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

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 in vitro 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-1 beta (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

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

Peptides containing LANA caspase cleavage sites inhibit IL-1β production.

<p>Cell permeable peptides with an N-terminal cellular HIV-1 Tat delivery sequence RKKRRQRRR containing the N-terminal caspase cleavage site of LANA (T-LP-Nterm) or the C-terminal caspase cleavage site of LANA (T-LP-Cterm) were tested as potential inhibitors of IL-1β production following treatment of THP-1 cells with LPS. ZVAD was used as a positive caspase inhibitor control. (A) Effect of ZVAD (50 μM), T-LP-Nterm (50 μM) and T-LP-Cterm (50 μM) on IL-1β in the supernatant from THP-1-treated cells. Δata is from five separate experiments (except for T-LP-Nterm, 4 experiments). ***, <i>P</i>< 0.0005, **** <i>P</i>< 0.0001 for two tailed paired Student’s t-test. The average IL-1β λεωελ ϕορ untreated and LPS treated THP-1 cells was 276 pg/ml and 2344 pg/ml, respectively. (B) Immunoblot analysis for cleaved (mature) caspase-1 and actin as a loading control. The fold change in cleaved caspase expression normalized to β-actin is shown just below the immunoblot using the LiCor system. (C) Immunoblot analysis for Pro-IL-1β and actin as a loading control. The fold change in Pro-IL-1β expression normalized to β-actin is shown just below the immunoblot using the LiCor system.</p

Caspases cleave FLAG-LANA <i>in vitro</i>.

<p>Caspase-1, 2, 3, 6, 7, 8, 9, 10 were tested for their ability to cleave LANA. HEK293T cells were transiently transfected with FLAG-LANA-pCMV, tagged with FLAG at the N-terminus as stated in Materials and Methods. Nuclear extracts containing LANA were harvested in the absence of protease inhibitors, treated with caspases for 16 hrs at 37°C and then analyzed by western blot. (A) FLAG-LANA treated with 0.15 units caspases μg^-1 protein and probed with antibody to FLAG, anti-rabbit secondary antibody conjugated to alkaline phosphatase and visualized using stabilized Western Blue substrate (Promega). DMSO vehicle (lane 1) and 50 μM ZVAD (lane 10) were used as controls. FLAG-LANA and the primary cleavage products containing FLAG (FLAG-LANAcp1 and FLAG-LANAcp3) are indicated. Other products that increased in intensity to some of the caspases (caspase-1 and caspase-10) were not further investigated since these forms appeared to be present in the control extracts as well. (B) Same as Fig 2A except that a lower amount (0.07 units) of the caspases was used with an incubation time of only 4 hrs. Full-length FLAG-LANA (FLAG-LANA) and the primary caspase cleavage product (FLAG-LANAcp1) are indicated. FLAG-LANA (calculated molecular weight of 127 kDa) migrated as a band of approximately 160 kDa using the NuPage LDS gel system (InVitrogen). Molecular weight markers in kDa are indicated to the left of each blot. The molecular weight of FLAG is 1 kDa.</p

Peptides containing LANA caspase cleavage sites inhibit caspase activity, decrease PARP cleavage and increase cell viability.

<p>Caspase activity assays were performed as described in Materials and Methods. Peptides containing the N-terminal caspase cleavage site of LANA (LP-Nterm) or the C-terminal caspase cleavage site of LANA (LP-Cterm) were dissolved in PBS and tested as potential caspase inhibitors at a concentration equal to the commercial caspase-1 substrate (Ac-YEVD-pNa, 200 μM, Sigma) or caspase-3 substrate (Ac-DEVD-pNa, 200 μM, Sigma). PBS was used as the negative control. (A) Effect of LP-Nterm and LP-Cterm on caspase-1 activity. (B) Effect of LP-Nterm and LP-Cterm on caspase-3 activity. In both A and B, the linear caspase activity rate (rfu/min) is plotted with the assay run in triplicate in two separate experiments with one representative experiment shown. Note: The increase in activity of caspase-1 by the LP-Nterm may be due to the presence of a cysteine in this peptide which could protect loss of activity of the enzyme due to oxidation over time. (C) HEP3B cells were pre-treated with DMSO, ZVAD, Tat-LP-Nterm (T-LPN) or Tat-LP-Cterm (T-LPC) peptide for two hrs and then exposed to DMSO alone (control) or etoposide for 16 hrs at 50 μm to induce apoptosis and PARP cleavage. Cell lysates were analyzed by western blot for cleaved PARP and β-actin (loading control). The percent inhibition of PARP cleavage as determined using the LiCor system is indicated below the blot. The immunoblot shown is a representative of 3 different experiments with Tat-LP-Nterm and Tat-LP-Cterm at 50 μM (Tat-LP-Cterm at 100 μM done once). (D) Cell viability following pretreatment with DMSO, ZVAD or T-LPC followed by DMSO or etoposide treatment. Cells were pretreated with DMSO (control), Tat-LP-Cterm or ZVAD (50 μM) for two hrs followed by etoposide treatment at 50 μM. To assess cell viability total cellular protein obtained from the adherent cells following 3 PBS washes was determined by BCA assay. The results shown are the average of 4 independent experiments. ** P< 0.01, * P< 0.05, for two tailed Student’s t-test.</p