Cellular siRNA Kinome Screen
To determine which cellular kinases are important for KSHV reactivation we performed a siRNA screen targeting the human cellular kinome. Over 720 siRNAs against all human protein and lipid kinases were included in the screen. Each well contained a pool of 4 siRNAs with different target sequences to a single cellular kinase. For the screen, we used KSHV-infected 293 (KSHV-293) cells that harbor latent KSHV and constitutively express GFP, while RFP is under the control of a lytic promoter and thus is only expressed upon viral reactivation (Vieira and O’Hearn, 2004
). A variety of confounding factors often leads to high false discovery rates among siRNA screens, an inevitable association of high throughput investigations. To lessen the false discovery rate we performed our primary screen in triplicate and used siRNAs that have been previously validated. Cells were reverse transfected with the siRNA pools and seventy hours post-siRNA transfection, GFP and RFP images and fluorescence intensities were acquired using a Cellomics ArrayScan VTI HCS Reader (). To ensure efficient siRNA transfection, an siRNA against Ubiquitin B (UBB) was included as a control siRNA in the screen as UBB knockdown is known to lead to cell death (Tiedemann et al., 2010
) (). The siRNA screen data was analyzed using the R statistical program environment (Team., 2008
). Statistically significant changes in viral reactivation (i.e. RFP intensity) were determined using both the median and mean RFP values for all the wells of the siRNA screen. shows a Waterfall plot of the Z-scores of the median RFP value for each of the siRNAs. A Z-score of 2 or higher was considered significant (). Additionally, lists the cellular kinases that when depleted showed a ≥ 2 standard deviation increase from the overall mean RFP intensity. One isoform of Tousled like kinase (TLK) named TLK2 stood out in both these analyses ( and ) because knockdown of TLK2 showed a Z-score of 15 based on median RFP value, and TLK2 knockdown led to a level of RFP expression that was 13 standard deviations above the mean RFP value for the screen. The next most significant kinases were only 3 standard deviations above the mean. These encompassed 7/720 (1%) of the siRNA targets and attests to the specificity and stringency of our screen. It is possible that some of the siRNAs in our screen do not sufficiently deplete their target protein and thus, potentially provide a false negative result. shows representative GFP and RFP images from the kinome screen. As can be seen in the RFP panels, knockdown of TLK1 shows no change in RFP expression compared to the GAPDH siRNA control in KSHV-293 cells, while TLK2 knockdown results in a large increase in RFP-positive cells indicative of viral reactivation. To rule out the possibility that viral reactivation following TLK2 knockdown is due to off-target effects of one or more of the TLK2 siRNAs, we transfected KSHV-293 cells with each of the 4 individual TLK2 siRNAs that target different regions of the TLK2 transcript. Cells were examined by microscopy (Supp. Fig. 1A
) and cell lysates were subjected to Western blots (Supp. Fig. 1B
). Three of the four siRNAs showed significant knockdown of TLK2 and robust viral reactivation.
Design of siRNA Screen and Analysis of Data
List of transfected siRNAs that yield increased RFP intensities at least 2 standard deviations from the mean when transfected into KSHV-293 cells
TLK2 Knockdown Leads to KSHV Reactivation in KSHV-293 Cells
To validate our screen results, we reverse transfected KSHV-293 cells with equivalent amounts of GAPDH siRNA, or a pool of 4 siRNAs against TLK1 and TLK2. Cells were imaged at 70 hours post-transfection for GFP and RFP expression; again knockdown of TLK2 robustly induced RFP expression (). Cell lysates were also harvested and Western blots were performed to look at kinase expression levels. As seen in , siRNA transfection led to dramatic decreases in the expression of the target protein.
TLK2 Plays a Role in KSHV Reactivation
To validate this result we also used a TLK2 siRNA from a different source and measured viral reactivation. We saw the same reactivation phenotype as described above (Supp. Fig. 2A and B
). In order to determine if TLK2 levels were increased in KSHV-293 latently infected cells compared to uninfected cells, we performed Western blots probing for TLK2 and found that expression levels of TLK2 were similar between both infected and uninfected cells (Supp. Fig. 2C
). We also tested whether knockdown of TLK2 affected cell viability of HEK-293 cells in the absence of KSHV. 293 cells were transfected with a non-targeting control (NTC) siRNA or siRNAs against TLK2 or UBB, and cells were examined by microscopy at 72 hours to gauge viability (Supp. Fig. 2D
). TLK2 knockdown had only a minor effect on cell viability, and cell death in the TLK2 knockdown cells was similar to the NTC siRNA transfected control cells, and was much less pronounced than the positive control cells transfected with UBB siRNA. We also examined the effect of TLK2 knockdown on HEK-293 cell proliferation using a MTS assay and saw that TLK2 knockdown did not cause an overall loss in proliferation as was seen following UBB knockdown over the time period examined (Supp. Fig. 2E
Depletion of TLK2 Leads to Expression of KSHV Lytic Genes
To determine if TLK2 knockdown resulted in increased viral lytic gene expression, we transfected KSHV-293 cells with a non-targeting control siRNA or siRNAs against TLK1 or TLK2. We also treated mock transfected KSHV-293 cells with 12-O-Tetradecanoyl-phorbol-13-acetate (TPA), a potent chemical inducer of KSHV lytic reactivation, as a positive control. RNA was harvested 54 hours post-transfection and quantitative real-time PCR (qPCR) was performed for three key viral lytic mRNAs: vIL-6, ORF57, and vGPCR (). TLK2 knockdown cells induced expression of all three viral lytic mRNAs, and to levels fairly similar to the TPA treated sample. The corresponding Western blots showing protein levels of the targeted genes are also depicted (). To investigate whether depletion of TLK2 led to an increase in viral genomes, KSHV-293 cells were transfected with siRNAs against GAPDH, TLK1, or TLK2, and total DNA was extracted 94 hours post-transfection. Viral genomes were quantitated by qPCR (). TLK2 siRNA transfected cells displayed an approximate 16-fold increase in viral genome copy number compared to the GAPDH depleted cells. Knockdown of TLK1, TLK2, and GAPDH was confirmed by Western blot analysis (). This demonstrates that knockdown of TLK2 results in increased viral lytic gene expression and increased viral genome replication.
The Major Viral Lytic Switch Protein, ORF50/RTA, is Activated Upon TLK2 Knockdown
The KSHV replication and transcription activator protein (RTA), encoded by ORF50, plays an essential role in the initiation of viral lytic gene expression. Since knockdown of TLK2 led to the expression of viral lytic genes that were regulated by KSHV ORF50, we wanted to determine if ORF50 levels were also increased. To determine if ORF50 promoter activity was increased following TLK2 knockdown, we performed a reporter gene assay using a luciferase construct under the control of the ORF50 promoter. 293 cells were transfected with siRNA against GAPDH, TLK1, or TLK2 and then 24 hours later the cells were transfected with an ORF50-promoter luciferase construct (Damania et al., 2004
). Luciferase expression was measured 48 hours post-transfection (). Depletion of TLK2 resulted in an approximate 10-fold increase in ORF50 promoter activity compared to GAPDH and TLK1 depleted cells. Knockdown of the targeted proteins was confirmed (). This indicates that TLK2 knockdown leads to the activation of the ORF50 promoter, even in the absence of other viral proteins. Next, we examined ORF50 transcript levels. KSHV-293 cells were transfected with a non-targeting control (NTC) siRNA or siRNAs against TLK1 or TLK2. Total RNA was harvested 54 hours post-transfection and qPCR for ORF50 mRNA was performed. As can be seen in , there was a 12-fold increase in ORF50 mRNA levels when TLK2 siRNA was transfected into KSHV-293 cells compared to the control siRNA. Additionally, lytic gene expression of two other viral genes, K1 and ORF36, were also increased in TLK2 siRNA transfected KSHV-293 cells ().
The Major Lytic Switch Protein, KSHV ORF50/RTA, is Activated Following TLK2 Knockdown
Depletion of TLK2 Leads to Complete Viral Reactivation and Production of Infectious Virus
To test the effect of TLK2 knockdown on overall viral gene expression, we performed genome-wide viral profiling using our KSHV qPCR array (Dittmer, 2003
). KSHV-293 cells were transfected with the non-targeting control siRNA or a siRNA targeting TLK1 or TLK2. As a positive control for viral reactivation, we treated untransfected cells with 0.1mM sodium butyrate, a known inducer of KSHV reactivation. Cells were incubated for 96 hours and then KSHV genome-wide transcription was measured. When TLK2 was depleted from the KSHV-293 cells there was upregulation of nearly all viral genes, similar to the sodium butyrate-treated cells, indicative of complete viral reactivation (; also see Supp. Fig. S3
). This was in contrast to the control siRNA and TLK1 siRNA treated cells which showed minimal levels of upregulated viral transcripts in KSHV-293 cells.
Genome-wide Upregulation of Viral Transcripts, Induction of Lytic Proteins, and Production of Infectious Progeny Virions Following TLK2 Knockdown
Complete KSHV replication results in virion production and the release of infectious virions from the cell which can subsequently infect naïve cells. In order to determine if TLK2 knockdown led to the production of infectious virions, we transfected KSHV-293 cells with siRNAs targeting GAPDH, TLK1, or TLK2. Both the cell lysates and supernatants were collected 96 hours post-transfection. The lysates were used to perform Western blots examining expression of two viral lytic proteins, an early lytic protein (vIL-6) and a late lytic protein (K8.1A), which were highly expressed only in the TLK2 siRNA transfected cells (), and not in the TLK1 or GAPDH siRNA transfected cells.
The supernatants collected from the KSHV-293 transfected cells were clarified to remove cellular debris and then used to infect naïve Vero cells to determine if infectious virus was present. The Vero cells were monitored by fluorescence microscopy for GFP-positive cells, indicative of viral infection. Pockets of GFP-positive cells can clearly be seen in the Vero cells treated with the supernatants from the TLK2 knockdown KSHV-293 cells () indicating that infectious virus was produced. These newly infected Vero cells were harvested and total intracellular DNA was extracted in order to perform a viral load assay. In agreement with our other data, naive Vero cells incubated with the supernatants from the TLK2 knockdown KSHV-293 cells displayed much higher levels of viral genomes than the GAPDH knockdown cells ().
TLK Depletion Can Also Reactivate KSHV From PEL
To examine the effect of TLK knockdown in KSHV-infected B cells, BCBL-1 PEL cells were transfected with a non-targeting control siRNA or siRNAs against TLK1 or TLK2. At 120 hours post-transfection, the cells were harvested and protein lysate was extracted and subjected to Western blot analysis against the viral lytic proteins, vIL-6 and K8.1A. As can be seen in , BCBL-1 cells transfected with the TLK2 siRNA showed viral reactivation as indicated by the increased expression of vIL-6 and K8.1A lytic proteins compared to the control siRNA-transfected cells. Cells transfected with TLK1 siRNA also showed a certain degree of viral reactivation in PEL but not to the same extent as TLK2 depletion, suggesting that depending on the particular cell type, one of the two TLK genes may be predominantly involved in modulating viral latency and suppressing reactivation. To investigate the effect of TLK2 depletion on a variety of PEL cell lines, we infected a panel of PEL cells (BC-3, JSC-1, VG-1) with either a control lentivirus or one targeting TLK2 for knockdown. Infections proceeded for 96 hours and then Western blots for vIL-6 were performed on the harvested lysates (). Viral reactivation was seen in each of the examined PEL cell lines. This data shows that depletion of TLKs in natural KSHV-infected PEL cells, leads to KSHV reactivation.
Knockdown of TLKs Leads to KSHV Reactivation in PEL and Reduction of Phospho-histone H3 Associated with the KSHV ORF50 Promoter
TLK2 Knockdown Leads to Decreased Phosphorylated Histone H3 Bound to the ORF50 Promoter
The expression and repression of genes, both cellular and viral, is tightly controlled by chromatin structure and modifications. It has been shown that TLKs can phosphorylate histone H3 at the serine 10 position (Li Y, 2001
). This histone modification has been shown to modulate transcription (Burkhart et al., 2007
, Goto et al., 1999
, Van Hooser et al., 1998
, Lefebvre, 2002
, Mahadevan et al., 1991
). In latently infected cells, the KSHV ORF50 promoter is associated with repressive histone, which is released upon reactivation (Gunther and Grundhoff, 2010
, Toth et al., 2010
). One mechanism by which TLK2 depletion could lead to viral reactivation in KSHV-293 cells is that the histone H3 associated with the ORF50 promoter is dephosphorylated leading to activation and expression of ORF50/RTA.
To determine if depletion of TLK2 led to less phosphorylated histone H3 associated with the ORF50 promoter, we performed a chromatin immunoprecipitation (ChIP) assay. KSHV-293 cells were transfected with either a non-targeting control siRNA or the TLK2 siRNA and incubated for 96 hours. ChIP analysis was performed using an anti-phospho histone H3 (Ser10) or control IgG antibody and the amount of phosphorylated histone H3 bound to the ORF50 promoter was determined by qPCR. Following TLK2 depletion there was an approximate 5.5-fold reduction in the amount of serine 10-phosphorylated histone H3 associated with the ORF50 promoter compared to the control siRNA (). The qPCR reactions were also run on an agarose gel to visualize the PCR products (). Since it is possible that the decreased association of phospho-histone H3 with the ORF50 promoter is due to a global decrease in histone H3 phosphorylation, we performed an immunofluorescence assay (IFA) to examine the status of total phosphorylated histone H3 (Ser10). KSHV-293 cells were depleted of TLK2 by siRNA or transfected with a control siRNA and 48 hours later, the cells were stained for pHistone H3 (Ser10). The overall level of phosphorylated histone H3 (Ser10) was unchanged upon TLK2 knockdown (Supp. Fig. 4A
). This was confirmed by Western blot as neither total levels of histone H3 nor phospho-histone H3 (Ser10) were altered upon TLK2 knockdown (Supp. Fig. 4B
). These data show that the viral reactivation phenotype seen following depletion of TLK2 is associated with less phosphorylated histone H3 bound to the ORF50 promoter and a subsequent increase in expression of ORF50/RTA.
TLKs Also Regulate Epstein-Barr Virus Reactivation From Latency
To determine if the actions of the Tousled-like kinases in maintenance of latency are specific to KSHV or if they also modulate reactivation of other related oncogenic gammaherpesviruses, we examined the effects of TLK depletion on the Epstein-Barr virus (EBV) lifecycle. We depleted TLK1 or TLK2 by siRNA in an EBV-infected gastric cancer cell line (AGS-EBV) (Zhou et al., 2005
) and measured the expression of signature lytic viral mRNAs and proteins (). Interestingly, in the case of EBV, TLK1 appears to have the predominant role in the regulation of EBV reactivation from latency. As can be seen in , depletion of either TLK1 or TLK2 led to induction of lytic EBV mRNA transcripts, but knockdown of TLK1 resulted in higher levels of all the lytic viral mRNAs tested. A similar effect was seen at the protein level; siRNA-mediated knockdown of either TLK1 or TLK2 led to increased expression of the viral lytic proteins EA-D and EA-R with the highest levels seen following TLK1 depletion (). To ensure that EBV reactivation in our system is specific to the TLKs, we also knocked down the cellular kinase STK38. Depletion of STK38 in the AGS-EBV cells showed no appreciable reactivation above background (). We next examined whether the reactivation phenotype observed in the AGS-EBV cells is also seen in the Burkitt’s lymphoma cell line, Akata. Akata-BX1 cells were infected with lentiviruses expressing either a scrambled shRNA, or a shRNA targeting TLK1 or TLK2. Cells were infected for 96 hours at which point cell lysates were harvested and EBV reactivation was measured by expression of the viral lytic proteins EA-D and EA-R, as well as the lytic transactivator protein, Zta. Similar to AGS-EBV cells, knockdown of both TLK1 and TLK2 led to viral reactivation as shown by increased expression of all three lytic proteins (). Also, cells depleted of TLK1 expressed higher levels of the lytic proteins than those which had TLK2 knocked down, further suggesting that TLK1 is the predominant Tousled-like kinase for maintenance of EBV latency and suppression of reactivation. The levels of viral reactivation in Akata and Akata-BX1 cells following TLK knockdown were also compared to reactivation following human IgG treatment, a known inducing agent of reactivation in Akata cells (Supp. Fig. 5
). These data indicate that the Tousled-like kinases not only play a role in KSHV reactivation, but also another gammaherpesvirus, EBV.
Knockdown of TLKs Leads to Reactivation of the Related Gammaherpesvirus, EBV