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. 2012 Apr;86(8):4404-19.
doi: 10.1128/JVI.06617-11. Epub 2012 Feb 15.

An alternative Kaposi's sarcoma-associated herpesvirus replication program triggered by host cell apoptosis

Alka Prasad  1 , Michael Lu, David M Lukac, Steven L Zeichner
Affiliations

An alternative Kaposi's sarcoma-associated herpesvirus replication program triggered by host cell apoptosis

Alka Prasad et al. J Virol. 2012 Apr.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to several neoplastic diseases: Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). KSHV replicates actively, via a controlled gene expression program, but can also remain latent. It had been thought that the transition from latent to lytic replication was controlled exclusively by the replication and transcription activator protein RTA (open reading frame 50 [ORF50] gene product). A dominant-negative (DN) ORF50 mutant, ORF50ΔSTAD, blocks gene expression and replication. We produced a PEL cell line derivative containing both latent KSHV genomes and an inducible ORF50ΔSTAD. We unexpectedly found that induction of apoptosis triggered high-level viral replication, even when DN ORF50ΔSTAD was present, suggesting that apoptosis triggers KSHV replication through a distinct RTA-independent pathway. We verified that apoptosis triggers KSHV replication independent of RTA using ORF50 small interfering RNA (siRNA) and also showed that caspase activity is required to trigger KSHV replication. We showed that when apoptosis triggers KSHV replication, the kinetics of late gene expression is accelerated by 12 to 24 h and that virus produced following apoptosis has reduced infectivity. KSHV therefore appears to replicate via two distinct pathways, a conventional pathway requiring RTA, with slower replication kinetics, producing virus with higher infectivity, and an alternative apoptosis-triggered pathway that does not require RTA, has faster replication kinetics, and produces virus with lower infectivity. The existence of a distinct apoptosis-triggered, accelerated replication pathway may have evolutionary advantages for the virus and clinical significance for the treatment of KSHV-associated neoplasms. It also provides further evidence that KSHV can sense and react to its environment.

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Figures

Fig 1
Fig 1
Expression of ORF50ΔSTAD in ORF50ΔSTAD BCBL-1 cells treated with DOX. (A) Expression of ORF50ΔSTAD RNA as a function of the concentration of DOX in ORF50ΔSTAD BCBL-1 cells. The ORF50ΔSTAD BCBL-1 cells have both latent copies of KSHV, including a wild-type ORF50, and a DOX-inducible ORF50ΔSTAD gene engineered into the cell line using the Invitrogen Flp-In T-REx system. The DOX concentrations are shown in micrograms per milliliter. In panel A, the data are plotted on a linear scale for DOX concentration. In panel B, the data are plotted on a logarithmic scale for DOX concentration to enhance visibility of the lower concentration data points. As the DOX concentration increases, ORF50ΔSTAD RNA expression reaches a plateau at a DOX concentration of about 0.5 μg/ml, with no subsequent increases in ORF50ΔSTAD RNA expression at higher DOX concentrations. ORF50ΔSTAD was determined using a real-time RT-PCR assay, normalized to an internal GAPDH standard. (C) Expression of ORF50ΔSTAD RNA ORF50ΔSTAD BCBL-1 cells treated with TPA. ORF50ΔSTAD expression (circles) reaches a plateau between 0.01 μg/ml and 0.1 μg/ml. Further increases in DOX concentration do not lead to increases in expression of ORF50ΔSTAD RNA. Expression of full-length wild-type ORF50 RNA (squares) does not increase in the TPA-treated cells when the DN ORF50ΔSTAD is induced with even the lowest tested concentrations of DOX, confirming that the DN ORF50ΔSTAD can block ORF50 expression.
Fig 2
Fig 2
KSHV production and cell viability in ORF50ΔSTAD BCBL-1 cells treated with increasing DOX. (A) KSHV virions, assayed as protected KSHV DNA. TPA induces KSHV virion production in the absence of DOX. With DOX at low concentrations, induction of ORF50ΔSTAD blocks KSHV virion production. However, at high DOX concentrations (≥5 μg/ml), DOX not only fails to block KSHV virion production triggered by TPA treatment but also is associated with the induction of high levels of KSHV virion production. (B) High DOX concentrations (≥5 μg/ml) are associated with a decrease in cell viability as measured by trypan blue exclusion. (C) The cells were examined with flow cytometry for apoptotic markers using propidium iodide (PI) and annexin V (Ann). At high DOX (≥5 μg/ml), most cells scored positive for PI and Ann staining, indicating apoptosis. Ann-/PI-, no staining for either annexin V or propidium iodide; Ann+, positive staining for annexin V alone; PI+, positive staining for propidium iodide alone; Ann+/PI+, positive staining for both annexin V and propidium iodide.
Fig 3
Fig 3
Induction of apoptosis in BCBL-1 and ORF50ΔSTAD BCBL-1 cells by DCPE. BCBL-1 and ORF50ΔSTAD BCBL-1 cells were examined at 0 h and at 24 h and 48 h after treatment with DOX (a low concentration [0.1 μg/ml]), TPA, and DCPE, and combinations of the agents, as indicated. DCPE had significant cytotoxic effects in both cell lines. TPA treatment was associated with mildly cytotoxic effects in both cell lines. However, in the ORF50ΔSTAD BCBL-1 cells, the toxic effects of TPA, which was likely due to induction of viral replication, was inhibited by the presence of ORF50ΔSTAD induced by DOX, which blocks viral replication.
Fig 4
Fig 4
Expression of wild-type full-length (FL) ORF50 and truncated DN ORF50ΔSTAD after treatment with inducers of ORF50 and ORF50ΔSTAD expression and inducers of apoptosis. ORF50ΔSTAD BCBL-1 cells were treated with TPA to induce ORF50 expression and viral replication via the normal pathway, with a low concentration of DOX (0.1 μg/ml) to induce ORF50ΔSTAD expression, with DCPE to induce apoptosis, and with various combinations of the agents. (A) Expression of full-length wild-type ORF50 and ORF50ΔSTAD RNA, measured by real-time RT-PCR assays. (B) Expression of ORF50 and ORF50ΔSTAD proteins, assessed using immunoblots employing an antipeptide antibody made against an amino-terminal ORF50 peptide. TPA induces expression of FL ORF50 RNA and protein. DOX induces expression of ORF50ΔSTAD RNA and protein. When ORF50ΔSTAD is present, the expression of FL ORF50 is blocked at both the RNA and protein level. The immunoblot in panel B was deliberately overexposed to show that there is a very small amount of residual FL ORF50 expression in the cells prior to induction, slightly visible in the untreated and DCPE-only lanes. BCBL-1 cells spontaneously experience a small amount of lytic KSHV replication even without treatment with an inducing agent, which is suppressed when ORF50ΔSTAD expression is induced with DOX. (Bottom) The blot was reprobed with an antiactin monoclonal antibody (the antiactin-probed blot is a much shorter exposure).
Fig 5
Fig 5
Effects of TPA, apoptosis triggered by DCPE, and induction of the ORF50ΔSTAD expression by a low concentration of DOX on KSHV DNA and virion production in BCBL-1 cells and ORF50ΔSTAD BCBL-1 cells. BCBL-1 and ORF50ΔSTAD BCBL-1 cells were subjected to several treatments: TPA to induce FL ORF50 expression and viral replication via the normal pathway, DOX (a low concentration of 0.1 μg/ml) to induce ORF50ΔSTAD expression, and DCPE to induce apoptosis, and combinations of the agents. Production of total and protected KSHV DNA was measured using a real-time PCR assay (25). (A) KSHV total DNA in BCBL-1 cells. (B) KSHV total DNA in ORF50ΔSTAD BCBL-1 cells. (C) KSHV virion protected DNA in BCBL-1 cells. (D) KSHV virion protected DNA in ORF50ΔSTAD BCBL-1 cells. (E) Effects of low concentrations of DOX on KSHV replication in ORF50ΔSTAD BCBL-1 cells. The levels of ORF50ΔSTAD induced by very low concentrations of DOX (0.001 μg/ml) completely suppress KSHV replication induced by TPA. (The levels of KSHV replication are below the baseline level for samples from cells treated with a low concentration of DOX, because ORF50ΔSTAD blocks the small amount of spontaneous KSHV replication that occurs in the cells.) TPA induces KSHV replication, and TPA-induced KSHV replication is blocked by ORF50ΔSTAD. DCPE also induces KSHV replication, but DCPE-induced KSHV replication is not blocked by ORF50ΔSTAD.
Fig 6
Fig 6
KSHV protein expression in cells treated with TPA or the apoptosis inducer DCPE. ORF50ΔSTAD BCBL-1 cells were treated either with TPA to induce KSHV replication via the conventional pathway or with DCPE to induce apoptosis or left untreated. The cells were studied with confocal immunofluorescence microscopy using as the primary antibody a monoclonal antibody against KSHV ORF K8.1A, a well-characterized KSHV late gene whose product is associated with the plasma membrane (red – ORF K8.1 detected using an anti-ORF K8.1A mouse monoclonal antibody and an Alexa Fluor 687-conjugated goat anti-mouse secondary antibody). Cells were counterstained with DAPI (blue). Both positive-control TPA treatment and treatment with the apoptosis inducer DCPE resulted in high levels of expression of ORF K8.1A in essentially all of the treated cells.
Fig 7
Fig 7
Schematic diagram of the apoptotic cascade and the sites of action of the general and specific caspase inhibitors used in this study. The figure illustrates some key features of the apoptotic cascade, highlighting sites of action of the caspase inhibitors used in this study (⊣). Abbreviations: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TRAIL-R, TRAIL receptor; TNFα, tumor necrosis factor alpha; TNF-R1, tumor necrosis factor receptor 1; IL-1, interleukin-1; IL-1R, IL-1 receptor; FADD, Fas-associated death domain protein; TRADD, TNF receptor-associated death domain; CASP8, caspase-8; PARP, poly(ADP) ribose polymerase; EndoG, endonuclease G; CytC, cytochrome c; ICE, mammalian interleukin-1 beta-converting enzyme; IRAK, interleukin-1 receptor-associated kinase; FLICE, FADD-like ICE; FLIP, FLICE-inhibitory protein; AIF, apoptosis-inducing factor; DFF40, DNA fragmentation factor, 40 kDa; CAD, caspase-activated DNase; IAP, inhibitor of apoptosis; TRAF2, TNF receptor-associated factor 2; MyD88, myeloid differentiation primary response gene (88); tBID, truncated BID; BH3, interactive domain death agonist.
Fig 8
Fig 8
Effects of the caspase inhibitors on apoptosis induced by DCPE and KSHV replication induced by treatment with DCPE. The effects of increasing concentrations of the general caspase inhibitor Z-VAD-FMK, the caspase-9 inhibitor Ac-LEHD-CHO, the caspase-6 and caspase-8 inhibitor Ac-VEID-CHO, and the caspase-3 inhibitor Ac-AAVALLPAVLLALLAPDGVD-CHO on cell apoptosis, as measured by flow cytometric assays for Annexin V and propidium iodide and the production of KSHV virions as protected KSHV DNA. Control treatments included cells not treated with any inducer (Untreated), in which the cells were treated with DMSO, the vehicle used to dissolve DCPE, or cells treated with TPA (as a positive control for the induction of viral replication). Four-component Gompertz plots were fitted to the dose-response data (Stata 11), except for the data obtained when the cells were treated with the caspase-6 inhibitor Ac-VEID-CHO, since no activity was observed for this inhibitor. The general Z-VAD-FMK and specific caspase-9 and caspase-3 inhibitors had similar dose-response curves for inhibition of apoptosis and viral replication. Inhibition of the end effector caspase-3 appears to be sufficient to block KSHV replication triggered in association with apoptosis.
Fig 9
Fig 9
The effects of transfecting the caspase-3-expressing plasmid pcasp3-Wt-GFP into ORF50ΔSTAD BCBL-1 cells were assessed using confocal microscopy. ORF50ΔSTAD BCBL-1 cells were transfected with either the negative-control plasmid pUC19 or pcasp3-Wt-GFP, which expresses functional wild-type caspase-3 as a fusion protein with GFP, and various chemical inducers. Nuclei are stained blue with DAPI, the KSHV late gene ORF K8.1 is stained red, Annexin V is stained magenta, and GFP is shown as green. TPA was given to induce KSHV replication via the orthodox pathway, a low concentration of DOX was given to induce expression of the DN ORF50DSTAD, and DCPE was given to induce apoptosis in the following combinations. The cells were treated with pUC19 alone (A), pUC19 plus DOX (B), pUC19 plus TPA (C), pUC19 plus TPA plus DOX (D), pUC19 plus TPA plus DOX plus DCPE (E), and pcasp3-Wt-GFP (F), The pcasp3-Wt-GFP lead induces both apoptosis and KSHV gene expression, indicating that the end effector caspase-3 is sufficient to induce KSHV replication.
Fig 10
Fig 10
Effects of silencing ORF50 on KSHV replication. (A) ORF50 silencing in BCBL-1 cells treated with TPA and DCPE to induce KSHV replication. BCBL-1 cells were transfected with an ORF50 siRNA and control GAPDH siRNA and then treated with TPA or DCPE to induce KSHV replication. RNAs were assayed for ORF50 RNA at 24 h via a TaqMan real-time RT-PCR assay (results normalized to actin control and a nontargeting siRNA pool). Transfection of ORF50 siRNA resulted in a decrease in ORF50 RNA expression in both cells treated with TPA (86% decrease) and DCPE (93% decrease). (B) ORF50 protein levels in normal BCBL-1 cells after induction by TPA and DCPE and ORF50 silencing. ORF50 siRNA knocks down expression of ORF50 when cells are treated with both TPA and DCPE. (Note that the DCPE lanes were exposed longer, because TPA is a potent inducer of ORF50 expression, while DCPE is not). (C) Effects on KSHV replication of silencing ORF50 in BCBL-1 cells induced with TPA or DCPE. Cells were transfected with siRNA and induced with either TPA or DCPE and assayed for KSHV virions using a real-time PCR assay for protected KSHV DNA after 48 h. Silencing ORF50 led to a statistically significant decrease in virus production when cells were induced with TPA (P = 0.004 [indicated by an asterisk in the figure]), but no statistically significant decrease when ORF50 was silenced in DCPE-treated cells.
Fig 11
Fig 11
KSHV gene expression kinetics during viral replication induced by high concentrations of DOX, by TPA, and by the apoptosis inducer DCPE. Expression of an early KSHV gene, ORF57 (triangles) and a late gene, ORF17 (circles), was assayed using TaqMan real-time RT-PCR assays at serial times after treatment. (A) ORF50ΔSTAD BCBL-1 cells were left untreated (green) or treated with a low concentration (0.1 μg/ml) of DOX (orange) or treated with a high concentration of DOX (red). Exposure to a high concentration (but not a low concentration) of DOX induced expression of the early gene ORF57 followed by expression of the late gene ORF17, but the kinetics of ORF17 expression was accelerated by ∼12 to 24 h over that previously described (25, 34). (B) ORF50ΔSTAD BCBL-1 cells were treated with TPA to induce viral replication via the normal pathway, with a low concentration of DOX to induce ORF50ΔSTAD expression, with DCPE to induce apoptosis, and with various combinations of the agents as follows: untreated (gray), low concentration of DOX (0.1 μg/ml) (lavender), TPA (blue), low concentration of DOX plus TPA (green), DCPE (yellow), low concentration of DOX plus DCPE (orange), and TPA plus DCPE (red). Real-time RT-PCR was used to assay for ORF57 RNA (triangles) and ORF17 RNA (circles). The key comparison is between ORF17 after induction with TPA (blue circles) versus ORF17 after induction with DCPE (yellow circles) or DCPE plus DOX (orange circles) or TPA plus DCPE (red symbols). ORF17 expression kinetics are accelerated after induction with the proapoptotic agent DCPE compared to kinetics observed following induction with TPA whether or not DN ORF50ΔSTAD is induced with DOX. Curves fitted were Gompertz four-component functions. The difference between ORF17 expression at 12 h following TPA induction (blue circles) compared to ORF17 expression following DCPE/apoptosis induction (red circles) was highly significant (P = 0.0003 by t test).
Fig 12
Fig 12
Infectivity of KSV virions made in the normal and apoptosis-associated pathways. Equal numbers of KSHV virions produced by cells in which KSHV replication was induced with TPA and by cells in which KSHV replication was induced in association with apoptosis induced by DCPE were added to activated HUVEC, followed by extensive washing after inoculation (no KSHV DNA was detectable by PCR in the last wash). After 48 h, supernatants from the HUVEC were assayed for KSHV virions using an assay for protected KSHV DNA (25). The virions produced by the normal, TPA-induced replication pathway displayed infectivity more than 5-fold greater than virions made following induction of host cell apoptosis. Values are means plus standard deviations (SD) (error bars). (P < 0.0001 by t test).
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