KSHV RTA Abolishes NFκB Responsive Gene Expression during Lytic Reactivation by Targeting vFLIP for Degradation via the Proteasome

Kaposi's sarcoma herpesvirus (KSHV) is a gamma-2 herpesvirus present in all cases of Kaposi's sarcoma, primary effusion lymphoma (PEL), and some cases of multicentric Castleman's disease. Viral FLICE inhibitory protein (vFLIP) is a latently expressed gene that has been shown to be essential for survival of latently infected PEL cells by activating the NFκB pathway. Inhibitors of either vFLIP expression or the NFĸB pathway result in enhanced lytic reactivation and apoptosis. We have observed a decrease in vFLIP protein levels and of NFκB activation in the presence of the KSHV lytic switch protein RTA. Whereas vFLIP alone induced expression of the NFĸB responsive genes ICAM1 and TNFα, inclusion of RTA decreased vFLIP induced ICAM1 and TNFα expression in both co-transfected 293T cells and in doxycycline induced TREx BCBL1 cells. RTA expression resulted in proteasome dependent destabilization of vFLIP. Neither RTA ubiquitin E3 ligase domain mutants nor a dominant-negative RAUL mutant abrogated this effect, while RTA truncation mutants did, suggesting that RTA recruits a novel cellular ubiquitin E3 ligase to target vFLIP for proteasomal degradation, allowing for inhibition of NFĸB responsive gene expression early during lytic reactivation.


Introduction
Kaposi's Sarcoma Herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), is the causative agent of Kaposi's Sarcoma (KS) and is associated with primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). As with all herpesviruses, KSHV has two phases of its lifecycle, latent infection and lytic replication. During latency, only five genes are expressed, and the genome is maintained as a multicopy episome tethered to the chromatin by the Latency-Associated Nuclear Antigen (LANA) [1,2]. Lytic replication is initiated via expression of the viral regulator of transcription activation (RTA) protein both directly in primary infection and as a lytic switch factor during reactivation [3]. Viral FLICE inhibitory protein (vFLIP) is a latently expressed protein that has homology to the cellular FLIPs which function as inhibitors to death receptor induced apoptosis. vFLIP has also been shown to activate NFkB by interacting with the IkB kinase complex (IKK) [4][5][6][7]. This occurs via activation of IKK followed by phosphorylation of IkB and subsequent translocation of NFkB into the nucleus [8,9]. Knockdown of vFLIP by siRNA in PEL cell lines results in apoptosis, consistent with its reported anti-apoptotic activity [10]. Chemical inhibition of NFkB by Bay11-7082 in PEL cells promotes lytic reactivation while activation of NFkB inhibits lytic promoters [11,12]. This data suggests that vFLIP induced NFkB activation is required for cell survival and maintenance of latent infection and must be negatively regulated in order for the virus to enter the lytic replication cycle.
Aside from being the major switch for reactivation of KSHV from latency, RTA has been assigned intrinsic E3 ubiquitin ligase activity [13]. RTA has been shown to specifically target IRF7 for proteasomal degradation as a mechanism to abrogate the interferon a/b response to viral infection [13]. In addition, RTA has been reported to degrade a number of known RTA repressors such as Hey1, LANA and NFkB (p65) [14,15]. More recently RTA has been shown to also recruit and stabilize the cellular ubiquitin ligase RAUL in order to target both IRF3 and IRF7 for proteasomal degradation [16].
Here we provide evidence that RTA targets vFLIP for proteasomal degradation resulting in downregulation of NFkB responsive gene expression early during lytic reactivation. RTA expression inhibited vFLIP induced NFkB activation as well as vFLIP-mediated NFkB responsive gene expression. In particular, vFLIP induced ICAM1 and TNFa gene expression in 293T cells and this gene expression was abolished upon expression of RTA. We observed similar transient down-regulation of ICAM1 and TNFa in doxycycline-induced TREx BCBL1-RTA cells. Expression of RTA resulted in dose dependent destabilization of vFLIP and this effect was abrogated by treatment with proteasome inhibitors. RTA and RAUL ubiquitin ligase mutants did not affect RTA induced vFLIP downregulation, while several RTA truncation mutants displayed a moderate defect in vFLIP degradation and were unable to down-regulate vFLIP induced TNFa and ICAM1. Taken together this data suggests that RTA downregulates vFLIP induced NFkB activation and associated gene expression through recruitment of another as of yet unidentified cellular ubiquitin ligase.

Immunoblot analysis
Cells were harvested by direct addition of 2.56 Laemmli Buffer followed by incubation at 1006C for 5 min. Lysates were run on a 10% SDS-PAGE or Any kD Mini-PROTEAN TGX Precast Gel (Biorad) and Tris-glycine running buffer. Proteins were transferred to a PVDF membrane using a semi-dry transfer system at 25 V for 20 min. Membranes were blocked in 5% non-fat dry milk in PBS for 1 hr followed by incubation with primary antibody overnight at 46C and secondary antibody for 1 hr at room temperature. Proteins were visualized by addition of ECL substrate and detection of chemiluminescent signal on x-ray film.
RNA extraction, reverse transcription, and real-time RT-PCR RNA was extracted using the Promega SV Total RNA Extraction Kit. DNase-treated RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (Fermentas) according to manufacturer's instructions. The resulting cDNA was used for qPCR performed on an ABI Prism 7000 Sequence Detection System using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Fermentas) as per manufacturer's specifications. vFLIP, ICAM1, TNFa, GAPDH, and Beta-actin were amplified using the following primers: B-actin F 59-CAT GTA CGT TGC TAT CCA GGC-39, R 59-CTC CTT AAT GTC ACG CAC GAT-39; GAPDH F 59-AAT CCC ATC ACC ATC TTC CAG-39, R 59-AAA TGA GCC CCA GCC TTC-39; ICAM1 F 59-CAA TGT GCT ATT CAA ACT GCC C-3, R 59-CAGCGTAGGGTAAGGTTCTTG-39; TNFa F 59-ACT TTG GAG TGA TCG GCC-39, R 59-GCT TGA GGG TTT GCT ACA AC-39; vFLIP F 59-GGATGCCCTAATGTCAATGC-39, R 59-GGCGATAGTGTTGGAGTGT-39. Relative gene expression was calculated using the DDCt method using GAPDH and bactin housekeeping genes for normalization. Data was analyzed for statistical significance using a fixed-effects one-way ANOVA with alpha set at 0.05 using JMP (Version 10.0). Error bars represent the standard error. Human NFkB Signaling Pathway RT 2 Profiler PCR Array was obtained from SABiosciences (QIAGEN). Samples were prepared, run and analyzed according to the manufacturers instructions.

Immunofluorescence assay
293T cells were grown and transfected in chamber slides as indicated. Cells were fixed with paraformaldehyde and permeablized with 0.3% Triton-X 100. Cells were stained with the indicated primary antibodies for 1 hr, followed by washing with PBS and staining with the appropriate fluorophore conjugated secondary antibody. Slides were washed and coverslips were mounted using Vector mounting media with DAPI. Cells were visualized using a Zeiss Axio Observer inverted fluorescence microscope.

RTA inhibits vFLIP induced NFkB activation
It is well established that vFLIP induces NF B activation [4][5][6][7]9] and that activation of NF B is required for maintenance of latency [11,12]. These data suggest that NF B associated gene expression might need to be down-regulated for the virus to enter the lytic cycle of replication. To determine whether RTA has an effect on NF B signaling, 293T cells were transfected with vFLIP and/or RTA and NFkB activity was assessed using a luciferase reporter containing five NFkB response elements. vFLIP expression induced a 15-fold NFkB activation as expected, however cotransfection with RTA reduced the activation three-fold (Fig. 1a). The role of RTA in blocking vFLIP induced NF B activation could be explained by one or more of the following mechanisms: RTA induced degradation of vFLIP or RTA-induced downregulation of components of the NF B pathway. To evaluate whether RTA was targeting components of the NFkB pathway, we assessed the effect of RTA on TNFa induced NFkB activation. While co-transfection with RTA consistently resulted in a dose dependent 2-3 fold decrease in vFLIP induced NFkB activation, RTA had no significant effect on NFkB activation stimulated by TNFa, suggesting that RTA was targeting an aspect of vFLIPinduced NF B activation, rather than a component of the signal transduction pathway (Fig. 1b). We were also unable to find any evidence that RTA down-regulated either LANA nor NF B protein levels or those of components of the IKK pathway in the absence of vFLIP (data not shown). This data suggests that RTA expression counteracts latency by inhibiting vFLIP-induced NFkB activation by targeting vFLIP functions.

RTA expression induces vFLIP degradation via the proteasome
RTA has previously been shown to participate in protein degradation either through its intrinsic ubiquitin ligase activity or through recruitment of the cellular ubiquitin E3-ligase RAUL [13,16]. We observed via immunofluorescence assay (IFA) that vFLIP localized predominantly to nuclear bodies in transfected 293T cells; however vFLIP expression was greatly reduced and difficult to detect at all by IFA in the presence of co-transfected RTA (Fig 2). This observation supported the hypothesis that RTA might be affecting vFLIP protein stability. To evaluate the role of RTA on vFLIP stability, 293T cells were transfected with myc-vFLIP in the presence and absence of RTA (Fig 3a). Cotransfection of RTA with vFLIP resulted in greatly decreased levels of vFLIP protein as detected by western blot (Fig 3a). This effect was dose dependent and inhibited by the proteasome inhibitor MG132 (Fig 3b and 3c) suggesting that RTA is inducing vFLIP degradation via the proteasome, rather than targeting vFLIP mRNA for degradation or inhibiting transcription.

RTA inhibits vFLIP induced upregulation of the NF B responsive genes ICAM1 and TNFa
To further examine the role of RTA on vFLIP function, we examined the affect of RTA on vFLIP induction of NF B responsive genes. We used an NFkB signaling pathway qPCR Array to identify genes whose transcripts were up-regulated by vFLIP in 293T cells and detected a number of genes that had been previously reported (Table 1) [19][20][21]. We chose to validate vFLIP-induced expression of TNFa and ICAM1 mRNA in the presence and absence of RTA in transfected 293T cells. TNFa was particularly interesting because it likely provides a positive feedback mechanism for maintaining latency since TNFa is a well characterized activator of NF B [22]. While both ICAM1 and TNFa expression were negligible in the empty vector transfected control cells, transfection of myc-vFLIP resulted in a 24 and 103fold increases in TNFa and ICAM1 expression respectively (Fig. 4a). However, co-transfection of RTA with myc-vFLIP resulted in degradation of vFLIP (Fig. 4d) and an 11 and 13-fold reduction in TNFaa (Fig. 4b) and ICAM1 (Fig. 4c) expression, respectively, bringing gene expression down to background levels. RTA alone had no effect on TNFa and ICAM1 transcript levels (Figs. 4b and c).
We observed results in doxycycline induced TREx BCBL1 RTA cells that are consistent with those obtained in transfected 293T cells. Cells were treated with doxycycline for 0, 6,12, 24 and 48 hrs. RTA protein was detected after 6 hrs, indicating efficient lytic induction (Fig. 5a). Cells were analyzed at corresponding time points for TNFa and ICAM1 expression. Interestingly, both TNFa and ICAM1 RNA levels experienced a significant drop at 6 hrs followed by a rebound by 24 hrs (Fig. 5b). This data suggests that vFLIP mediated NFkB activity is transiently inhibited after RTA induction. This is consistent with previous reports of vFLIP  vFLIP was detected with anti-myc antibody (Millipore) followed by a rhodamine conjugated secondary antibody (Jackson ImmunoResearch). RTA was detected with our own antibody against RTA followed by a FITC conjugated secondary antibody. Nuclei were visualized using a DAPI containing mounting media. doi:10.1371/journal.pone.0091359.g002 expression during lytic replication and NF B activation later in lytic replication [12,[23][24][25].
To exclude the possibility that RTA was inhibiting vFLIP at the transcriptional level rather than at a post-translational level, we examined vFLIP transcript levels in doxycycline induced TREx BCBL1 RTA cells. As was previously reported, vFLIP mRNA was sufficiently expressed in TREx BCBL1 RTA cells (average DCT in uninduced cells was 5.960.037) and expression actually increases upon lytic induction, suggesting that RTA protein expression is not inhibiting vFLIP transcription [12,23,24,26] (Fig. 5c).

RTA induces vFLIP degradation through recruitment of a novel cellular ubiquitin ligase
RTA has been reported to participate both directly and indirectly in the degradation of several inhibitors of lytic replication [13][14][15][16]. To evaluate whether RTA is directly promoting the degradation of vFLIP, we employed two mutants in the ubiquitin E3-ligase domain of RTA, M1 and M3, in which C141 has been mutated to S and H145 to L respectively. These mutants cannot degrade IRF7 and M3 is also defective for degradation of Hey-1 and K-RBP [13][14][15]. However, in 293T cells co-transfected with vFLIP and either RTA mutant (M1 or M3), vFLIP protein levels were just as efficiently reduced as with wild-type RTA (Fig. 6a, lanes 2, 3 and 4), suggesting that while RTA is promoting vFLIP degradation, the degradation does not require the intrinsic ubiquitin E3-ligase activity of RTA. As well as having intrinsic E3 ubiquitin ligase activity targeting IRF7, RTA was previously shown to also recruit and stabilize the cellular HECT-ubiquitin E3-ligase RAUL leading to degradation of both cellular IRF3 and IRF7 [16]. To evaluate the possible role of RAUL in RTA induced degradation of vFLIP, we employed a dominant negative mutant of RAUL that has a C1051A mutation at the catalytic site in its HECT domain [17]. In the presence of RTA and endogenous RAUL, vFLIP is efficiently degraded (Fig. 6b, lane 4), however upon addition of the mutant myc-tagged RAUL ubiquitin E3 ligase in the presence of RTA, we did not observe any significant increase in vFLIP levels (Fig. 6b, lane 5). This data suggests that RTA-induced vFLIP degradation does not occur via a RAUL-dependent mechanism.
Amino acids 11-150 in RTA are required for vFLIP degradation Since RTA is not using its intrinsic ubiquitin ligase activity or RAUL to destabilize vFLIP, we hypothesized that RTA must be recruiting another cellular ubiquitin ligase. Therefore, to determine whether a specific domain of RTA is required for RTA induced degradation of vFLIP we used RTA truncation mutants to assess their effects on vFLIP stability. The results with RTA mutants R03(1-377), R04(1-273), R06(151-548), and R11(D11-272) are compared with wild-type RTA(1-691) in Fig. 7a. All of these mutants were previously shown to still bind to C/EBPaa and/or DNA, suggesting that despite their truncation they are still folding well enough to maintain some of their functional domains [18]. Co-transfection of R06 RTA(151-548) and R11(D11-272) with vFLIP resulted in a moderate defect in vFLIP degradation (Fig. 7b, compare lanes 4 to 7 and 8). The R03(1-377) and R04(1-273) proteins were not detected on western blots because they are missing the epitope recognized by our RTA antibody (located at amino acids 527-539). We feel confident, however, that expression of RTA was sufficient because vFLIP was completely degraded. RTA 11-149 retained the ability to induce vFLIP degradation, though not as efficiently as full length RTA (Fig. 7c). This data suggests that a region within amino acids 11-149 is likely to be critical for forming a stable interaction with either vFLIP or a cellular protein required for vFLIP destabilization or both. This region contains the ubiquitin ligase domain but is distinct from the region reported to bind IRF7.

Discussion
It is well established that RTA activates immediate-early, early and late gene expression by transactivating multiple viral promoters. More recently RTA has been assigned intrinsic ubiquitin E3-ligase activity as well as been shown to interact with and stabilize the cellular ubiquitin E3-ligase RAUL. RTA has been shown to influence the stability of a number of cellular factors that may act as inhibitors of lytic reactivation including K-RBP, LANA, IRF7, IRF3 and Hey1. vFLIP is a latently expressed protein that promotes latency and has been reported to inhibit lytic reactivation in a cell type specific context through NF B activation and suppression of the AP1 pathway (27). NFkB activation has been shown to inhibit lytic reactivation and   inhibition of NFkB signaling has been shown to promote lytic reactivation [11,12,27]. Our data suggests that RTA expression inhibits vFLIP induced NFkB activation, possibly as a mechanism to counteract promotion of latency by vFLIP, and may thus be necessary for either reactivation and/or the primary lytic infection process. RTA had no effect on TNFa induced NFkB activation suggesting that RTA is specifically targeting vFLIP, rather than components of the NF B pathway. We observed a dramatic decrease in vFLIP protein levels in the presence of RTA in co-transfection experiments. This effect was inhibited by treatment with MG132, suggesting that RTA is targeting vFLIP for degradation via the proteasome. RTA does not appear to affect vFLIP transcription, because vFLIP transcripts were shown to increase after doxycycline induction of RTA in TREx BCBL1-RTA cells.
Several RTA deletion mutants exhibited a defect in vFLIP degradation, supporting our postulation that RTA is promoting the degradation of vFLIP. We evaluated the possibility that degradation was occurring through the ubiquitin E3-ligase activity of RTA. We did not see any significant defect in proteolysis of vFLIP in the presence of the RTA ubiquitin ligase mutant. This suggests that RTA is recruiting a cellular protein to degrade vFLIP. This is a common strategy for viruses; they often degrade host proteins involved in the immune response. In fact, RTA has been shown to recruit and stabilize the cellular ubiquitin E3-ligase RAUL to target IRF3 and IRF7 for degradation leading to downmodulation of the interferon response [16]. We evaluated the role of RAUL in RTA induced degradation of vFLIP but observed no significant stabilization of vFLIP in the presence of a dominant negative RAUL ubiquitin E3-ligase mutant. This data suggests that RTA likely interacts with another ubiquitin ligase, possibly in the region of amino acids 11-150. We also demonstrated that RTA inhibits expression of two vFLIP induced NF B responsive genes, TNFa and ICAM1, in both transfected 293T cells and TREx BCBL1-RTA cells, likely through degradation of vFLIP. This data provides further evidence for the role of RTA in inhibition of NF B early in lytic reactivation.
Overall, we have identified a novel previously unsuspected mechanism involved in activation of KSHV lytic replication in the presence of an established latent state. In addition to activating multiple viral lytic cycle gene promoters, the viral immediate-early regulatory proteins evidently must also need to remove viral and cellular blocks to or repressors of the induction of the lytic replication process. Specifically, we present evidence here that the KSHV lytic cycle trigger protein RTA abrogates latent state NFkB signaling by targeting the key KSHV-encoded latent state protein vFLIP for proteasomal degradation.