Tousled-like kinases modulate reactivation of gammaherpesviruses from latency

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to human malignancies. The majority of tumor cells harbor latent virus, and a small percentage undergo spontaneous lytic replication. Both latency and lytic replication are important for viral pathogenesis and spread, but the cellular players involved in the switch between the two viral life-cycle phases are not clearly understood. We conducted a small interfering RNA (siRNA) screen targeting the cellular kinome and identified Tousled-like kinases (TLKs) as cellular kinases that control KSHV reactivation from latency. Upon treatment of latent KSHV-infected cells with siRNAs targeting TLKs, we saw robust viral reactivation. Knockdown of TLKs in latent KSHV-infected cells induced expression of viral lytic proteins and production of infectious virus. TLKs were also found to play a role in regulating reactivation from latency of another related oncogenic gammaherpesvirus, Epstein-Barr virus. Our results establish the TLKs as cellular repressors of gammaherpesvirus reactivation.

Figure 1. Design of siRNA Screen and Analysis of Data

A) Schematic of cellular kinome siRNA screen. siRNAs from the Dharmacon SMARTpool kinase siRNA library were loaded into 384-well plates in triplicate. KSHV-293 cells were added to each well containing siRNA and incubated for 70 hours at 37°C. Both RFP and GFP images were then taken for 5 fields per well. B) Control siRNAs demonstrate efficient siRNA transfection in our screen. GAPDH and Ubiquitin B (UBB) siRNAs were reverse transfected at 25nM each into 2500 KSHV-293 cells/well in a 384-well plate. At 70 hours post-transfection brightfield, GFP, and RFP pictures were taken on a fluorescent microscope. C) Statistical analysis of primary hits. A Waterfall plot was used to analyze the data acquired from the screen. It shows the Z-score of the median RFP intensity of each siRNA. D) Reactivation by TLK2 knockdown in KSHV-293 cells. GFP and RFP images of a representative field taken during the screen are shown for the wells containing siRNA targeting GAPDH, TLK1, and TLK2. See also Fig. S1.

Figure 2. TLK2 Plays a Role in KSHV Reactivation

A) Knockdown of TLK2 reactivates KSHV. KSHV-293 cells were reverse transfected with a final siRNA concentration of 25nM. A single GAPDH siRNA or a pool of 4 siRNAs against TLK1 or TLK2 were used. At 70 hours post-transfection, images were taken on a fluorescent microscope. B) siRNAs efficiently knock down target. Cellular lysates from the samples imaged in Fig. 2A were harvested and Western blots were performed for TLK1, TLK2, GAPDH, and the loading control tubulin. C) Viral lytic mRNAs are expressed. KSHV-293 cells were either mock transfected or reverse transfected with 50nM of the non-targeting control siRNA or the pooled TLK1 or TLK2 siRNAs. The mock transfected sample was treated with 25 ng/mL of TPA at the time of transfection. Levels of viral lytic transcripts, vIL-6, Orf57, and vGPCR, were measured by qPCR at 54 hours post-transfection. Values are normalized to the control siRNA. D) Western blots were performed for the indicated proteins for the experiment described in 2C. E) KSHV-293 cells were reverse transfected as described above in 2C. At 96 hours post-transfection, DNA was harvested and viral load was determined by qPCR. Primers for Orf57 were used as the indicator of viral genome copies. F) Western blots were performed for the indicated proteins to confirm knockdown in the experiment described in 2E. Non-specific bands are indicated by an asterisk “*”. Error bars represent standard deviation from the mean. Data were analyzed using a two-tailed type II Student’s t test for significance. See also Fig. S2.

Figure 3. The Major Lytic Switch Protein, KSHV ORF50/RTA, is Activated Following TLK2 Knockdown

A) The KSHV ORF50 promoter is activated by TLK2 knockdown. HEK-293 cells were transfected with GAPDH siRNA or a pool of TLK1 or TLK2 siRNAs (50nM) for 24 hours followed by transfection with an ORF50 luciferase reporter construct. Lysate was harvested 48 hours after ORF50-luciferase transfection and a luciferase assay was performed. Values are normalized to protein levels determined by Bradford assay. B) Western blots were performed for TLK1, TLK2, and tubulin to confirm knockdown. C) Increase in ORF50 mRNA. Levels of ORF50 viral transcript were measured by qPCR as described in 2C. D) Induction of other viral lytic genes. KSHV-293 cells were reverse transfected with GAPDH siRNA or a pool of TLK1 or TLK2 siRNAs (50nM) for 96 hours. Total RNA was harvested and reverse transcriptase PCR was performed on the RNA (+/− reverse transcriptase (RT)) and PCR products were run out on agarose gels. M, Marker lane. Error bars represent standard deviation from the mean. Data were analyzed using a two-tailed type II Student’s t test for significance.

Figure 4. Genome-wide Upregulation of Viral Transcripts, Induction of Lytic Proteins, and Production of Infectious Progeny Virions Following TLK2 Knockdown

A) TLK2 knockdown induces KSHV reactivation. KSHV-293 cells were either treated with 0.1mM sodium butyrate or transfected with 50nM of a single control siRNA or siRNA targeting TLK1 or TLK2 for 96 hours. RNA was isolated and a KSHV viral array was performed to determine viral transcript levels. This array has multiple qPCR primer pairs for each annotated KSHV ORF. A heat map for the viral array is shown. Higher transcript levels are indicated by red, lower levels by blue. Each primer was scaled independently, such that the median across all experiments for this primer is indicated by white color. Genes on the vertical axis and samples on the horizontal axis were clustered by similarity of their transcription profile using a Euclidian based distance matrix and Ward’s algorithm. The corresponding dendrograms (tree) are shown outside the heatmap. The branch length corresponds to relative similarity of samples and genes, e.g. induced (NaB) and TLK2 siRNA treated samples form the right branch and cluster together. Control siRNA and TLK1 siRNA treated samples form the left main branch and cluster together. B) Viral lytic proteins are expressed. KSHV-293 cells were reverse transfected with GAPDH siRNA or a pool of TLK1 or TLK2 siRNAs (50nM). Cell lysates were harvested 96 hours post-transfection and Western blots were performed for the lytic proteins vIL-6 and K8.1A, cellular TLK1, TLK2, and GAPDH, and the loading control, tubulin. C) Infectious virus is produced. Cell supernatants were harvested from the same samples as in 4B and used to infect 80,000 Vero cells/well. Images were taken 96 hours post-infection. D) Viral load assay. Cell-associated DNA was harvested from the Vero cells described in 4C above at 96 hours post-infection and intracellular viral genome copy number was determined for the whole cell population. Error bars represent standard deviation from the mean. Data were analyzed using a Student’s t test for significance. See also Supp. Fig. S3.

Figure 5. Knockdown of TLKs Leads to KSHV Reactivation in PEL and Reduction of Phospho-histone H3 Associated with the KSHV ORF50 Promoter

A) Depletion of TLKs reactivates KSHV in PEL cells. BCBL-1 cells were transfected with single siRNAs (150mM) against TLK1, TLK2, or a non-targeting control (NTC) and cell lysates were harvested 120 hours post-transfection. Western blots were performed for the viral lytic proteins, vIL-6 and K8.1A, as well as TLK1, TLK2, and tubulin. B) Reactivation in a panel of PEL lines. BC-3, JSC-1, and VG-1 cells were infected with lentivirus expressing either a scrambled control shRNA or shRNA targeting TLK2. Cell lysates were collected 96 hours post-infection and Western blots were performed for vIL-6, TLK2, and tubulin. C) Chromatin immunoprecipitation assay. KSHV-293 cells were reverse transfected with 50nM of either a non-targeting control siRNA or a siRNA against TLK2 prior to plating. A ChIP assay was performed 96 hours post-transfection as described in the Experimental Procedures using either an antibody against phosphorylated histone H3 (Ser10) or mouse IgG. The level of phospho-histone H3 bound to the ORF50 promoter was determined by qPCR using promoter-specific primers. Values are given as a relative enrichment compared to the IgG control. Error bars represent standard deviation from the mean. D) Visualization of the qPCR products. PCR products from 5C were run on a 2% agarose gel. Non-specific bands are denoted by an asterisk “*”. See also Supp. Fig. S4.

Figure 6. Knockdown of TLKs Leads to Reactivation of the Related Gammaherpesvirus, EBV

A) EBV lytic mRNAs are expressed upon knockdown of the TLKs. AGS-EBV cells were reverse transfected with 50nM of the non-targeting control siRNA or single TLK1 or TLK2 siRNAs. Levels of viral lytic transcripts BMRF1, BLLF1b, and BCLF1 were measured by qPCR at 90 hours post-transfection. Values are normalized to the control siRNA. Error bars represent standard deviation from the mean. B) EBV lytic proteins are induced upon knockdown of the TLKs. AGS-EBV cells were reverse transfected with 50nM of a single siRNA against TLK1, TLK2, or the non-targeting control and cell lysates were harvested 120 hours post-transfection. Western blots were performed for the EBV lytic proteins EA-D and EA-R, as well as TLK1, TLK2, and tubulin. C) Reactivation is specific to the TLKs. AGS-EBV cells were reverse transfected with 50nM of the non-targeting control siRNA or a single siRNA targeting TLK1 or a control kinase, STK38. Cell lysates were harvested 96 hours post-transfection and Western blots were performed for EA-D, EA-R, TLK1, STK38, and tubulin. D) Reactivation in Burkitt’s lymphoma cells. Akata-BX1 cells were infected with lentiviral particles expressing either a non-targeting control (NTC), TLK1, or TLK2 shRNA. 96 hours later cells were harvested and Western blots were performed for the indicated proteins. Asterisks “*” denote non-specific bands. See also Supp. Fig. S5.