Download DNA-dependent protein kinase interacts functionally with the RNA

Journal of General Virology (2011), 92, 1710–1720
DOI 10.1099/vir.0.029587-0
DNA-dependent protein kinase interacts
functionally with the RNA polymerase II complex
recruited at the human immunodeficiency virus
(HIV) long terminal repeat and plays an important
role in HIV gene expression
Shilpi Tyagi,1,2 Alex Ochem2 and Mudit Tyagi1,3
Correspondence
Mudit Tyagi
[email protected]
1
National Center for Biodefense and Infectious Diseases, George Mason University, Biomedical
Research Laboratory, 10650 Pyramid Place, MS 1J5, Manassas, VA 20110, USA
2
International Centre for Genetic Engineering and Biotechnology (ICGEB), Wernher and Beit
Building (South), Anzio Road, Observatory 7925, Cape Town, South Africa
3
Department of Molecular Biology and Microbiology, Case Western Reserve University,
Adelbert Road, Cleveland, OH 44106, USA
Received 8 December 2010
Accepted 25 March 2011
DNA-dependent protein kinase (DNA-PK), a nuclear protein kinase that specifically requires
association with DNA for its kinase activity, plays important roles in the regulation of different DNA
transactions, including transcription, replication and DNA repair, as well as in the maintenance of
telomeres. Due to its large size, DNA-PK is also known to facilitate the activities of other factors by
providing the docking platform at their site of action. In this study, by running several chromatin
immunoprecipitation assays, we demonstrate the parallel distribution of DNA-PK with RNA
polymerase II (RNAP II) along the human immunodeficiency virus (HIV) provirus before and after
activation with tumour necrosis factor alpha. The association between DNA-PK and RNAP II is
also long-lasting, at least for up to 4 h (the duration analysed in this study). Knockdown of
endogenous DNA-PK using specific small hairpin RNAs expressed from lentiviral vectors resulted
in significant reduction in HIV gene expression and replication, demonstrating the importance of
DNA-PK for HIV gene expression. Sequence analysis of the HIV-1 Tat protein revealed three
potential target sites for phosphorylation by DNA-PK and, by using kinase assays, we confirmed
that Tat is an effective substrate of DNA-PK. Through peptide mapping, we found that two of
these three potential phosphorylation sites are recognized and phosphorylated by DNA-PK.
Mutational studies on the DNA-PK target sites of Tat further demonstrated the functional
significance of the Tat–DNA-PK interaction. Thus, overall our results clearly demonstrate the
functional interaction between DNA-PK and RNAP II during HIV transcription.
INTRODUCTION
Phosphorylation is the most common and one of the most
important mechanisms of acute and reversible regulation
of protein function (Hill & Treisman, 1995; Hunter, 1995).
Specifically, human immunodeficiency virus (HIV) transcriptional elongation, which is the main functional activity
of the Tat protein, is totally dependent on the association
of Tat with kinases able to phosphorylate the carboxyterminal domain (CTD) of human RNA polymerase II
(RNAP II). In the absence of Tat, the RNAP II complexes
present at the HIV promoter are dysfunctional and
elongate poorly. However, once Tat is synthesized, it
recruits positive transcription elongation factor b (P-TEFb)
at the HIV LTR and enhances the processivity of RNAP II,
thus fostering increased levels of full-length transcripts
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(Feinberg et al., 1991; Kao et al., 1987; Karn, 1999). The
recruitment of P-TEFb (a complex of cyclin T1/2 and
CDK9) through its interactions with Tat and the
transactivation response region (TAR) results in activation
of the CDK9 kinase and the consequent hyperphosphorylation of the CTD of RNAP II at serine 2.
The CTD of mammalian RNAP II contains 52 tandemly
arranged heptapeptide repeats of the Tyr-Ser-Pro-Thr-SerPro-Ser (YSPTSPS) motif. Phosphorylation of serine
residues Ser-5 and Ser-2 of these repeats is mainly correlated
with transcriptional initiation and elongation capacity of
RNAP II, respectively (Dahmus, 1995; Orphanides et al.,
1996). It is well-established that paused RNAP II contains
predominantly hypophosphorylated CTDs, whereas processively elongating polymerase possesses hyperphosphorylated
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DNA-PK plays a role in HIV transcription
CTDs (O’Brien et al., 1994). These findings clearly
demonstrate the critical role of cellular protein kinases in
HIV-1 transcription. More than half a dozen kinases are
known to phosphorylate CTD of RNAP II, and the
involvement of two kinases, namely CDK9 and CDK7, in
CTD phosphorylation during HIV gene expression has
already been well established (Dahmus, 1996; Isel & Karn,
1999; reviewed by Majello & Napolitano, 2001). One of the
kinases that is known to phosphorylate the RNAP II CTD is
the DNA-dependent protein kinase (DNA-PK), a nuclear
kinase that specifically requires association with DNA for its
activity (Anderson & Carter, 1996; Carter et al., 1990; Dvir
et al., 1993; Lees-Miller et al., 1990). Human DNA-PK
comprises two components: a 450 kDa catalytic subunit
(DNA-PKcs) (Hartley et al., 1995), which is a serine/
threonine kinase, and a regulatory component known as Ku
autoantigen (Gottlieb & Jackson, 1993). Ku is a heterodimer
composed of subunits of 70 kDa (Reeves & Sthoeger, 1989)
and 80 kDa (Yaneva et al., 1989). The 70 kDa subunit
possesses ATPase and DNA helicase activities (Tuteja et al.,
1994). The critical role of DNA-PK in the non-homologous
end joining (NHEJ) DNA-repair pathway is well-established
(Lees-Miller et al., 1995; Taccioli et al., 1994). The NHEJ
pathway is essential for the maintenance of chromosomal
integrity in developing lymphocytes and to repair DNA
double-strand breaks that occur during V(D)J recombination. Impaired V(D)J recombination results in severe
combined immunodeficiency disease (SCID), which is
represented by undeveloped B and T lymphocytes (Blunt
et al., 1995; Meek et al., 2004; Nussenzweig et al., 1996;
Taccioli et al., 1994). Curiously, whilst some studies have
suggested an important role for DNA-PK during HIV
integration into the host-cell genome (Daniel et al., 1999;
Jeanson et al., 2002; Nunnari et al., 2005), others have
indicated that the kinase is dispensable (Ariumi et al., 2005;
Baekelandt et al., 2000; Kilzer et al., 2003; Li et al., 2001).
Thus, the exact role of DNA-PK during HIV integration into
the host-cell genome has not received a universal consensus.
The DNA-binding component of DNA-PK is the heterodimeric Ku factor, one of the most abundant DNA-endbinding proteins in human cells (de Vries et al., 1989). Ku
binds mainly to the dsDNA ends of a variety of DNA
structures; however, it does not bind to closed circular DNA
(Dynan & Yoo, 1998; Yoo & Dynan, 1998), underscoring the
absolute need for free DNA ends for its binding activity.
There have been several published critical observations that
DNA-PK can phosphorylate the RNAP II CTD. DNA-PKcs
and Ku are components of the RNAP II holoenzyme
(Maldonado et al., 1996). Ku specifically recognizes stem–
loop structures and, in particular, binds to HIV-1 TAR
RNA (Kaczmarski & Khan, 1993). Moreover, Tat interacts
in vitro with DNA-PK (Chun et al., 1998). Recent work
from the Rice group (Liu et al., 2010) has reported the
interaction of Ku70 with the minor 55 kDa isoform of
CDK9, and this interaction was suggested to play a role in
the post-integration repair and/or circularization of viral
LTR circles. Furthermore, An et al. (2011) have also
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reported an interaction between DNA-PK and cyclin T2,
another subunit of the P-TEFb complex, thus strengthening the notion of a possible role for DNA-PK in HIV
transcription. All of these findings prompted us to
investigate the role of DNA-PK in HIV transcription and
to determine whether the HIV-1 Tat protein itself could be
a substrate for DNA-PK phosphorylation.
RESULTS
Recruitment of DNA-PK at the HIV LTR
As it is known that DNA-PK phosphorylates the CTD of
RNAP II (Dvir et al., 1993; Trigon et al., 1998), we
performed chromatin immunoprecipitation (ChIP) assays
to investigate the possible presence of this enzyme at the
HIV LTR. These assays were performed by using a Jurkat
T-cell-based clone, the E4 clone, which carries latent HIV
provirus in its genome (Pearson et al., 2008; Tyagi & Karn,
2007) (Fig. 1a). Assays were performed before and after
activating latent provirus with tumour necrosis factor
alpha (TNF-a). The immunoprecipitated DNA was analysed by using two primer sets, one amplifying the
promoter region of the LTR (2116 to +4 with respect
to the transcription start site), which provides a measure of
the factors involved in transcriptional initiation. The
second primer set used amplifies the nucleosome-2 region
of the LTR (+286 to +390 with respect to the
transcription start site), which quantifies those factors
involved in the elongation phase of HIV transcription.
As shown in Fig. 1(b), latent proviruses have low but
detectable levels of RNAP II at the promoter region of the
LTR (2116 to +4). However, following induction with
TNF-a, RNAP II levels increase rapidly at the promoter,
reaching at least fivefold higher than the pre-induction
levels. Parallel to RNAP II recruitment, we also found a
proportional jump in the levels of DNA-PK at the
promoter, clearly suggesting a significant role for DNAPK in HIV transcription. As a control, we used P80, a
subunit of the transcription factor (TF) IIH complex,
which plays an important role during the initial phase of
HIV transcription (Kim et al., 2006). Nuclear factor kappa
B (NF-kB), upon activation by TNF-a, induces the
recruitment of TFIIH at the HIV promoter and enhances
the phosphorylation of the RNAP II CTD through CDK7
kinase, another component of the TFIIH complex (Kim
et al., 2006). Consistent with that observation, we detected
the enrichment of P80 at the promoter region of LTR after
TNF-a stimulation, thus confirming the validity of our
assay conditions.
Analogous to the observations at the promoter region, at
the nucleosome-2 region we found parallel enrichment in
the levels of RNAP II and DNA-PK upon LTR transactivation by TNF-a. However, enhanced recruitment of P80
following TNF-a activation was not observed at the
nucleosome-2 region, confirming our previous results that
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S. Tyagi, A. Ochem and M. Tyagi
Fig. 1. DNA-PK is part of the RNAP II complex
involved in HIV transcription. (a) Genetic map
of the virus present in E4 Jurkat cells, along
with locations of all primers employed in the
study. (b) Quantitative ChIP assays showing
recruitment of different transcription factors.
The ChIP assays were performed before and
after activating a latently infected E4 Jurkat cell
line with 10 ng TNF-a ml”1 for 30 min. Left
panel, promoter region; right panel, nucleosome-2 region of LTR. (c) Profile ChIP assays
showing distribution of RNAP II (left panel) and
DNA-PK (right panel) along the HIV provirus
before and after stimulating cells with TNF-a
for 3 h.
TFIIH does not play any role during the elongation phase
of transcription (Kim et al., 2006). Thus, these results
provide direct evidence for the recruitment of DNA-PK at
the HIV LTR during HIV transactivation.
DNA-PK is a component of the RNAP II complex
involved in HIV transcription
In order to confirm the association between RNAP II and
DNA-PK at the HIV provirus, we performed highresolution ChIP assays to measure the distribution of both
DNA-PK and RNAP II along the HIV provirus (Fig. 1c). In
the absence of TNF-a stimulation, the latent proviruses
carry both DNA-PK and RNAP II at extremely low levels.
However, TNF-a activation for 3 h results in a sixfold
increase in the amount of RNAP II at the promoter. The
major part of the transcription complex was shifted to the
downstream regions, as we detected even higher levels of
RNAP II at the nucleosome-1 region of the LTR. The
nucleosome-2 region, which is a marker of the elongation
phase of transcription, also exhibits a fourfold increase in
RNAP II levels. We also analysed several other downstream
regions of the provirus, such as the 59 and 39 regions of the
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env gene, as well as a reporter GFP gene (approx. 4.5 kb
downstream). We found notably higher levels of DNA-PK
and RNAP II up to the 59 region of the env gene (approx.
1.7 kb downstream), but lower levels further into the
downstream regions. However, levels of both proteins
remained significantly higher than on their latent counterparts, demonstrating ongoing transcriptional elongation.
The parallel distribution pattern of both proteins along the
HIV provirus clearly demonstrates the alliance between
RNAP II and DNA-PK, thus validating our hypothesis that
DNA-PK is part of a larger RNAP II transcription complex
involved in HIV gene expression.
Furthermore, these results provide direct evidence for the
enrichment of DNA-PK at the LTR during HIV transactivation. Thus, the parallel recruitment of DNA-PK and
RNAP II at the HIV LTR clearly suggests an important role
for DNA-PK in HIV gene expression.
DNA-PK phosphorylates all three serine residues
of CTD
Phosphorylation of residue Ser-5 of the RNAP II CTD is
linked with the initiation phase of HIV transcription (Kim
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Journal of General Virology 92
DNA-PK plays a role in HIV transcription
et al., 2002; Yamamoto et al., 2001), whilst phosphorylation of Ser-2 of the CTD is found to be correlated with the
elongation phase of HIV gene expression (Kim et al., 2006;
Price, 2000; Saunders et al., 2006). Earlier reports that
DNA-PK is a CTD kinase (Dvir et al., 1993; Trigon et al.,
1998) and our finding that DNA-PK is present at both the
promoter and downstream regions of the LTR (Fig. 1)
clearly suggest its role during both the initiation and the
elongation phases of transcription. We performed phosphorylation studies using synthetic peptides with various
amounts of purified DNA-PK in order to establish the role
of this protein complex during specific phases of HIV gene
expression and to determine the exact target site(s) of
DNA-PK phosphorylation. We synthesized three peptides
containing two hepta repeats of CTD with double
mutations of serine residues to alanine at positions 5 and
7 (Fig. 2a), positions 2 and 7 (Fig. 2b) or positions 2 and 5
(Fig. 2c). Phosphorylation reactions were performed; the
phosphorylated peptides were separated by electrophoresis
and quantified (see Methods).
Interestingly, DNA-PK was able to phosphorylate all three
peptides. However, consistent with previous studies (Arias
et al., 1991; Dvir et al., 1993; Trigon et al., 1998), serine
residues at positions 2 (Fig. 2a) and 7 (Fig. 2c) of the hepta
peptide were better substrates than serine 5 (Fig. 2b). These
results suggest clearly that DNA-PK could be involved in
both the initiation and elongation phases of transcription,
albeit more significantly during elongation. The amount of
CTD phosphorylation was directly proportional to the
amounts of DNA-PK used. Utilizing DNA-PK purchased
from Promega, we also observed some faint bands in
control reactions without DNA, probably indicating a less
pure enzyme preparation. Nevertheless, specific DNAdependent CTD phosphorylation by DNA-PK is quite
evident, with a clear dose-dependent outline. Taken
together, our results demonstrate that DNA-PK interacts
dynamically with the HIV LTR and suggest its role during
both phases of transcription.
DNA-PK plays a significant role in HIV gene
expression
In order to confirm the functional significance of the
recruitment of DNA-PK to the HIV LTR and to
understand its role in HIV gene expression, we performed
knockdown experiments by using small hairpin RNA
(shRNA) to knock down endogenous DNA-PK specifically
(Fig. 3). Jurkat T cells were infected with lentiviral vectors
carrying luciferase reporter genes either under the control
of the HIV LTR promoter (pHR9P-Luc) or under an
internal human cytomegalovirus (CMV) immediate-early
promoter (pHR9P-SIN18-CMV-Luc), as negative control.
These cells were then superinfected with lentiviral vectors
expressing either shRNA sequences against DNA-PK or
control scrambled shRNA sequences (Fig. 3a). In Fig. 3(b),
in addition to lentiviral vectors expressing shRNAs, we also
included lentiviral vectors expressing HIV Tat from the
CMV promoter. Upon specific knockdown with shRNA
against DNA-PK, the endogenous DNA-PK levels were
reduced by .85 % in all instances (Fig. 3a, b) and led to
remarkable repression of HIV transcription.
By doing luciferase assays, we detected a clear dosedependent inhibition of the HIV LTR promoter (Fig. 3a,
lanes 2 and 3), whereas luciferase expression under the
Fig. 2. DNA-PK phosphorylates all three serine residues of CTD. In vitro kinase assays using purified HeLa cell DNA-PK,
purchased from Promega, were done in the absence (lane 1) or presence (lanes 2–4) of DNA. Reactions were performed using
three peptides, each carrying two hepta repeats of CTD with mutations in serine residues as follows: (a) mutated in Ser-5 and
-7; (b) mutated in Ser-2 and -7; (c) mutated in Ser-2 and -5. Reactions were performed in the presence of 1 mCi [c-32P]ATP.
The phosphorylated peptides were separated by electrophoresis using phosphate buffer–polyacrylamide gel at pH 6.0 (Trigon
et al., 1998) and quantified by densitometry.
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S. Tyagi, A. Ochem and M. Tyagi
against DNA-PK (lanes 4 and 5) or control scrambled shRNA (lane
3). After 36 h, cells were treated with TNF-a (lanes 2–5). Gag
(p24) production in the supernatants of cells was measured by
ELISA after 51 h. The data represent the results of three separate
experiments; bars indicate SD.
CMV promoter was inhibited only weakly upon DNA-PK
knockdown (lanes 5 and 6). Similar results were obtained
when assays were done in the presence of Tat (Fig. 3b). As
Tat is able to recruit CDK9 (another CTD kinase), in the
presence of Tat we obtained a comparatively lower, but still
significant impact of DNA-PK knockdown on HIV
transcription (Fig. 3b). Considering that the CMV promoter
was not affected due to reduced DNA-PK levels, whilst HIV
gene expression was heavily reduced, these results clearly
demonstrate the specific role of DNA-PK in HIV gene
expression. Furthermore, this analysis also demonstrates
that cellular levels of DNA-PK are high enough to contribute
significantly to facilitating HIV transcription.
DNA-PK is required for efficient HIV replication
In order to demonstrate the requirement of DNA-PK for
HIV replication, latently infected Jurkat J1.1 cells, which
carry replication-competent HIV, were superinfected with
lentiviral vectors carrying either shRNA sequences for
DNA-PK, to deplete this kinase, or scrambled shRNA
sequences as control. Thirty-six hours after this superinfection, cells were stimulated by treatment with TNF-a.
Subsequently, after 51 h, the Gag p24 antigen was analysed
in the supernatant by ELISA (Fig. 3c). We could detect
fairly reduced levels of p24 in those cells expressing shRNA
against DNA-PK. These data substantiate that loss of DNAPK appreciably reduces HIV replication, and validate the
importance of DNA-PK not only in HIV gene expression,
but also in HIV replication.
Tat is a substrate for DNA-PK phosphorylation
Fig. 3. Knockdown of endogenous DNA-PK restricts HIV gene
expression and replication in Jurkat T cells. (a) Jurkat cells were
infected with the indicated m.o.i. of lentiviral vectors expressing
shRNA from the Pol III promoter, either against DNA-PK (lanes 2,
3, 5 and 6) or control scrambled shRNA (lanes 1 and 4). Twentyfour hours later, cells were superinfected with lentiviral vectors
expressing a luciferase gene under the control of either the LTR
promoter (lanes 1–3) or the CMV promoter (lanes 4–6). (b) Jurkat
cells were coinfected with lentiviruses expressing Tat under the
control of the CMV promoter (lanes 2–4) alongside two different
m.o.i.s of lentiviral vectors expressing either shRNA against DNAPK (lanes 3 and 4) or control scrambled shRNA (lane 2). After
24 h, cells were superinfected with lentiviruses expressing a
luciferase gene under the control of the HIV LTR promoter (lanes
1–4). (c) Latently infected Jurkat J1.1 T cells were superinfected
with different m.o.i.s of lentiviral vectors expressing either shRNA
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As DNA-PK is known to interact directly with HIV Tat
(Chun et al., 1998), we investigated whether the Tat protein
is a substrate for DNA-PK and the potential effects of its
phosphorylation state on HIV transcription. We analysed
the amino acid sequence of Tat (HXB2, HIV-1 strain) and
discovered three putative target sites of DNA-PK phosphorylation (Fig. 4a). In fact, each of the serine residues at
positions 16, 62 and 75 is found to be followed by glutamine,
a typical amino acid configuration of the most common
target sites of DNA-PK (Bannister et al., 1993).
Considering that DNA-PK specifically requires DNA to
phosphorylate its substrates, we employed DNase- and
RNase-treated recombinant glutathione S-transferase
(GST)–Tat protein, to eliminate possible nucleic acid
contamination, in our phosphorylation assays (Ochem
et al., 2008). As shown in Fig. 4(b), Tat is an efficient
substrate for DNA-PK in the presence of DNA, both when
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DNA-PK plays a role in HIV transcription
peptides, each 20 aa long, spanning the entire Tat sequence
and overlapping by 10 aa. Phosphorylation assays were
carried out in the presence or absence of DNA, and the
results are shown in Fig. 5. The peptides encompassing the
serine residues at positions 16 or 62 were phosphorylated by
DNA-PK in the presence of DNA (lanes 2, 4, 12 and 13),
whereas the peptide containing the DNA-PK target site at
position 75 was not (lane 16). Thus, our results demonstrate
that, of three putative sites, only two are actually functional,
which is not surprising, as a similar pattern of DNA-PK
phosphorylation has also been observed for other proteins
(Anderson & Lees-Miller, 1992).
Physiological significance of Tat phosphorylation
by DNA-PK
Fig. 4. HIV-1 Tat is an excellent substrate for DNA-PK. (a)
Schematic representation of potential DNA-PK target sites (serine/
threonine followed by glutamine) in the HIV-1 Tat protein. (b)
Phosphorylation of undigested (1, 2) or thrombin-digested (3, 4)
GST–Tat protein phosphorylation by DNA-PK was examined in the
absence (1, 3) and the presence (2, 4) of DNA.
fused to the GST moiety (lane 2) and after its separation by
proteolytic cleavage with thrombin (lane 4). Interestingly,
there was no phosphorylation of Tat (lanes 1 and 3) in the
absence of DNA, clearly demonstrating the specificity and
purity of our DNA-PK protein preparation.
Serine residues at positions 16 and 62 are the
functional target sites for DNA-PK
phosphorylation
To map the position(s) of the phosphorylation site(s) in
the HIV-1 Tat protein precisely, we utilized a set of eight
Serine at position 62 lies immediately adjacent to the basic
region of Tat, suggesting that phosphorylation of Tat at
this site could modulate its HIV-1 LTR promoter
transactivation activity in different ways. These include
by affecting its interaction with TAR sequences, inducing
aberration in its nuclear localization (Karn, 1999; Tyagi
et al., 2001), or both. The serine residue at position 16 lies
in the N-terminal domain, which is also an essential
domain for efficient Tat transactivation activity (Karn,
1999), further suggesting the impact of Tat phosphorylation on HIV gene expression. The wild-type and mutated
GST–Tat proteins were used in a standard LTR transactivation assay using HL3T1 cells, a HeLa fibroblast-based cell
line harbouring an integrated LTR–CAT construct. We
generated Tat constructs mutated from serine to alanine at
either position 16 or 62, or at both positions. As shown in
Fig. 6(a), all three mutant proteins were modestly impaired
in LTR transactivation compared with wild-type Tat.
To confirm the functional significance, further similar
experiments were performed in Jurkat-pHR9P-Luc cells, a
T-cell line (a natural HIV target cell line) carrying HIV
provirus with luciferase as reporter (Kim et al., 2006;
Tyagi & Karn, 2007). Cells were transfected with 100 ng
of plasmids expressing different Tat constructs (mutant
Fig. 5. Two of three target sites for DNA-PK
phosphorylation are functional. Eight different
peptides, comprising 20 aa each and spanning the whole Tat sequence, each with 10 aa
overlapping the preceding peptide, were
phosphorylated by purified DNA-PK in the
absence (”) and presence (+) of DNA.
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S. Tyagi, A. Ochem and M. Tyagi
Fig. 6. Mutation in the DNA-PK target site
partially impaired Tat transactivation activity. (a)
HL3T1 cells were treated with the indicated
amounts of recombinant wild-type (Wt) or
mutated Tat proteins along with 100 mM
chloroquine. CAT assays were performed after
48 h and results represent the mean of several
independent assays. (b) Jurkat-pHR9P-Luc
cells were transfected with 100 ng plasmid
expressing different Tat proteins (wild-type
and mutants) under control of the CMV
promoter. All transfections were adjusted to
the same amount with backbone plasmid. The
graph shows the luciferase activity obtained
from duplicate experiments, repeated three
times. Bars indicate SD.
or wild-type) under control of the CMV promoter (Fig.
6b). The graph shows the luciferase activity obtained
from duplicate experiments, repeated three times. As
expected, similar to the CAT assays (Fig. 6a), the Tat
mutants clearly demonstrated modestly impaired transactivation capability. Nevertheless, this effect was reproducible in different experiments, as evidenced by short
error bars. Hence, comparable results were obtained when
Tat was expressed endogenously (Fig. 6b) or provided
exogenously (Fig. 6a).
Altogether, our results show clearly that DNA-PK facilitates
HIV-1 transcription essentially by phosphorylating the
RNAP II CTD, and that Tat phosphorylation also
contributes significantly to HIV replication.
DISCUSSION
In this study, we have demonstrated for the first time the
recruitment of DNA-PK at the HIV LTR. The parallel
recruitment of both DNA-PK and RNAP II at the HIV LTR
demonstrates clearly that DNA-PK is an integral part of a
larger RNAP II complex assembled at the HIV LTR. Our
observation is supported by another study that also
reported similar conclusions for another gene (An et al.,
2011). Although the precise role of DNA-PK in the RNAP
II complex recruited at the LTR remains the subject of our
future, more detailed investigation, the results obtained in
this study represent the elucidation of a potentially novel
mechanism underlying HIV-1 gene expression.
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The observations that DNA-PKcs and Ku (p70/p80) are
components of an RNAP II holoenzyme (Maldonado et al.,
1996) encouraged us to determine whether, along with
RNAP II, DNA-PK is also present in its functional
holoenzyme form at the HIV LTR. As expected, levels of
RNAP II at the latent provirus were highly restricted but,
upon proviral reactivation by TNF-a treatments, the levels
of RNAP II were increased severalfold, suggesting the
induction of HIV gene expression (Fig. 1a). Interestingly,
alongside this increase, the levels of DNA-PK also rose in
parallel upon TNF-a activation. Furthermore, our analysis
of DNA-PK levels at different LTR regions, such as the
promoter region, where recruitment identifies factors
involved in transcriptional initiation, and the nucleosome-2 region, which indicates factors involved in
transcriptional elongation, revealed similar increases in
the levels of DNA-PK at both regions following TNF-a
stimulation. Thus, these data argue strongly for the
involvement of DNA-PK during both the initiation and
the elongation phases of transcription. In addition, the
observed enrichment of DNA-PK at the LTR during HIV
transcriptional activation clearly suggests its important role
in HIV transcription.
By running high-resolution ChIP assays, we found a
parallel distribution of both RNAP II and DNA-PK along
the HIV provirus both before and after reactivation of
latent provirus (Fig. 1b). In addition, we also found their
association during both the initiation and the elongation
phases of transcription. Thus, our results confirm that
DNA-PK is a component of a larger RNAP II complex that
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DNA-PK plays a role in HIV transcription
is involved in both the initiation and the elongation phases
of HIV transcription.
Furthermore, our analysis of the phosphorylation pattern
of the CTD by DNA-PK unravelled the facilitatory roles of
this kinase complex in HIV transcription. Phosphorylation
of Ser-5 at the RNAP II CTD is linked to transcriptional
initiation (Kim et al., 2002; Yamamoto et al., 2001), whilst
Ser-2 hyperphosphorylation is found to be correlated with
the elongation phase of transcription (Kim et al., 2006;
Price, 2000; Saunders et al., 2006). The CTD of RNAP II is
a known phosphorylation target of several kinases,
including DNA-PK, and it is also known that knockdown
of any individual kinase does not confer complete
aberration of RNAP II function (Trigon et al., 1998;
Zhang & Corden, 1991). Phosphorylation of the CTD by
DNA-PK in the absence of the kinase consensus target
sequences appears to be in line with previous studies
reporting that certain proteins can be phosphorylated at
other sites by this DNA-dependent protein kinase
(Anderson & Lees-Miller, 1992). Although phosphorylation of the serine residues at positions 2 and 5 of the CTD
by DNA-PK emphasizes its role during both the initiation
and the elongation phases of transcription, the preferential
phosphorylation of the residue in position 2 over that in
position 5 clearly suggests a more prominent role for the
kinase during elongation. These results are also supported
by similar observations in the context of other cellular
promoters (Arias et al., 1991), where the presence of DNAPK in pre-initiation complexes and resultant CTD
phosphorylation have been reported.
Our results from employing shRNA to deplete endogenous
DNA-PK clearly define the physiological relevance of
DNA-PK in both HIV gene expression and replication. In
fact, upon DNA-PK knockdown, remarkably we found not
only a significant dose-dependent specific reduction in
HIV gene expression, but also a parallel reduction in HIV
replication. Furthermore, the shRNA knockdown experiments demonstrated clearly that cellular levels of DNA-PK
are sufficiently high to contribute significantly to facilitating HIV replication.
Overall, these results demonstrate that DNA-PK is an
integral part of RNAP II complexes recruited at the HIV
LTR and that DNA-PK plays significant roles in HIV
replication.
Prompted by an earlier observation that Tat forms direct
contacts with DNA-PK (Chun et al., 1998), we explored the
possibility of its phosphorylation by DNA-PK.
Interestingly, upon sequence analysis of the Tat protein
from the HXB2 strain of HIV-1, we discovered three
preferred DNA-PK target sites. The results of our
subsequent phosphorylation assays (shown in Figs 4 and
5) demonstrate clearly that HIV Tat is a bona fide substrate
of DNA-PK. As other authors have also observed, DNA-PK
does not always recognize all sequences conforming to the
target consensus sequences (reviewed by Smith & Jackson,
1999). We believe that the arginine residues of the
http://vir.sgmjournals.org
neighbouring basic domain of Tat might adversely affect
the phosphorylation process at position 75 by DNA-PK.
Previously, positively charged amino acids have been
suggested to negatively affect DNA-PK recognition on
neighbouring serines or threonines (Bannister et al., 1993),
probably due to conformational constraints. By analogy
with the observation made by the Nekhai group
(Ammosova et al., 2006), protein sequence analyses
revealed that the serine residues at positions 16 and 62
are fairly conserved among Tat proteins from different
HIV-1 isolates, but the same is not true in the case of HIV2 and simian immunodeficiency virus isolates.
We determined the physiological relevance of Tat phosphorylation by DNA-PK in HIV transcription by comparing the transactivation capabilities of wild-type Tat and of
Tat mutants carrying alanine instead of serine residues. The
modestly, but reproducibly lower HIV transactivation
activity of mutant Tat proteins, provided either exogenously (Fig. 6a) or expressed endogenously (Fig. 6b), clearly
demonstrates the wider role of DNA-PK in regulating HIV
gene expression and further reinforces the role of Tat
phosphorylation in HIV transactivation. However, it
should be noted that the only phenotype so far studied
for these mutants has been LTR transactivation.
Understanding the possible extent to which these mutations could interfere with other, non-transcriptional
functions of the Tat protein, including its neurotoxicity,
would require further investigation.
Interestingly, DNA-PK is not the only protein kinase able
to phosphorylate the Tat protein; at least three other
kinases [CDK2, protein kinase C (PKC) and the interferoninduced dsRNA-activated kinase (PKR)] have also been
shown to phosphorylate HIV Tat (Ammosova et al., 2006;
Brand et al., 1997; Endo-Munoz et al., 2005; Holmes,
1996). In addition, the Nekhai group has also confirmed
the presence of the phosphorylated form of Tat inside cells
(Ammosova et al., 2006).
Altogether, our results demonstrate that DNA-PK is an
integral part of RNAP II complexes recruited at the HIV
LTR and is able to facilitate both the initiation phase and
(primarily) the elongation phase of HIV transcription by
phosphorylating both Ser-2 and Ser-5, but mainly Ser-2
residues of the CTD of RNAP II. The influence of this
protein kinase complex on initiation and elongation is also
facilitated significantly by phosphorylation of the Tat
protein by DNA-PK.
These results are supported by a recent study (An et al.,
2011) that reported the interaction of DNA-PK with cyclin
T2, a subunit of P-TEFb, during transcription-coupled
repair in the dihydrofolate reductase gene and suggested
that this repair pathway could be a general feature of
normal transcription. Furthermore, the Rice laboratory has
also reported the interaction of Ku with CDK9, another
subunit of P-TEFb (Liu et al., 2010). CTD phosphorylation
induced by P-TEFb is well recognized to play an important
role during HIV transcriptional elongation. Clearly, these
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1717
S. Tyagi, A. Ochem and M. Tyagi
observations suggest an additional role of DNA-PK in HIV
transcription, probably by regulating P-TEFb recruitment.
Thus these observations substantiate our results and
suggest that, besides modulating CTD phosphorylation,
Tat phosphorylation and P-TEFb recruitment, DNA-PK
also facilitates HIV replication by supporting transcriptioncoupled repair during HIV gene expression.
Finally, the successful uncovering of the different pathways
that significantly affect HIV replication may provide
additional important and valuable targets for potential
drug molecules in the search and development of novel
anti-HIV-1 therapeutics.
METHODS
Cells and transfection. HL3T1 cells, kindly donated by B. Felber
(Felber & Pavlakis, 1988), are a HeLa-derived cell line containing an
integrated LTR–CAT construct. HL3T1 cells were grown in
Dulbecco’s modified Eagle’s medium (Invitrogen); Jurkat cells were
maintained in RPMI medium (Invitrogen). Both media were supplemented with 10 % FCS, 2 mM glutamine and 50 mg gentamicin ml21.
Cells were transfected by standard procedures. All transfections were
adjusted to the same amount of DNA by compensating with empty
plasmid. CAT and luciferase assays were performed 48 h after
transductions or transfections. The corresponding results represent the
mean values obtained in several (at least three) independent experiments.
HIV replication assays. Latently infected J1.1 cells (26106) were
superinfected with vesicular stomatitis virus G protein-pseudotyped
lentiviruses carrying either shRNA sequences against DNA-PK or
scrambled control shRNA sequences. After 36 h superinfection, cells
were treated in triplicate with 10 ng TNF-a ml21 and incubated for an
additional 51 h. Subsequently, virus levels in the supernatant were
determined by measuring the HIV-1 p24 Gag protein by ELISA,
following the protocol suggested by the manufacturer (ZeptoMetrix).
Biosciences; 3000 Ci (111 TBq) mM21; 10 mCi (370 MBq) ml21]
in a DNA-PK reaction buffer (Gottlieb & Jackson, 1993).
Phosphorylation was initiated by adding either 1 mg (in-housepurified) or 200–400 U (commercial; Promega) DNA-PK to the
reaction mixtures. Control reactions were carried out in the absence
of DNA. Reaction mixtures were incubated for 1 h at 37 uC and the
reaction was stopped by boiling in denaturing SDS-PAGE gel loading
buffer. The phosphorylated products were analysed by electrophoresis
either on a 4–20 % gradient Tris/glycine SDS-PAGE gel or on
phosphate buffer–polyacrylamide gel at pH 6.0 (Trigon et al., 1998).
Protein phosphorylation was quantified by densitometry.
ACKNOWLEDGEMENTS
We are grateful to the laboratory of Professor Jonathan Karn for
providing several cell lines and constructs. We thank Drs Zachariev
and Guarnaccia at ICGEB for synthesizing CTD peptides. We also
appreciate the support of the late Professor Arturo Falaschi. This
work is supported by AmFAR (the Foundation for AIDS Research)
and Case Western Reserve University grants to M. T.
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