Cell Host & Microbe
Tousled-like Kinases Modulate Reactivation
of Gammaherpesviruses from Latency
Patrick J. Dillon,1,2Sean M. Gregory,1,2Kristen Tamburro,1,2,3Marcia K. Sanders,1,2Gary L. Johnson,1,4
Nancy Raab-Traub,1,2Dirk P. Dittmer,1,2,3and Blossom Damania1,2,3,*
1Lineberger Comprehensive Cancer Center
2Department of Microbiology and Immunology
3Curriculum in Genetics
4Department of Pharmacology
University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
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 pathogen-
esis 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 reactiva-
tion 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.
tivation from latency of another related oncogenic
gammaherpesvirus, Epstein-Barr virus. Our results
establish the TLKs as cellular repressors of gamma-
Kaposi’s sarcoma-associated herpesvirus (KSHV) is linked to
a number of human malignancies, including Kaposi’s sarcoma
(KS), primary effusion lymphoma (PEL), and multicentric
Castleman disease (MCD) (Chang et al., 1994, Songyang et al.,
1994; Stu ¨rzl et al., 1997; Cesarman et al., 1995; Soulier et al.,
1995). In the majority of infected cells, KSHV remains latent;
however, a small percentage of cells can undergo spontaneous
lytic replication at any given time (Zhong et al., 1996; Poyet et al.,
2001). This low level of viral reactivation is believed to be impor-
tant for persistence and tumorigenesis (Grundhoff and Ganem,
2004). Both latency and lytic replication are important phases
for viral pathogenesis and spread, but the cellular players
involved in the switch between the two phases of the viral life
cycle are not clearly understood.
Incellculture,reactivation ofKSHVoccursfollowing treatment
with chemical compounds such as the phorbol ester 12-O-
tetradecanoyl-phorbol-13-acetate (TPA/PMA) and the histone
deacetylase inhibitor sodium butyrate. We previously reported
that activation of Toll-like receptors 7 and 8 in PEL cells led to
KSHV reactivation (Gregory et al., 2009). In an overexpression
system, using transient transfection of kinase complementary
DNAs (cDNAs), other investigators showed that both the Pim
and Ras family kinases are involved in KSHV reactivation (Cheng
et al., 2009; Yu et al., 2007).
To determine the cellular kinases that control KSHV reactiva-
tion from latency, we performed a small interfering RNA (siRNA)
screen of the cellular kinome in the absence of any chemical
inducers. This allowed us to assess which kinases are needed
for the virus to maintain latency or to induce reactivation. A
siRNA library containing siRNAs against 720 different human
kinases was used for the screen. We identified the Tousled-
like kinases (TLKs) as cellular kinases involved in reactivation
Originally described in the plant Arabidopsis thaliana, the
Tousled gene encodes a nuclear serine/threonine kinase that is
essential for flower and leaf development (Roe et al., 1993,
1997). Two mammalian homologs, TLK1 and TLK2, show 84%
sequence similarity to each other (Takahata et al., 2009). The
TLKs are regulated by cell-cycle-dependent phosphorylation,
their activity is tightly linked to DNA replication with maximal
activity during S phase, and they are sensitive to DNA-damaging
agents and inhibitors of DNA replication (Takahata et al., 2009).
TLKs are also involved in chromatin assembly. TLK1 and TLK2
bind to and phosphorylate the human chromatin assembly
factors Asf1a and Asf1b (Sillje ´ and Nigg, 2001). The TLKs have
been implicated in numerous replicative and transcriptional
processes, including chromosome condensation and segrega-
tion (Sunavala-Dossabhoy et al., 2003; Hashimoto et al., 2008),
gene silencing (Wang et al., 2007), and DNA repair (Sunavala-
Dossabhoy et al., 2005; Canfield et al., 2009).
In this study, we identified TLKs as modulators of KSHV reac-
tivation. Our results show that depletion of TLK2 in KSHV latently
infected epithelial cells leads to robust viral reactivation. Knock-
infectious progeny virions. Depletion of TLK2, and to a lesser
extent TLK1, also leads to KSHV viral reactivation from latently
204 Cell Host & Microbe 13, 204–214, February 13, 2013 ª2013 Elsevier Inc.
infected B cells. Moreover, we found that knockdown of TLK1,
and to a lesser extent TLK2, results in reactivation of another
gammaherpesvirus family member, Epstein-Barr virus (EBV).
Our results indicate that the TLKs are key regulators of KSHV
and EBV reactivation, and their expression is required for the
maintenance of viral latency.
Cellular siRNA Kinome Screen
To determine which cellular kinases are important for KSHV re-
activation, we performed a siRNA screen targeting the human
cellular kinome. More than 720 siRNAs against all human protein
and lipid kinases were included in the screen. Each well con-
tained a pool of four 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 green fluorescent protein (GFP), whereas red fluores-
cent protein (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 lead to
high false-discovery rates in siRNA screens, which is an inevi-
table result of high-throughput investigations. To lessen the
false-discovery rate, we performed our primary screen in tripli-
cate and used robust statistical analysis. We reverse transfected
thecells withthe siRNApools,and 70hrpost-siRNAtransfection
acquired GFP and RFP images and fluorescence intensities
using a Cellomics ArrayScan VTI HCS Reader (Figure 1A). To
ensure efficient siRNA transfection, an siRNA against Ubiquitin
B (UBB) was included as a control siRNA in the screen, because
UBBknockdown isknownto leadtocelldeath(Tiedemannetal.,
statistical program environment (http://www.R-project.org). We
determined statistically significant changes in viral reactivation
(i.e., RFP intensity) using both the median and mean RFP values
for all the wells of the siRNA screen. Figure 1C shows a waterfall
plot of the Z scores of the median RFP value for each of the
siRNAs. A Z score of R2 was considered significant (Figure 1C).
Additionally, Table 1 lists the cellular kinases that when depleted
showed a R2 SD increase from the overall mean RFP intensity.
One isoform of TLK, TLK2, stood out in both of these analyses
(Figure 1C; Table 1) because knockdown of TLK2 showed a Z
score of 15 based on the median RFP value, and TLK2 knock-
down led to a level of RFP expression that was 13 SDs above
the mean RFP value for the screen. The next most significant
kinases were only 3 SDs above the mean. These comprised
seven outof 720(1%) of the siRNA targets, attesting to the spec-
ificity and stringency of our screen. It is possible that some of the
siRNAs in our screen did not sufficiently deplete their target
proteinandthuscouldpotentially provideafalse-negative result.
Except for the TLKs, we did not confirm any of the hits listed in
Table 1. Figure 1D shows representative GFP and RFP images
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
(B) Control siRNAs demonstrate efficient siRNA transfection in our screen.
GAPDH and UBB siRNAs were reverse transfected at 25 nM each into 2,500
KSHV-293 cells/well in a 384-well plate. At 70 hr posttransfection, bright-field,
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 Figure S1.
Cell Host & Microbe
TLKs Modulate Gammaherpesvirus Reactivation
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Cell Host & Microbe
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214 Cell Host & Microbe 13, 204–214, February 13, 2013 ª2013 Elsevier Inc.