Caspase-7 Cleavage of Kaposi Sarcoma-associated Herpesvirus ORF57 Confers a Cellular Function against Viral Lytic Gene Expression*

Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 is a viral early protein essential for KSHV multiplication. We found that B cells derived from cavity-based B cell lymphoma with lytic KSHV infection display activation of caspase-8 and cleavage of ORF57 in the cytoplasm by caspase-7 at the aspartate residue at position 33 from the N terminus. Caspase-7 cleavage of ORF57 is prevented by pan-caspase inhibitor z-VAD, caspase-3 and caspase-7 inhibitor z-DEVD, and caspase-7 small interfering RNAs. The caspase-7 cleavage site 30DETD33 in ORF57 is not cleavable by caspase-3, although both enzymes use DEXD as a common cleavage site. B cells with lytic KSHV infection and caspase-7 activation exhibited a greatly reduced level of ORF57. A majority of the cells expressing active caspase-7 appeared to have no detectable ORF57 and vice versa. Upon cleavage with caspase-7, ORF57 was deficient in promoting the expression of viral lytic genes. Inhibiting caspase-7 cleavage of ORF57 in KSHV+ BCBL-1 cells by z-VAD, z-DEVD, or caspase-7 small interfering RNA led to increased expression of viral lytic genes and production of cell-free virus particles. Collectively, our data provide the first compelling evidence that caspase cleavage of ORF57 may represent a cellular function against lytic KSHV infection.

binding to cell death receptors on the cell surface. The intrinsic or mitochondrion-mediated pathway depends on signals within the cell, such as DNA damage, growth factor withdrawal, oxidative stress, or viral infection. Both pathways lead to a cascade of caspase activation: initiator caspases activate effector caspases, which in turn cleave a broad spectrum of intracellular substrates, leading to cell death. KSHV 2 (also called human herpesvirus 8 or HHV8), like other herpesviruses, establishes long term infection in the host, which is essential for development of all forms of Kaposi sarcoma (5,6), primary effusion lymphoma, and multicentric Castleman disease (7,8). KSHV exhibits two viral life cycles: latent and lytic infection. Only a few viral genes are expressed during latency, the role of which is to maintain the viral genome and ensure survival of infected cells (9). Lytic infection is associated with the expression of viral lytic genes and the production of infectious virus. KSHV ORF57 (also known as Mta or KS-SM) is a viral lytic gene that is essential for virus replication and the production of infectious virions in viral lytic infection (10,11). It also promotes viral RNA splicing and the stability of a subset of viral lytic transcripts (12). During our previous investigations into the expression and function of KSHV ORF57 during infection, we consistently detected ORF57 as a doublet on Western blotting. We postulated that the lower band of ORF57 is a cleavage product of full-length ORF57, because the ORF57 gene contains only one ORF that does not encode a product with this size difference from the full-length protein in HEK-293 cells by transient transfection. Here we report that the KSHV ORF57 protein is cleaved by cellular caspase-7 in viral lytic infection and that inhibition of the caspase cleavage of ORF57 leads to increased expression of ORF57-dependent viral transcripts and, consequently, the increased production of cell-free virus particles.
UV Irradiation and Induction of Apoptosis in HeLa Cells-Because UV irradiation induces activation of both caspase-8and caspase-9-mediated cell apoptosis (18 -20), HeLa cells at 24 h after transfection were washed with cold phosphate-buffered saline and exposed to UV light (254 nm, 200 mJ/cm 2 ) in Stratalinker (Stratagene, La Jolla, CA) as described (21). After irradiation, the cells were covered with fresh medium and incubated for an additional 4 h before harvesting.
Caspase Inhibitors-All of the peptide-based caspase inhibitors, z-VAD-fmk, z-DEVD-fmk, and z-VEID-fmk, and negative inhibitor control z-FA-fmk were obtained from EMD (Gibbstown, NJ). Each inhibitor or inhibitor control was used at a final concentration of 50 M in culture medium as indicated in each study. Cells grown in the inhibitor-containing medium at a time point as scheduled were collected for protein and RNA detection.
Preparation of Cytoplasmic Extracts for Caspase Digestion-The cytoplasmic extracts from apoptotic or virus-infected cells were prepared as described (22) with some modifications. After washes with cold phosphate-buffered saline, the cell pellets were resuspended in 5 volumes of ice-cold hypotonic buffer A (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol) and incubated for 15 min on ice. The swollen cells were disrupted by three freeze and thaw cycles. The nuclei were pelleted by 10 min of centrifugation at 10,000 ϫ g at 4°C. The resulting cytoplasmic extracts were used in caspase cleavage assays.
RNA Interference-The expression of individual endogenous caspases was knocked down by RNA interference with using siGenome SMARTpool siRNAs (Dharmacon, Lafayette, CO). ON-TARGETplus siCONTROL (nontargeting siRNA 1; Dharmacon) served as a negative control. Mission lentiviral transduction particles (Sigma) containing a viral vector expressing small hairpin RNA (shRNA) were used to knock down the expression of caspase-7 in BCBL-1 cells. Briefly, BCBL-1 cells were simultaneously transduced with three different stocks of viral particles containing three different shRNAs against human caspase-7 (TRCN03521, TRCN03522, and TRCN03523) at a multiplicity of infection of 1 by using ExpressMag Super magnetic kit (Sigma) or transduced with MISSION nontargeting (NT) shRNA control transduction particles at the same multiplicity of infection. Puromycin (1 g/ml) was added for selection 24 h after transduction. The surviving cells after 2 weeks of selection were induced with VA for 20 h for KSHV lytic infection and were analyzed by Western blotting.
In Vitro Caspase Cleavage Assays-All human active recombinant caspases were purchased from EMD and BIOMOL (Plymouth Meeting, PA). A 20-l cleavage reaction containing 200 ng of substrate protein and 2.5 units of active caspase dissolved in cleavage buffer (100 mM NaCl, 50 mM HEPES, pH 7.4, 10 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 0.1% CHAPS) was incubated for 4 h at 37°C. The reaction was stopped with an equal amount of 2ϫ SDS sample buffer and was immunoblotted. When cytoplasmic extracts were used as a source of active caspases, 10 l of cytoplasmic extracts was mixed with an equal volume of 2ϫ cleavage buffer containing substrate protein.
Immunodepletion-Before immunodepletion of individual endogenous caspases, the cytoplasmic extracts (100 g) were supplemented with NaCl to a final concentration of 100 mM. The specific antibodies (5 g) and 50 l of prewashed protein G-agarose beads (Upstate, Billerica, MA) were added to the cytoplasmic extracts and incubated overnight at 4°C. The immunocomplexes were removed by centrifugation, and the supernatants were used in caspase cleavage assays. The immunodepletion efficiency for caspase-3 or caspase-7 was measured by Western blotting. The extracts incubated with the beads only or with beads covered with nonspecific mouse IgG served as negative controls for the depletion.

Caspase-7 Cleavage of KSHV ORF57
AlexaFluor647, AlexaFlour546 or AlexaFlour488 (Molecular Probes, Carlsbad, CA). RTA-Myc fusion in TREx BCBL-1 RTA stable cells was detected with monoclonal anti-Myc antibody conjugated with fluorescein isothiocyanate (Sigma). The cell nuclei were stained with 4Ј,6Ј-diamino-2-phenylindole. The specific signal was imaged by epifluorescence or confocal microscopy and quantified by using an Image J software.
Flow Cytometry-After 15 h of induction with VA, BCBL-1 cells with stable expression of caspase-7 shRNA or NT shRNA were washed and fixed with 1.6% paraformaldehyde for 15 min. Intracellular staining for ORF57 and ORF59 was performed using eBioscience staining kit (catalog number 00-5523-00) based on Foxp3 staining protocol from eBioscience (San Diego, CA). Rabbit anti-ORF57 polyclonal and mouse anti-ORF59 monoclonal antibodies were labeled, respectively, with AlexaFluor488 and AlexaFlour546 fluorescent dyes using appropriate Zenon labeling kits (Invitrogen). The obtained fluorescence-activated cell sorter data were analyzed with FlowJo v8.8.6 software (Tree Star Inc., Ashland, OR).
Virus Production-Viral replication in BCBL-1 cells (10 6 /ml x 20 ml) was induced by VA in the presence of DMSO (vehicle), 25 M pan-caspase inhibitor z-VAD-fmk, caspase-3-and caspase-7-specific inhibitor z-DEVD, inhibitor negative control z-FA-fmk, or 1 mM phosphonoacetic acid. The culture medium was collected at 96 h after induction and cleared of cell debris by low speed centrifugation (5,000 ϫ g) for 10 min at 4°C, followed by filtration through 0.45-m filters. The cell-free virus particles were then pelleted by high speed centrifugation (30,000 ϫ g) for 1 h at 4°C and washed once with cold phosphate-buffered saline. The virus pellets were resuspended in an equal amount of SDS sample buffer. The amount of virus particles in each sample was determined by the level of KSHV tegument protein ORF45 as assessed by Western blot with anti-KSHV ORF45 (a gift from Yan Yuan).

Induction of KSHV Lytic Infection in KSHV ϩ B Lymphocytes Activates Initiator Caspase-8 and Production of a Caspase-Cleaved KSHV ORF57-
In cell lysates of KSHV ϩ BCBL-1 and JSC-1 cell lines after reactivation of the virus with a low dose of chemical inducers (Fig. 1A), we noticed KSHV ORF57 expressed as a doublet on Western blotting. The lower band was assumed to be a cleavage product of full-length ORF57 and was missing in HEK-293 cells by transient transfection of ORF57 cDNA. Although the proportion of cleavage varied among the examined chemical inducers and between the two cell lines, production of the lower band of ORF57 appeared proportionally to be higher in JSC-1 cells, in particular when butyrate and 12-O-tetradecanoylphorbol-13-acetate were used as inducers. 12-O-Tetradecanoylphorbol-13-acetate was a much stronger inducer in JSC-1 cells than in BCBL-1 cells (Fig.  1A), whereas VA was a better inducer than butyrate for the induction of ORF57 in both cell types. Because VA is not toxic to a noninfected Burkitt lymphoma B cell line (23), we used VA for further analyses.
When BCBL-1 cells were induced with VA in the presence or absence of pan-caspase inhibitor z-VAD-fmk, production of the lower band of ORF57 was strongly reduced in the presence of z-VAD-fmk, which inhibits the caspase cleavage of poly-(ADP-ribose) polymerase (PARP; Fig. 1B). To exclude the possible nonspecific effects of chemical inducers, this observation was further verified in TREx BCBL-1 RTA cells (14), which inducibly express RTA (ORF50), a potent transactivator for KSHV lytic induction (24). RTA induction by dox in these cells led to the production of both full-length and cleaved ORF57, along with the production of cleaved PARP (Fig. 1C). Caspase inhibitor z-VAD-fmk inhibited the cleavage, indicating that VA-or RTA-induced cleavage of ORF57 is mediated by active caspases during viral lytic induction (Fig. 1, B and C). To confirm the connection between caspase activation and ORF57 cleavage, we transfected HeLa cells with a vector expressing full-length ORF57 with a FLAG tag on its N terminus. We observed that ORF57, like PARP, was actively cleaved in HeLa cells when activation of initiator caspase-8 (supplemental Fig. S1) and apoptosis was induced by UV irradiation as reported (18,19,25), and the cleavage was reduced by z-VADfmk (Fig. 1D). These data suggest that caspase activity is needed for ORF57 cleavage.
Because activation of initiator caspase-8 triggers the extrinsic apoptosis pathway, which differs from the intrinsic apopto-sis pathway initiated by activation of initiator caspase-9 (2), we examined whether lytic KSHV induction activates a particular apoptosis pathway. We found that only initiator caspase-8 was triggered in BCBL-1 cells with lytic KSHV induction by VA, along with cleavage of expressed ORF57 (Fig. 1E). Consistent with this, RTA expression in TREx BCBL-1 RTA cells led to the production of active caspase-8, but not active caspase-9, from its inactive full-length procaspase, along with the induced production of cleaved PARP and ORF57 ( Fig. 1, C and F).
Mapping the Aspartate Residue at Position 33 from the N Terminus of ORF57 as a Caspase Cleavage Site-To understand where the cleavage takes place on ORF57 protein, HeLa cells were transfected with a vector expressing ORF57 tagged with either an N-or C-terminal FLAG ( Fig. 2A) and were UV-irradiated to induce cell apoptosis. The cleavage products of the ORF57 fusions in the cells were then analyzed by anti-ORF57 or anti-FLAG Western blotting. Because the anti-ORF57 antibody was derived from immunization with a N-terminal peptide spanning amino acid residues 119 -132 of ORF57, any cleavage product containing this region should be recognized by this anti-ORF57 antibody. Using this strategy by anti-FLAG Western blotting, we showed that the cleavage takes place in the N-terminal region of ORF57, upstream of the antibody recognition site (Fig. 2B, compare the anti-ORF57 and anti-FLAG Western blotting of FLAG-ORF57).
Next, we analyzed the cleavage efficiency of ORF57 in UVirradiated HeLa cells by introducing D-to-A point mutations into each of the four putative caspase cleavage sites in the N-terminal region of ORF57 that were predicted by using a software program, CASVM (26). We identified the aspartic acid residue at position 33 from the N terminus of ORF57 responsible for the cleavage (Fig. 2, A and C). A cleavage product of ORF57 was obtained only from wild-type (wt) ORF57, and not from mutant D33A ORF57 (Fig. 2D), when wt and mutant D33A ORF57 expressed from HEK-293 cells were separately incubated with BCBL-1 extracts bearing VA-induced active caspases (Fig. 2E). Together, these data identify the caspase cleavage site of ORF57 as 30 DETD 33 followed by an alanine at the cleavage site P 1 ' position (27). Thus, the cleaved ORF57 lacks the first 33 residues from the N terminus of ORF57.
Identification of Cellular Caspase-7 as an Essential Caspase in the Cleavage of KSHV ORF57-Because the mapped caspase cleavage site on ORF57 resembles a cleavage site for caspase-2, -3, and -7 (27), we performed a series of caspase digestions with (FLAG-ORF57) were UV-irradiated to induce apoptosis and were then blotted for full-length (FL) and cleaved ORF57 fusions with anti-ORF57 and anti-FLAG antibodies. C, identification of the aspartic acid residue at position 33 from the N terminus of ORF57 as a caspase cleavage site by point mutational analysis. Four putative caspase cleavage sites at the N terminus of ORF57 were mutated (Asp 3 Ala) individually or in combination in ORF57-FLAG expression vectors. The mutants were expressed in HeLa cells exposed to UV and blotted with anti-FLAG. PARP cleavage served as an internal control for apoptotic induction and sample loading. D, cleavage of wt or mutant D33A ORF57 by VA-induced BCBL-1 cell extracts containing active caspases. Each form of ORF57-FLAG fusion was expressed from HEK-293 cells, affinity-purified with anti-FLAG beads, and digested with BCBL-1 extracts prepared 48 h after VA treatment. Western blot was performed with an anti-FLAG antibody. E, ORF57 cleavage activity of the cytoplasmic extracts isolated from BCBL-1 cells with or without VA induction for 48 h. The cytoplasmic extracts were mixed with purified ORF57-FLAG proteins, and the cleavage of ORF57 was assessed by Western blot with an anti-FLAG antibody. none, no extract addition in the cleavage assay.
Caspase-7 Cleavage of KSHV ORF57 FLAG fusion expressed from baculovirus. Caspase-7 was identified as a caspase primarily responsible for the cleavage of ORF57 (Fig. 3A). Even though both caspase-3 and caspase-7 utilize a cleavage site composed of the amino acid residues DEXD (27), found in the classic caspase-3 substrate PARP, and the caspase-3 used in the cleavage assay was active (supplemental Fig. S2), caspase-7 was the primary candidate that cleaved ORF57. Although ORF57 could be cleaved to a much lower extent by caspase-10 and caspase-5 (Fig. 3A), titration assays demonstrated that significantly more caspase-10 compared with caspase-7 was needed to cleave ORF57 (Fig. 3B). All other caspases tested showed no cleavage activity for ORF57. ORF57 digestion by caspase-7 and less efficiently by caspase-10, but not by caspase-3, was also verified with FLAG-tagged ORF57 expressed from mammalian cells HEK-293 (Fig. 3C), indicating that protein modification of ORF57 in mammalian cells does not affect caspase-7 cleavage. When purified wt ORF57-FLAG and mutant D33A ORF57-FLAG fusions expressed from HEK-293 cells were compared in caspase-7 cleavage assays, only the wt ORF57 was cleaved (Fig. 3D), confirming the mapped caspase cleavage site (Fig. 2C) as the site for caspase-7 cleavage of ORF57.

Cellular Caspase-7 Plays a Key Role in ORF57 Cleavage in BCBL-1 Cells during KSHV Lytic Infection-
We next examined the expression kinetics of KSHV ORF57 and caspase-7 in VA-treated BCBL-1 cells. Both the cleavage product of ORF57 and the production of active caspase-7 from its catalytically inactive procaspase appeared 24 h after VA induction, similar to the time course of cleaved PARP appearance (Fig. 3E, left panels). We verified the production of active caspase-7 and the cleavage of ORF57 and PARP during KSHV lytic infection in doxinducible TREx BCBL-1 RTA cells (Fig. 3E, right panels). Both the cleavage of ORF57 and the production of active caspase-7 appeared only in the cytoplasm (Fig. 3F), in contrast to PARP, which is cleaved in the nucleus by caspase-3 (28).
We further verified the role of caspase-7 in ORF57 cleavage in BCBL-1 cells by using four additional approaches. First, we used z-DEVD-fmk, a caspase-3/7 inhibitor (29,30), in addition to the pan-caspase inhibitor z-VAD-fmk and found that ORF57 cleavage was reduced in VA-treated BCBL-1 cells when production of active caspase-7 decreased (Fig. 4A). Because z-VEID-fmk, a caspase-3/6 inhibitor (31), had no effect on ORF57 cleavage (Fig. 4A), we ascribed the reduced ORF57 cleavage in the presence of z-DEVD-fmk to the inhibition of active caspase-7. Second, depletion of caspase-7 from the cytoplasmic extracts of VA-induced BCBL-1 cells reduced the cleavage of ORF57, but depletion of caspase-3 did not (Fig. 4B). Third, knocking down caspase-7 expression in UV-irradiated HeLa cells (Fig. 4C, left panels) or in BCBL-1 cells (Fig. 4C, right panels) by RNA interference prevented the cleavage of ORF57, whereas knocking down caspase-3 expression in HeLa cells (Fig. 4C, left panels) had no effect on cleavage. Fourth, actinomycin D (32) induced the cleavage of ORF57 in caspase-3-deficient MCF-7 cells (Fig.  4D). Collectively, the data indicate that caspase-7 does play a major role in ORF57 cleavage in these cells.
Inverse Correlation between ORF57 Expression and Active Caspase-7 Production in KSHV Lytic Infection-We wished to determine whether caspase-7 activation occurred in direct correlation with ORF57 expression. Despite the existence of a few spontaneous apoptotic cells with active caspase-7, which have latent KSHV infection, virus reactivation in BCBL-1 cells dramatically increased the number of active caspase-7-positive cells, consistent with the results from Western blotting. We also observed an inverse correlation between active caspase-7  Fig. S4) expression in BCBL-1 cells with lytic KSHV infection. This relationship is more remarkable in dox-induced TREx BCBL-1 RTA cells in which the majority of dox-induced cells expressing ORF57 did not display active caspase-7 or vice versa (Fig. 5B). This was also observed for active caspase-3 and caspase-8 (supplemental Fig. S3). Further analysis showed that among the ORF57-positive BCBL-1 cells, only 19% of them at 24 h of induction were positive for active caspase-7. The number of double positive cells increased to 43% in the BCBL-1 cells induced by VA for 48 h (Fig. 5A). In contrast, the number of TREx BCBL-1 RTA cells at 24 h of induction stained for active caspase-7 and also positive for ORF57 was 56% (Fig. 5B and supplemental Fig. S3). However, all of the double positive cells, regardless of whether they were collected at 12 h (Fig. 6A) or 24 h (Fig. 6B) of induction, showed a prominent reduction of ORF57. The cells with active caspase-7 staining and apoptotic nuclear bodies exhibited much more reduced expression of ORF57 (Fig. 6B), when compared with the cells without apoptotic nuclear bodies (Fig. 6). In the apoptotic cells, a reduced expression of ORF57 could be further diminished by active caspase-7, and the resulting cells appeared to have no detectable ORF57 (compare cell 1 with cell 2 in Fig. 6B, single color versus overlay).
Given the fact that ORF57 expression and active caspase-7 production is inversely correlated, we investigated whether the cells expressing active caspase-7 ever underwent KSHV reactivation. We costained BCBL-1 TREx RTA cells induced with 1 g of dox for coexpression of RTA, ORF57, and active caspase-7 and analyzed by confocal microscopy. As shown in Fig. 7, more than 99% of ORF57 ϩ cells counted coexpressed RTA, but the majority of the ORF57 ϩ or RTA ϩ cells expressed no active caspase-7 or vice versa. There were only 24% of active caspase-7 ϩ cells coexpressing both RTA and ORF57, with 10% coexpressing RTA only and none for coexpression of ORF57 alone. Because RTA expresses earlier than ORF57, RTA coexpression with active capase-7, but not with ORF57, during KSHV lytic induction suggests that the cells are either at the early stage of induction or at a stage in preventing further reactivation by active caspase-7. Nevertheless, these data confirmed that the cells positive only for active caspase-7 did not undergo full KSHV reactivation, or their viral reactivation was completely diminished by active caspase-7 at an early stage of the reactivation.   a K-bZIP protein. We analyzed the effect of caspase cleavage of ORF57 on the expression of these two genes in HEK-293 cells by cotransfecting assays with increasing amounts of full-length ORF57, a truncated (⌬1-33 amino acids) mutant that mimics caspase-cleaved ORF57, or a D33A mutant (Fig. 2). Although all three versions of ORF57 appeared as nuclear proteins (data not shown), the truncated (⌬1-33 amino acids) mutant was deficient in promotion of ORF56 expression (Fig. 8A) or K8␣ (K-bZIP) production (Fig. 8B) from K8␤ transcripts (17). The reduction of K8␣ production from K8␤ transcripts indicates that the truncated mutant is deficient in promoting viral RNA splicing (13). When coexpressed with full-length ORF57, the truncated mutant in a small amount was suppressive for the full-length ORF57 activity in promotion of K8␤ RNA splicing (supplemental Fig. S5).

Caspase Cleavage of ORF57 Attenuates Its Function in Promoting Viral Lytic Gene Expression and Virus Production-
We also assessed the functional deficiency of caspase-cleaved ORF57 in a Bac36-⌬57 stable cell line, which contains an ORF57-null KSHV genome and expresses no ORF57 and thus has reduced expression of a subset of viral lytic genes (ORF56, ORF59, K8, etc.) upon lytic induction (11). Three versions of ORF57 (full-length, truncated ⌬1-33, and D33A) were all efficiently expressed in Bac36-⌬57 stable cells after transfection (Fig. 8C). When KSHV ORF59 expression was used as a readout, both full-length and D33A, and not the truncated ⌬1-33, were able to efficiently induce the expression of ORF59, a viral DNA polymerase processivity factor. As predicted, the D33A, which is resistant to caspase-7 cleavage, worked even better (ϳ2.7-fold) than the fulllength ORF57 in the complement assay to restore ORF59 expression from Bac36-⌬57 stable cells (Fig. 8C).
Whether caspase cleavage of ORF57 affects virus production was examined in BCBL-1 cells with lytic induction. As shown in Fig. 9A, BCBL-1 cells induced with VA in the presence of the pan-caspase inhibitor z-VAD-fmk showed a great increase in the expression of ORF59 and PAN RNA (polyadenylated nuclear RNA with unknown function) and an intermediate increase in the expression of K8 and RTA. There were also more ORF59 ϩ cells in the presence of z-VAD-fmk but not in the presence of the inhibitor negative control z-FA-fmk or DMSO, despite a similar level of ORF57 expression among the three groups (supplemental Fig. S6). Together, these data suggest that caspase cleavage of ORF57 during viral lytic infection permits the host cells to restrain the expression of viral lytic genes and to control virus production.
To quantify cell-free virus particles in the culture medium of BCBL-1 cells induced with VA in the presence or absence of z-VAD-fmk, we enriched the cell-free virus particle in the cul- ture medium by high speed centrifugation after filtration and measured the amount of virus particles by Western blotting of the virus-associated KSHV tegument protein ORF45 (33). As predicted, cell-free virus production greatly increased in the cells only when the caspase cleavage of ORF57 was blocked by z-VAD-fmk (Fig. 9B). In contrast, the cells treated with phosphonoacetic acid, a viral DNA polymerase inhibitor, showed no production of cell-free virus particles, whereas the cells treated with DMSO or z-FA-fmk released only a minimal amount of cell-free virus particles into the culture medium. Because there was little change in cell viability (data not shown) among the different treatment conditions at the time when the culture medium was collected, we conclude that the increased production of cell-free virus particles from the cells receiving z-VADfmk resulted from the prevention of ORF57 cleavage.
Because z-VAD-fmk is a pan-caspase inhibitor and has pleiotropic effects, we utilized z-DEVD-fmk, a caspase-3/7-specific inhibitor (Fig. 9C), and caspase-7 siRNA (Fig. 9, D and E) to further define the role of caspase-7 activation in KSHV gene expression and virus production. In both studies, we observed that inhibition of caspase-7 activity promoted the expression of ORF59, as monitored by Northern blot (Fig. 9C) or by flow cytometry (Fig. 9, D and E) and the production of cell-free virus particles as determined by ORF45 immunoblot (Fig. 9C). In particular, we observed by fluorescence-activated cell sorting a remarkable reduction of the cells with cleaved ORF57 (cells with ORF57 ϩ staining only) along with a nearly 2-fold increase of the cells with ORF57 ϩ ORF59 ϩ (double positive) staining when the VA-induced BCBL-1 cells with caspase-7 knockdown were compared with the cells with NT siRNA treatment (Fig. 9,   (20,50,100, and 200 ng) of full-length, caspase-cleaved (⌬1-33 amino acids (aa)), or D33A ORF57-FLAG plus a constant amount (300 ng) of plasmid pVM9 expressing a KSHV ORF56-FLAG fusion protein (A) or K8␤-expressing vector pST3 (B) and being adjusted with an empty vector to a total amount of 500 ng of plasmid DNA for each transfection. The cells samples were blotted, respectively, with anti-FLAG and anti-ORF57 antibodies (A) or with anti-K8 and anti-ORF57 (B). Tubulin served as sample loading. C, caspase-cleaved ORF57 is unable to efficiently restore KSHV ORF59 expression in Bac36-⌬57 stable cells. The Bac36-⌬57 stable cells in a 6-well plate transfected either with 500 ng of an empty FLAG vector or a plasmid expressing full-length, ⌬1-33, or D33A ORF57 for 24 h were induced with 3 mM butyrate for additional 24 h. The expression of ORF57-FLAG and ORF59 was then assessed, respectively, by anti-ORF57 (red) and anti-ORF59 (red) immunofluorescence staining. Green fluorescent protein (GFP, green) expression indicates the presence of the Bac36-⌬57 genome in the stable cells. The cell nuclei were stained with 4Ј,6Ј-diamino-2-phenylindole (blue). Total ORF59 ϩ cells on the right from each complement assay were obtained by microscopy from five fields.
D and E). These data clearly indicate an important role of caspase-7 cleavage of ORF57 in regulation of the expression of ORF57 targets.

DISCUSSION
KSHV ORF57 encodes a viral nuclear protein essential for viral gene expression and virus multiplication during viral lytic infection. In this report, we have demonstrated that KSHV ORF57 contains a classical caspase cleavage site, 30 DETD 33 , in its N terminus and can be cleaved by cellular caspase-7 during virus lytic induction (Fig. 1). Although the cleaved ORF57 retains three intact NLSs (15), it remains mainly in the cytoplasm of infected cells. The consequence of caspase-7 cleavage of ORF57 in lytic KSHV infection is reducing a subset of viral lytic gene expression and virus production. However, when ⌬1-33 ORF57 with an added N-terminal methionine was overexpressed as a FLAG-tagged protein, with the FLAG on its C terminus, it can be translocated into the nucleus, but its ability to promote the expression of viral lytic genes (ORF56, ORF59, and K8) remains attenuated in cotransfection assays (Fig. 8). It is unclear how the N-terminal truncation contributes to the attenuated function of ORF57. Computer predictions using NetPhos (34) and Scansite 2.0 (35) suggest that the first 33 N-terminal amino acid residues also contain several putative phosphorylation sites that could potentially be important to various ORF57 functions, as has been reported for its homologue Epstein-Barr virus EB2 (36). Mutation of these putative sites leads to disruption of ORF57 functions (data not shown). Nevertheless, our data clearly indicate that caspase-7 cleavage of ORF57 appears to be a cellular function against virus infection in viral lytic infection. Viral lytic infection leads to cell apoptosis (23) and production of active caspases. As reported in this study, KSHV lytic infection activates caspase-8-mediated apoptosis pathway, resulting in the activation of caspase-3 and -7 in the infected B cells. However, it remains to be understood how this apoptosis pathway can be induced during viral lytic infection, which is beyond our focus in the present study. Caspase-3 and -7 share 53% sequence identity, high structural similarity, and nearly identical substrate preference, with both enzymes targeting the same DEXD motif (27). Gene knock-out experiments have shown that caspase-3 and -7 have similar phenotypes (37). However, given the overlap between caspase-3 and -7 in their substrate specificity, it is remarkable that caspase-7, but not caspase-3, has a specific role in the cleavage of KSHV ORF57 at a classical DETD2A cleavage site. Recent studies have suggested that substrates can have a substantial preference for caspase-7 over caspase-3 or vice versa. The reported substrates with specificity for caspase-7, but not for the closely related caspase-3, are Nogo-B (SSTD2S) (38), claspin (DEYD2G) (39), and Ataxin-7 (PKMD2G and FDPD2I) (40). A substrate preference for caspase-3 over caspase-7 is found in protein phosphatase-1 inhibitor-3 (DTVD2X) (41) and in excitatory amino acid transporter EAAT2 (DTID2S) (42). These data suggest that caspase-7 and caspase-3 have distinct functions. Moreover, caspase-3 and -7 preferentially function in different compartments of apoptotic (28) and virus-infected cells, with caspase-3 acting in the nucleus and caspase-7 in the cytoplasm. The preferential cleavage of ORF57 by caspase-7 in the cytoplasm of infected cells provides a great advantage for the cells to attenuate ORF57 immediately after its translation. Because ORF57 functions mainly in the nucleus, caspase-7 cleavage of ORF57 in the cytoplasm enables the infected cells to take action even before ORF57 gets into the nucleus, an attractive strategy to prevent KSHV replication and virus production.
The insensitivity of the characterized caspase-7 cleavage site 30 DETD 33 to caspase-3 might be evolutionarily conserved. After the expression of ORF57 in lytic infection, KSHV expresses K7, a caspase-3-specific inhibitor (43), and vIRF3, which preferentially suppresses caspase-3 and only slightly suppresses caspase-7 (44). Therefore, having an ORF57 that is resistant to caspase-3 cleavage, a key mediator of apoptosis in mammalian cells with promiscuous substrates, could allow the virus to have enough ORF57 at early infection to promote the expression of the ORF57 targets.
Caspase-10 showed some activity for ORF57 cleavage in vitro. However, we were unable to determine its role in vivo because of the lack of a specific antibody (45). We are assuming that caspase-10, as well as caspase-5, which displayed a little activity in vitro, plays only minimal roles in ORF57 cleavage in vivo, because both immunodepletion of caspase-7 from cell extracts and siRNA-mediated knockdown of caspase-7 expression from cells dramatically reduced ORF57 cleavage.
KSHV also expresses protein inhibitors of caspase-8 and caspase-9 during viral latent infection. Notably, all of the latent proteins are expressed during viral lytic infection, when they presumably execute a similar function to prevent the lytically infected cells from apoptosis. However, activation of initiator caspase-8 and induction of caspase-8-mediated apoptosis pathway during lytic KSHV infection suggests that these viral caspase inhibitors do not function efficiently in lytic infection. KSHV latent vFLIP (ORF71/K13) is a caspase-8 inhibitor but also inhibits caspase-9 activity (46) to prevent death receptormediated apoptosis. Thus, vFLIP is essential for the survival of infected lymphoma cells (47). KSHV latent K15 is a HAX-1 (HS-1 associated protein-1)-binding protein that acts as a potent inhibitor of Bax-induced apoptosis (48) by inhibiting caspase-9 (49). By doing so, KSHV equips itself to develop persistent and latent infection for cell proliferation and transformation by protecting latently infected cells from apoptosis. In KSHV lytic infection, these proteins, together with lytic K7, vIRF3, and vBcl-2, presumably prevent viral lytic infection-induced apoptosis of the infected cells to ensure a complete viral life cycle and virus production. These could be the cells represented by predominant expression of ORF57 and/or RTA but no active caspase-7 (Figs. 5-7 and supplemental Figs. S3 and S4). However, KSHV lytic infection does induce caspase-8-mediated apoptosis pathway, which activates caspase-7 as feedback in fighting against virus infection. This leads to caspase-7 cleavage of ORF57 in the infected cells very early in the lytic infection. The consequence of this battle is to limit the expression of this essential viral protein as shown in the cells mainly with active caspase-7 staining (Figs. 5-7 and supplemental Figs. S3 and S4) and to delay virus productivity even before the virus to complete its life cycle. We have the following two observations to support this scenario: (a) the majority of cells with active caspase-7 expression alone underwent apoptosis, with a fragmented nucleus and without, or at least suppressed, KSHV reactivation, whereas other cells with ORF57 and/or RTA coexpression did not exhibit apoptotic nuclear morphology and greatly diminished the presence of active caspase-7 (Figs. 6 and 7); and (b) specific reduction of caspase-7 by RNA interference or a peptide-based inhibitor z-DEVD could prevent ORF57 from cleavage and enhance the expression of ORF57 targets in more cells with KSHV lytic infection (Fig. 9, C-E).