCR after 48 hours in normoxia(N) or hypoxia(H). mRNA levels are normalized to that of 18S ribosomal RNA and are expressed as fold change relative to cells containing shScr under normoxia. (<b>C and D</b>) Protein levels of HIF-1α measured by Western blot analysis of nuclear extracts after 24 hours in culture. β-actin is shown as a loading control. (<b>C</b>) Normoxic levels of HIF-1α levels in the absence or presence of 50μM cobalt chloride (CoCl₂), a hypoxia mimic that prevents oxygen-induced degradation of HIF-1α. (<b>D</b>) HIF-1α levels under normoxia or hypoxia.</p

Effect of HIF-1α knockdown on the expression of KSHV latent genes in PELs.

<p>(<b>A</b>) Schematic of multicistronic KSHV latency locus showing the location of latent genes and hypoxia-response elements (HREs) in the promoter region. (<b>B</b>) mRNA levels of latent genes in BCBL-1 cells within and (<b>C</b>) outside of the latency locus as measured by RT-qPCR and normalized to that of 18S RNA. RNA levels for each gene are expressed as fold change relative to shScr cells under normoxia (N). Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between shScr and shHIF-1 cells are indicated. *<i>P</i> ≤0.05, **<i>P</i> ≤ 0.01 (2-tailed t-test).</p

HIF-1α knockdown leads to reduced lytic replication of KSHV.

<p><b>(A)</b> mRNA levels of RTA, vIL-6, and ORF26 (a late lytic gene) of KSHV in BCBL-1 cells measured by RT-qPCR. Error bars represent standard deviation from at least 3 experiments. (<b>B)</b> Western blot showing protein levels of intracellular RTA, vIL6, and β-actin in the cell lysates as well as secreted vIL6 in the supernatant after 48 hours in normoxia (N) or hypoxia (H). (<b>C)</b> Protein levels of ORF45 in concentrated virus particles released in the supernatants after 72 hours. *Unspecific band. Western blots were done on lysates from three independent experiments and representative blots are shown.</p

Effect of HIF-1α suppression on growth of PELs.

<p><b>(A)</b> Growth rate of shScr or shHIF-1 BCBL-1 and BC-3 cells measured by counting live cells every 24 hours in normoxia. (<b>B)</b> Proliferation rate of shScr or shHIF-1 cells measured by MTS assay at 72 hours in normoxia using Promega’s CellTiter 96 Aqueous One Solution assay, which measures the amount of NADH or NADPH produced by metabolically active cells. (<b>C)</b> Colony forming efficiency of shHIF-1 cells relative to shScr cells. Error bars represent standard deviations from at least 3 independent experiments. Results shown in B and C are fold changes compared to shScr cells. Statistically significant differences between shScr and shHIF-1 cells are indicated. *<i>P</i> ≤0.05, **<i>P</i> ≤ 0.01.</p

Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses

<div><p>Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi’s sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman’s disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase-3 processing of LANA. This indicates that these are the principal sites that are susceptible to caspase cleavage. Using peptides spanning the identified LANA cleavage sites, we show that caspase activity can be inhibited <i>in vitro</i> and that a cell-permeable peptide spanning the C-terminal cleavage site could inhibit cleavage of poly (ADP-ribose) polymerase and increase viability in cells undergoing etoposide-induced apoptosis. The C-terminal peptide of LANA also inhibited interleukin-1beta (IL-1β) production from lipopolysaccharide-treated THP-1 cells by more than 50%. Furthermore, mutation of the two cleavage sites in LANA led to a significant increase in IL-1β production in transfected THP-1 cells; this provides evidence that these sites function to blunt the inflammasome, which is known to be activated in latently infected PEL cells. These results suggest that specific caspase cleavage sites in KSHV LANA function to blunt apoptosis as well as interfere with the caspase-1-mediated inflammasome, thus thwarting key cellular defense mechanisms.</p></div

Changes in KSHV LANA following treatment of BCBL-1 cells with varying concentrations of H₂O₂ and sequence analysis of LANA identifying potential caspase cleavage sites.

<p>(A) BCBL-1 cells were cultured in media at 500,000 cells per ml to which was added H₂O₂ at 100 and 200 μM (diluted into PBS) or PBS as a control for 20 hrs. Nuclear and cytoplasmic extracts (10 μgs per sample) were prepared and analyzed by western blot for LANA using a monoclonal antibody that is directed towards the C-terminal region of LANA (Leica), and with antibodies directed toward TBP (rabbit) and HSP90 (mouse). Blots were then incubated with appropriate secondary antibodies (goat anti-mouse or rabbit IR800 secondary antibody) and analyzed using the LiCor system. Full length LANA (LANA-fl) and three faster migrating forms of LANA (arrows) are indicated. Molecular weight markers in kDa are indicated to the left of the blot. Full length LANA (calculated molecular weight of 126 kDa based on the amino acid sequence) migrates at approximately 165 kDa in the Nupage LDS gel system. HSP90 is shown as a cytoplasmic loading control and TBP as a nuclear loading control. (B) The 1095 LANA amino acid sequence translated from the KSHV genome derived from BCBL-1 cells (Genbank U93872) was analyzed for potential caspase cleavage sites utilizing the caspase webserver Cascleave (<a href="http://sunflower.kuicr.kyoto-u.ac.jp/~sjn/Cascleave/webserver.html" target="_blank">http://sunflower.kuicr.kyoto-u.ac.jp/~sjn/Cascleave/webserver.html</a>). Two sites with high probability scores were located within the N-terminal domain of LANA, and one was located in the C-terminal domain. Shown in bold and underlined are these three putative caspase cleavage sites; cleavage by caspases is predicted to occur at the carboxyl side of aspartate as indicated by an asterisk (*). (C) The two peptides that were found to be cleaved by caspase-1 and/or -3 are shown and denoted as LP-Nterm and LP-Cterm (with Cascleave scores of 0.980 and 0.840, respectively). (D) Reverse phase HPLC of 1mM LP-Nterm (MW 1919 Da) following treatment with PBS control (top panel), caspase-1 at 2.5 units/μl (middle panel), or caspase-3 at 0.25 units/μl (bottom panel). The new peak (bottom panel) represented the expected mass of 1182 Da for the N-terminal product if cleaved after the aspartic acid as shown (the C-terminal product GRECGPH was not detected under these assay conditions). (E) Reverse phase HPLC of 1mM LP-Cterm (MW 2040 Da) following treatment with vehicle control (top panel), caspase-1 at 2.5 units/μl (middle panel), or caspase-3 at 0.25 units/μl (bottom panel). The caspase-1 and 3 cleavage products identified by mass spectrometry are indicated in the middle and lower panels. The new peaks generated represented the expected masses of 1127 Da (10 minute peak) and 931 Da (12 minute peak) (F) Depiction of full length LANA and the five different forms (cp1-cp5) of BCBL-1 LANA expected to be generated following caspase cleavage at the 2 caspase cleavage sites identified from peptide analysis.</p

Caspase inhibitors block oxidative stress-induced changes in LANA.

<p>BCBL-1 (A, B) or BC-3 (C, D) cells were treated with PBS vehicle control or 100 μM H₂O₂ in the presence of 50 μM Z-FA-FMK (- ctrl), a negative control peptide (lanes 1 and 2), ZVAD-FMK (ZVAD), a pan-caspase inhibitor (lanes 3 and 4), a specific inhibitor of caspase-1 (C1 inh) (lanes 5 and 6) or a specific inhibitor of caspase-3/7 (C3/7 inh) (lanes 7 and 8). Nuclear (A, C) and cytoplasmic (B, D) extracts were harvested and LANA expression was analyzed by western blot and probed with antibody to FLAG, anti-rabbit secondary antibody conjugated to alkaline phosphatase and visualized using stabilized Western Blue substrate (Promega). Results are representative of 3 separate experiments for controls and ZVAD and 2 separate experiments for the C1 and C3/7 inhibitors. Molecular weight markers are shown to the left. To the right of the blots the different forms of LANA are indicated and include the location for full length LANA (LANA-fl) and three forms of LANA migrating below full length LANA with presumptive designations as LANAcp3, LANAcp2 and LANAcp5 based on their mobility.</p

Mutation of both LANA caspase cleavage sites leads to increased IL-1β production.

<p>THP-1 cells were matured overnight by treatment with TPA and then transiently transfected with FLAG-tagged forms of LANA-DM, WT-LANA, LANA-Nmut, or LANA-Cmut. The next day cells were treated with LPS and the level of IL-1β in the supernatants measured 20 hrs later. Cell extracts were also made for protein analysis and immunoblots. (A) IL-1β levels as determined by ELISA following transfection with the LANA plasmid constructs and treatment with LPS. The average level of IL-1β detected following transfection with vector alone (no LPS) was 2131 pg/ml (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005064#ppat.1005064.s003" target="_blank">S3 Fig</a>). (B) Immunoblot for cleaved caspase-1 and actin showing the relative levels of active casapse-1 compared to vector alone for the LPS-treated cells using the LiCor system. Data shown in (A) are the average +/- the standard deviation from 4 independent experiments. ** <i>P</i>< 0.01, *** P<0.005 for two-tailed Student’s t-test. Note that the average values for the single mutants were not statistically significantly different (p>0.05) when compared to wild type.</p