Journal of General Virology (2011), 92, 1519–1531
DOI 10.1099/vir.0.030494-0
Recruitment of cyclin-dependent kinase 9 to
nuclear compartments during cytomegalovirus late
replication: importance of an interaction between
viral pUL69 and cyclin T1
Sabine Feichtinger,1 Thomas Stamminger,1 Regina Müller,1 Laura Graf,1
Bert Klebl,23 Jan Eickhoff23 and Manfred Marschall1
Correspondence
1
Manfred Marschall
2
Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, Erlangen, Germany
[email protected].
GPC Biotech AG, Martinsried, Germany
uni-erlangen.de
Received 17 January 2011
Accepted 24 March 2011
Cyclin-dependent protein kinases (CDKs) are important regulators of cellular processes and are
functionally integrated into the replication of human cytomegalovirus (HCMV). Recently, a
regulatory impact of CDK activity on the viral mRNA export factor pUL69 was shown. Here,
specific aspects of the mode of interaction between CDK9/cyclin T1 and pUL69 are described.
Intracellular localization was studied in the presence of a novel selective CDK9 inhibitor, R22,
which exerts anti-cytomegaloviral activity in vitro. A pronounced R22-induced formation of nuclear
speckled aggregation of pUL69 was demonstrated. Multi-labelling confocal laser-scanning
microscopy revealed that CDK9 and cyclin T1 co-localized perfectly with pUL69 in individual
speckles. The effects were similar to those described recently for the broad CDK inhibitor
roscovitine. Co-immunoprecipitation and yeast two-hybrid analyses showed that cyclin T1
interacted with both CDK9 and pUL69. The interaction region of pUL69 for cyclin T1 could be
attributed to aa 269–487. Moreover, another component of CDK inhibitor-induced speckled
aggregates was identified with RNA polymerase II, supporting earlier reports that strongly
suggested an association of pUL69 with transcription complexes. Interestingly, when using a
UL69-deleted recombinant HCMV, no speckled aggregates were formed by CDK inhibitor
treatment. This indicated that pUL69 is the defining component of aggregates and generally may
represent a crucial viral interactor of cyclin T1. In conclusion, these data emphasize that HCMV
inter-regulation with CDK9/cyclin T1 is at least partly based on a pUL69–cylin T1 interaction, thus
contributing to the importance of CDK9 for HCMV replication.
INTRODUCTION
Human cytomegalovirus (HCMV) is a worldwide human
pathogen belonging to the beta subgroup of the family
Herpesviridae. Although primary HCMV infection of the
immunocompetent host typically remains asymptomatic,
severe disease can occur upon infection of immunonaı̈ve
and immunocompromised individuals such as neonates,
transplant recipients and cancer or AIDS patients
(Mocarski et al., 2007). The efficiency of HCMV replication is dependent on the interplay between viral and hostcell functions (Marschall et al., 2011). In particular, several
regulatory steps in the course of HCMV infection depend
on the activity of cellular cyclin-dependent protein kinases
(CDKs), mainly CDK1, -2, -7 and -9 (Bain & Sinclair,
3Present address: Lead Discovery Center GmbH, Dortmund, Germany.
Supplementary co-immunoprecipitation data are available with the online
version of this paper.
030494 G 2011 SGM
2007; Fortunato et al., 2000; Jault et al., 1995; Kapasi &
Spector, 2008; Kapasi et al., 2009; Rechter et al., 2009;
Sanchez & Spector, 2006; Sanchez et al., 2004; Tamrakar
et al., 2005). HCMV-induced upregulation of CDK1, -2, -7
and -9 along with the corresponding cyclin subunits is
explained by the fact that CDK activity is required at
various stages of virus replication, such as the regulation of
gene expression and the modification as well as localization
of viral proteins (Bain & Sinclair, 2007; Kapasi et al., 2009;
Rechter et al., 2009). CDKs are heterodimeric serine/
threonine kinases that become activated upon binding to
their regulatory cyclin subunits. CDKs are involved in
the regulation of multiple cellular processes and can be
subdivided into two major functional groups: cell cycleassociated and transcription-regulating CDKs. Whilst
CDK1 and -2 mostly participate in cell-cycle regulation,
CDK7 and -9 represent prototypes of transcriptionregulating CDKs, which act by phosphorylating the
C-terminal domain (CTD) of the large subunit of RNA
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S. Feichtinger and others
polymerase II (RNAPII; Majello & Napolitano, 2001;
Reinberg et al., 1998). In humans, the CTD consists of 52
repeats of the consensus heptapeptide sequence Tyr-SerPro-Thr-Ser-Pro-Ser and is susceptible to differential phosphorylation levels in the event of transcription (Meinhart
et al., 2005). Transcriptional activity is highly dependent on
the phosphorylation pattern of the serines at positions 2
and 5. Once the hypophosphorylated form of RNAPII has
been recruited to the pre-initiation complex, phosphorylation of the CTD at the serine residue at position 5
(mediated by CDK7/cycH/MAT-1, which is part of the
transcription factor II H transcription complex) promotes
the initiation of transcription. However, productive transcription requires further phosphorylation of CTD at serine
residue 2 (mediated by CDK9/cyclin T1, also known as
positive transcription elongation factor b).
Virus-specific regulation and reprogramming of CDK
activity has been demonstrated by numerous studies on
HCMV and other herpesviruses (Biglione et al., 2007; DaiJu et al., 2006; Davido et al., 2003; Diwan et al., 2004; Jault
et al., 1995; Kapasi et al., 2009; Kapasi & Spector, 2008;
Kudoh et al., 2003, 2004; Pumfery et al., 2006; Rechter
et al., 2009; Salvant et al., 1998; Sanchez & Spector, 2006;
Sanchez et al., 2003, 2004; Schang, 2002, 2005a, b;
Tamrakar et al., 2005; Wang et al., 2001). These findings
have intensified the research on pharmacological CDK
inhibitors (PCIs) and their putative use in antiviral therapy
(Bresnahan et al., 1997; Herget & Marschall, 2006;
Marschall & Stamminger, 2009; Schang et al., 2006). Classical PCIs are low-molecular-mass compounds (,500 Da)
that consist of flat hydrophobic heterocyclic compounds.
They typically bind by hydrophobic interactions and
hydrogen bonds to the target kinases (Hardcastle et al.,
2002), resulting mostly in binding of the ATP-binding
pocket of their target CDKs, thus inhibiting kinase activity
by competing with the ATP co-substrate. In many HCMVbased studies, the CDK inhibitor roscovitine has been
proven as a useful tool to investigate virus–CDK interactions in detail. Roscovitine is a purine analogue classified as
a PCI preferentially inhibiting CDK1, -2, -5, -7 and -9 (de
la Fuente et al., 2003; Fischer & Gianella-Borradori, 2003;
Schang, 2002, 2005b) that, interestingly, binds with highest
affinity to the active form of target CDKs (De Azevedo
et al., 1997; Knockaert et al., 2002).
For HCMV, studies using roscovitine have clearly demonstrated inhibition of virus replication at several stages. As the
time point of drug addition is relevant, it is possible to
distinguish between effects due to the presence of roscovitine immediately after infection and due to addition of the
drug at 6–8 h post-infection (p.i.) (Kapasi & Spector, 2008;
Kapasi et al., 2009; Sanchez & Spector, 2006; Sanchez et al.,
2004; Tamrakar et al., 2005). Instant inhibition of CDK
activity strongly impairs the entire immediate-early (IE)
gene expression (mRNA and protein levels) with subsequent
consequences on the expression of early and late genes.
When the addition of roscovitine is delayed until 6–24 h p.i.,
other effects are observed, including a specific decrease in
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IE2 protein (IE2p86) and the major tegument protein,
pp150. In addition, pUL69, a viral mRNA export factor and
multifunctional protein, accumulates in a hyperphosphorylated form, in which its localization changes from a
homogeneous distribution within viral nuclear replication
centres towards a speckled aggregation throughout the
nucleus (Rechter et al., 2009; Sanchez & Spector, 2006;
Tamrakar et al., 2005). pUL69 has been characterized as a
nuclear mRNA export factor (Lischka et al., 2001, 2006;
Toth et al., 2006), a transcriptional activator (Winkler &
Stamminger, 1996; Winkler et al., 1994) and an inducer of
cell-cycle arrest (Hayashi et al., 2000; Lu & Shenk, 1999).
Previously, we demonstrated that pUL69 is phosphorylated
by CDKs (Rechter et al., 2009) and by viral protein kinase
pUL97 (Thomas et al., 2009). Phosphorylation is a determinant of the mRNA export activity of pUL69, as concluded from the finding that both CDK and pUL97
inhibitors can suppress this activity in a reporter assay
(Rechter et al., 2009; Thomas et al., 2009). In the present
study, we characterized the interaction between CDK9/
cyclin T1 and pUL69 and analysed the significance of this
regulatory association. A scenario addressing the molecular
mechanism and functional consequences of this mutual
virus–cell inter-regulation is discussed.
RESULTS AND DISCUSSION
HCMV replication is positively regulated by CDK9
activity
The influence of CDK9 activity on HCMV replication was
analysed by the use of a novel, selective CDK9 inhibitor
termed R22. This compound, belonging to the chemical
class of aminopyrimidines, showed a marked selectivity
towards CDK9/cycT1 activity in vitro (IC50 0.04±0.01 mM).
Other CDK/cyclin complexes showed little sensitivity to R22
(Fig. 1a). A selectivity panel with a series of non-CDK
human protein kinases illustrated the selective inhibitory
potency of R22, with the exceptions of GSK-3b and EGFR,
which indicated some additional modest level of inhibition
(Fig. 1b). When assayed for a putative anti-cytomegaloviral
activity, R22 exerted a concentration-dependent inhibition
of HCMV replication in primary human foreskin fibroblasts (HFFs; Fig. 1c), as expressed by an IC50 value of
6.07±0.38 mM. It should be noted that the IC50 of 0.04 mM
for CDK9 in vitro was measured in a biochemical assay at the
Km [ATP] of ~30 mM. Under intracellular conditions, R22
has to compete with much higher concentrations of ATP in
a range between 1 and 5 mM, meaning that the intracellular
CDK9 inhibition with a measured IC50 of 6.07 mM is in the
expected range (according to the Cheng–Prusoff equation,
predicting a micromolar efficacy range in vivo; Cheng &
Prusoff, 1973). This finding suggests a strong impact of
CDK9 on the replication efficiency of HCMV. As far as the
controversially discussed role of EGFR in HCMV entry and
replication is concerned (Chan et al., 2009; Isaacson et al.,
2007; Marschall & Stamminger, 2009), we cannot fully
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Journal of General Virology 92
Inter-regulation between HCMV pUL69 and cyclin T1/CDK9
Fig. 1. Anti-HCMV effect of CDK9 inhibitor R22 and inhibitor-induced nuclear relocalization of pUL69 and CDK9. The CDK9
inhibitor R22 was analysed for selectivity among CDK/cyclin complexes (a) or other human protein kinases (b). Data from the in
vitro kinase assays are presented as IC50 values or the percentage of inhibition at 10 mM (% no inhibitor). Assays were
performed at least in triplicate or reproduced by independent confirmation settings. Grey-shaded fields, inhibition ¢50 % (cutoff); yellow-shaded fields, inhibition ¢90 %. CDK/cyc, cyclin-dependent protein kinase/cyclin complex; Pim-1, proviral insertion
site serine/threonine kinase 1; PLK1, polo-like kinase 1; v-Abl Gag, tyrosine kinase Abl fused to Mo-MuLV Gag; p70S6K,
70 kDa ribosomal serine/threonine protein S6 kinase; c-Met, receptor tyrosine kinase Met; c-Src, Src-family tyrosine kinase;
c-Raf, Ras-binding kinase 1; SRPK1, serine/arginine-specific protein kinase; RSK1, ribosomal S6 kinase 1; ROCK2, Rho
serine/threonine kinase 2; PDGFRb, platelet-derived growth factor receptor b; ERK1, extracellular signal-regulated kinase 1;
Kit, tyrosine kinase c-kit; Jnk1a1, c-Jun N-terminal kinase 1a1; InsR, insulin receptor; IKKb, IkB kinase b; GSK-3b, glycogen
synthase kinase 3 b; EGFR, epidermal growth factor receptor; CK1a, casein kinase 1 a; Akt/PKBa, serine/threonine kinase, also
known as protein kinase B a. (c) HFFs were infected with HCMV AD169 (m.o.i. of 0.25) and treated immediately with R22 at
different concentrations as indicated. Cells were lysed at 7 days p.i. and subjected to GFP fluorometry. Determinations were
performed in quadruplicate. Ganciclovir (GCV) served as an antiviral reference drug. (d) HFFs were infected with HCMV
AD169 (m.o.i. of 1) and treated with roscovitine (Rosco; 10 mM) or R22 (10 mM), both added at 24 h p.i. Intracellular
localization of pUL69 and CDK9 was determined by indirect immunofluorescence analysis at 72 h p.i. Nuclei were stained with
DAPI.
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S. Feichtinger and others
exclude an additional EGFR-based anti-cytomegaloviral
effect of R22. However, our previous studies with established
EGFR inhibitors (e.g. AG1478) indicated that a pronounced
anti-cytomegaloviral effect of these inhibitors can only be
measured after pre-incubation of the cells prior to virus
addition (Herget et al., 2004). In the experiment described
in Fig. 1(c), R22 was not pre-incubated but was added
immediately after virus adsorption, therefore arguing
against an EGFR-based mode of inhibition. In addition,
according to the Cheng–Prusoff equation, all other kinases
analysed are predicted to possess a low affinity to R22,
which renders it very unlikely that other kinases are
responsible for the antiviral effect caused by R22. This
issue was addressed further by supplementary experiments
investigating the CDK9 selectivity of R22 (e.g. analyses on
putative R22 effects towards the CDK1-mediated phosphorylation of Eg5 Thr-927 as well as CDK2- and CDK4/
6-mediated substrate phosphorylations), not providing
any indication for off-target effects of R22 (J. Eickhoff,
unpublished data). Thus, inhibition of CDK9 activity was
considered to be responsible for the R22-mediated block
of HCMV replication.
CDK9 and cyclin T1 (but not CDK-1, -2 or -7) have been
identified (Rechter et al., 2009). To address the question of
whether individual speckled aggregates vary in their protein
composition or whether pUL69, CDK9 and cyclin T1 are
constantly detectable, co-staining of multiple proteins in
individual cells was performed by confocal immunofluorescence analysis. As shown in Fig. 2 (panels s–v and y–b),
each aggregate analysed was invariably composed of pUL69,
CDK9 and cyclin T1. Of note, a slight difference in
localization of the proteins was detected in HCMV-infected
cells in the absence of an inhibitor (Fig. 2, panels f–q).
Whilst in smaller replication centres the co-localization
between pUL69, CDK9 and cyclin T1 was observed mainly
at the periphery (Fig. 2, panel k), these co-localization areas
were distributed throughout enlarged replication centres
(Fig. 2, panel q). The reason for this phenomenon is
unexplained so far. Under conditions of CDK inhibition, the
three proteins co-localized mainly in the centre of speckled
aggregates (Fig. 2, panels w and c). No difference was noted
between various aggregates, i.e. pUL69, CDK9 and cyclin T1
were found consistently and were perfectly co-localized.
Direct interaction between pUL69 and cyclin T1
Nuclear speckled aggregates are formed in the
presence of CDK9 inhibitor R22
Co-localization between pUL69, CDK9 and cyclin
T1 in R22-induced aggregates is consistently a
three-component pattern in individual speckles
The strict co-localization patterns observed suggested an
interaction between pUL69 and the CDK9/cyclin T1
complex. This idea was supported by previously published
data obtained with a yeast two-hybrid system indicating a
pUL69–cyclin T1 interaction (Rechter et al., 2009). To
validate these findings, co-immunoprecipitation analyses
were performed with proteins from HCMV-infected primary
fibroblasts and plasmid-transfected 293T cells (Fig. 3). In the
transient transfection system, all three proteins could be
overexpressed to amounts suitable for this analysis; however,
some co-expressions clearly influenced each other, leading to
variations in expression levels (Fig. 3b). Importantly, the
amounts of effectively immunoprecipitated proteins were
nevertheless very similar among samples, as shown for the
example of pFLAG-UL69 (Fig. 3a, lanes 2 and 3 and 7–9).
Detection of the proteins was achieved by the use of tagspecific antibodies or antibodies recognizing both the
recombinant and the endogenous version of the protein
(pAb-cycT1). As a striking result, cyclin T1, either in the
absence or presence of co-expressed CDK9–haemagglutinin
(HA), could be co-immunoprecipitated with an antibody
recognizing pFLAG-UL69 (Fig. 3a and b, lanes 8 and 9).
CDK9–HA alone was not co-immunoprecipitated together
with pFLAG-UL69 (Fig. 3a, lane 7). All controls (Fig. 3a,
lanes 1–6 and 10–13) demonstrated the reliability and
specificity of the assay. In the reciprocal setting, transiently
expressed pFLAG-UL69 could also be co-immunoprecipitated when using a precipitation antibody recognizing
endogenous cyclin T1 (pAb-cycT1; see Supplementary Fig.
S1, available in JGV Online). Thus, pUL69 specifically binds
to cyclin T1 in transiently transfected cells.
Nuclear speckled aggregates induced by CDK inhibitors
appear to contain a group of proteins, among which pUL69,
This result was confirmed with proteins co-immunoprecipitated from lysates of HCMV-infected cells. HFFs were
The intracellular localization of CDK9 in HCMV-infected
fibroblasts was analysed by indirect immunofluorescence
using a confocal laser-scanning microscope. In particular,
we wanted to clarify whether the inhibitor R22 was able to
induce speckled aggregates containing viral pUL69, CDK9
and other proteins, as typically obtained by treatment with
roscovitine (Rechter et al., 2009; Sanchez & Spector, 2006).
As an important finding, R22 was as efficient as roscovitine
in inducing speckled aggregates (Fig. 1d). HCMV-infected
cells treated with R22 or roscovitine showed the typical
speckled co-localization between pUL69 and CDK9 (Fig.
1d, panels i–p). In the absence of inhibitor, CDK9 was
detected in a homogeneous localization over the entire
nucleus in mock-infected cells (Fig. 1d, panels a–d) or was
recruited into virus replication centres during late times of
HCMV replication (Fig. 1d, panels e–h), as described
previously (Rechter et al., 2009). In control settings, inhibitors of other protein kinases (including EGFR inhibitor
AG1478) did not induce alterations of the nuclear localization of pUL69 or CDK9 (Rechter et al., 2009; Thomas
et al., 2009; S. Feichtinger & M. Marschall, unpublished
data). Thus, inhibition of CDK9 disturbed the normal
nuclear localization of viral and cellular proteins such as
pUL69 and CDK9 and thus may interfere with an important step of virus–cell inter-regulation.
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Journal of General Virology 92
Inter-regulation between HCMV pUL69 and cyclin T1/CDK9
Fig. 2. Immunofluorescence analysis of the localization of CDK9, cyclin T1 (cycT1) and pUL69 in parallel. AD169-infected HFFs
(m.o.i. of 1) were treated with the broad CDK inhibitor roscovitine (Rosco) or with the CDK9 inhibitor R22 from 24 h p.i. The
intranuclear distribution of CDK9, cyclin T1 and pUL69 was monitored at 72 h in parallel using protein-specific antibodies. The inset
images show enlargements of representative areas of the replication centres or speckled aggregates. Nuclei were stained with DAPI.
infected with HCMV AD169 (m.o.i. of 1 and 2) and
subsequently treated with roscovitine or left untreated (Fig.
3c, d). In both cases, pUL69 could be co-immunoprecipitated with cyclin T1. Roscovitine treatment augmented the
amount of co-immunoprecipitated pUL69 to some extent
(Fig. 3c, upper panel, lanes 4 and 6). Cyclin T1 and CDK9
were found to be upregulated in HCMV-infected cells.
Interestingly, CDK9 was to some extent also detectable in the
cyclin T1–pUL69 co-immunoprecipitates (Fig. 3c, second
panel, lanes 3–6). Due to the upregulation of CDK9,
however, the experiment did not allow us to conclude
whether this interaction of the three proteins was intensified
in a non-competitive way in HCMV-infected compared with
mock-infected cells. Nevertheless, the data suggest the
formation of a functional three-component complex on
the basis of a direct pUL69–cyclin T1 interaction.
of the positive colonies remained close to the detection
limit (Fig. 3e, panels 4, 7 and 11). Interestingly, when using
N-terminally truncated versions of pUL69, the interaction
was positive for aa 269–744 (Fig. 3e, panels 7–8) but
negative for aa 380–744 (Fig. 3e, panels 9 and 10). In
addition, a C-terminally truncated construct (aa 1–487;
Fig. 3e, panels 11 and 12) also showed cyclin T1 interaction
activity. Further C-terminally truncated versions of pUL69
were negative (data not shown). This result indicated the
importance of pUL69 region aa 269–487 for cyclin T1
interaction.
The domain of pUL69 responsible for interaction with
cyclin T1 was narrowed down by experiments using the
yeast two-hybrid system (Fig. 3e). The pUL69–cyclin T1
interaction was detectable in independent experiments, but
the signal intensity in filter lift assays was low so that most
The association of pUL69–cyclin T1–CDK9 aggregates
with further cellular proteins was investigated. As the
CDK9–cyclin T1 complex is a regulatory component of
transcription complexes, the question of an association
with RNAPII was addressed. For this purpose, RNAPII-
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RNAPII is associated with virus replication
centres and CDK inhibitor-induced nuclear
aggregates
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S. Feichtinger and others
Fig. 3. Co-immunoprecipitalion (CoIP) and yeast two-hybrid analyses demonstrating the interaction between CDK9, cyclin T1 and
pUL69. (a, b) CoIP with proteins from transiently transfected cells: 293T cells were co-transfected with CDK9-HA, cyclin T1 (cycT1)
and pFLAG-UL69 in various combinations as indicated and lysed at 48 h post-transfection. (a) pFLAG-UL69 was
immunoprecipitated from cell lysates followed by detection of possible co-precipitated interactors by SDS-PAGE and Western
blot (WB) analysis using tag- or protein-specific antibodies. (b) Expression of individual proteins was controlled. RFP, Red
fluorescent protein. (c, d) CoIP with proteins from HCMV-infected cells: interaction between cyclin T1 and pUL69 was confirmed in
HCMV-infected cells (m.o.i. of 1 or 2) in the absence or presence of roscovitine (10 mM). (c) After precipitation of cyclin T1 from cell
lysates at 72 h p.i., immunoprecipitates were studied for pUL69 and CDK9. Staining of IE1 was used as specificity control. pAbg,
Goat polyclonal antibody. (d) Expression of proteins of interest was monitored by WB analysis. (e) The yeast two-hybrid system was
used to narrow down the protein domains responsible for the interactions identified by CoIP analysis. Signals obtained by staining of
yeast colonies in a filter lift assay were categorized as strongly positive (4), intermediate or weakly positive (+) and negative (”).
Constructs pVA3 and pTD1 expressing p53 and simian virus 40 large T antigen (SV40-T) were used as positive controls (Clontech).
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Journal of General Virology 92
Inter-regulation between HCMV pUL69 and cyclin T1/CDK9
specific antibodies were used to detect RNAPII in its
differentially phosphorylated forms of the CTD. The
putative co-localization of RNAPII with viral nuclear
compartments in HCMV-infected cells was analysed by
confocal laser-scanning microscopy. When using an antibody recognizing both the unphosphorylated and phosphorylated forms of CTD of RNAPII, co-localization with
virus replication centres was detected (mAb-RNAPII; Fig.
4a, panels e–h). Even more strikingly, a distinct colocalization of RNAPII with pUL69 in inhibitor-induced
speckled aggregates could be demonstrated (Fig. 4a, panels
j–m and o–r). The inset images show that both proteins were
found throughout these structures within replication centres
(Fig. 4a, panel i), whilst in speckled aggregates the proteins
co-localized mainly at their periphery (Fig. 4a, panels n and
s). When specifically detecting the unphosphorylated form,
a strong signal was found within virus replication centres
(mAb-RNAPII-0; Fig. 4b, panels e–h), but association with
speckled aggregates could only be detected to a minor extent
(Fig. 4b, panels i–p). Further investigation of the localization
of the phosphorylated forms did not clearly reveal distinct
and reliable staining patterns but showed variability between
separate experiments (mAb-RNAPII-5P and mAb-RNAPII2P; data not shown). Therefore, the association of
phosphorylated RNAPII subforms within speckled aggregates could not be safely defined by this experimental
setting. However, the present data argue for co-localization
of RNAPII with virus replication centres at late times of
infection, and, as shown with the non-phospho-specific
mAb-RNAPII, an association of RNAPII with CDK
inhibitor-induced aggregates could also be demonstrated.
pUL69 represents the defining component of
nuclear speckled aggregates
To address the question of which of the associated proteins
might determine their recruitment to CDK inhibitorinduced speckled aggregates, we performed infection experiments with a bacterial artificial chromosome (BAC)-derived
recombinant of HCMV AD169 lacking a functional UL69
gene (HB5DUL69). This virus does not express pUL69 (Fig.
5c) and develops a replication cycle that is inefficient and
retarded in the kinetics of viral gene expression (R. Müller, S.
Feichtinger & T. Stamminger, unpublished data). Despite
these defects, HB5DUL69 formed normal virus replication
centres in the nuclei of infected fibroblasts, as characterized
by the specific organization of viral DNA processivity factor
pUL44, used here as a marker protein (Fig. 5a, b, panels e–
h). In addition, in the presence of roscovitine, the formation
of virus replication centres appeared mostly unaffected (Fig.
5a, b, panels i–l). As a control, a BAC-derived recombinant
HCMV lacking UL27 was analysed in parallel (TB40/
EDUL27; Le et al., 2008), showing an unaltered speckled
aggregation of pUL69 comparable to HCMV AD169
(data not shown). As an important finding, however,
HB5DUL69-infected cells were totally devoid of roscovitine-derived speckled aggregates. Neither CDK9 nor cyclin
T1 was detectable in the location of any speckled structures
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in the late phase of virus replication (Fig. 5a, b, panel j).
These findings strongly argue for pUL69 as the defining
component of nuclear aggregates upon inhibition of CDK
activity. Interestingly, in the case of HCMV HB5DUL69,
cyclin T1 and CDK9 were detectable within replication
centres even in the absence of pUL69 (Fig. 5a, b, panels f
and j). Thus, although pUL69 appears to be the key
component for the formation of speckled aggregates,
another as yet undefined viral protein seems to be required
for the recruitment of cyclin T1 and CDK9 to nuclear
replication centres.
Conclusions
This study provides the first evidence for (i) the direct
interaction between pUL69 and cyclin T1 in HCMVinfected fibroblasts, (ii) the specific role of CDK9 activity
for the correct nuclear localization of viral and cellular
proteins, (iii) the initial supposition that inhibition of CDK9
may be sufficient to induce a speckled aggregation of the
pUL69–cyclin T1–CDK9 complex and to block virus
replication in fibroblasts, (iv) the association of further
proteins such as RNAPII with these aggregates and (v) the
importance of pUL69 for cyclinT1–CDK9 interaction as the
defining component of CDK inhibitor-induced aggregates.
In this work, we show direct binding of cyclin T1 to pUL69
in transfected 293T cells and in HCMV-infected fibroblasts.
The fact that cyclin T1 and not CDK9 acts as the binding
partner of pUL69 is in line with several findings of CDK9/
cyclin T1-associated proteins. Brd4 recruits CDK9 via its
binding to cyclin T1 to the paused RNAPII at acetylated
promoter regions to enhance transcription elongation (Yang
et al., 2008). CDK9 localization seems generally dependent
on cyclin T1-binding in both uninfected and HCMVinfected fibroblasts (Kapasi et al., 2009; Napolitano et al.,
2002). Along with previous results (Rechter et al., 2009), the
identification of a cyclin T1–pUL69 interaction indicates
recruitment of CDK9 to pUL69 via cyclin T1 in order to
enable pUL69 phosphorylation and, consequently, the
modulation of pUL69 activity.
Our studies with roscovitine and other CDK inhibitors on
the localization pattern of viral and cellular nuclear
proteins supported the idea of aggregation of a selective
panel of proteins in the observed speckled nuclear
structures. The specificity of proteins studied indicated
that pUL69, CDK9, cyclin T1 and RNAPII could be
identified as constant components in speckled aggregates,
whereas other proteins, such as HDAC2, CDK7, cyclin B1,
SC-35, pp65, pp71, IE2p86, pUL26, pUL44, pUL50,
pUL84, pUL97 and pUL112–113, were found not to be
associated. These proteins either remained in their typical
localization or were recruited to virus replication centres
but were never detectable in speckled aggregates (Rechter
et al., 2009; data not shown).
The phenomenon of pUL69 speckled aggregation induced
by roscovitine seems not to be confined to HCMV
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S. Feichtinger and others
Fig. 4. Distribution pattern of cellular RNAPII in relation to pUL69. HFFs were infected with AD169 (m.o.i. of 1) and cultured
with roscovitine or R22 from 24 h p.i. At 72 h, cells were used for immunofluorescence staining for the detection of pUL69 and
different forms of the CTD of RNAPII. Detection antibodies were used that either recognized the unphosphorylated as well as
the phosphorylated forms (a) or selectively recognized the unphosphorylated form of the CTD of RNAPII (b). Inset images show
enlargements of representative areas of the replication centres or speckled aggregates.
laboratory strain AD169 but has also been detected for
AD169-derived virus variants carrying ganciclovir/cidofovir
resistance and for other viral strains (e.g. Towne; Rechter
et al., 2009). Here, we additionally analysed clinical HCMV
isolates (29A and 5B) and a novel multidrug-resistant
HCMV (A834P; Scott et al., 2007). All viruses showed
pronounced aggregation of pUL69, which was very similar
1526
to the pattern described for the AD169 reference strain (data
not shown). Thus, the known differences in the coding
capacity of these viruses do not provide functions that
render pUL69 independent from roscovitine- or R22induced aggregation. Several lines of evidence indicate that
CDK inhibitor-induced speckled aggregates may represent
nuclear structures in which the normal function of
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Journal of General Virology 92
Inter-regulation between HCMV pUL69 and cyclin T1/CDK9
Fig. 5. Localization of CDK9 and cyclin T1 (cytT1) in cells infected with a recombinant UL69-deleted virus (HCMV HB5DUL69).
HFFs were infected with HB5DUL69 for 9 days (until the onset of visually detectable cytopathic effects induced by the
decelerated virus replication), followed by roscovitine treatment for 48 h. Localization of CDK9 (a) and cyclin T1 (b) was visualized
using specific antibodies. In addition, viral pUL44 was stained with a pUL44-specific antibody as a marker for nuclear replication
centres. (c) A control staining of HB5DUL69-infected cells with a specific antibody confirmed the absence of pUL69 expression.
associated proteins is suppressed. This suppression may add
to the strong inhibitory effect that CDK inhibitors exert
on HCMV replication. CDK-specific regulation can be
http://vir.sgmjournals.org
manifested at several, so far poorly characterized regulatory
steps of the HCMV replication cycle, such as protein
phosphorylation, modulation of protein activity, protein
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S. Feichtinger and others
interactions and transport pathways. The data presented in
this study support the evolving concept that CDK9/cyclin T1
activity is highly associated with the regulatory functions
of pUL69 and thus with the efficiency of HCMV replication. Further detailed aspects of the molecular mechanism
of CDK9/cyclin T1–HCMV inter-regulation need to be
elucidated in future studies.
METHODS
CDK inhibitors and reference compounds. Roscovitine was
purchased from Calbiochem and CDK inhibitor R22 (aminopyrimidine) was provided by GPC Biotech AG. R22 is the product of a series
of medicinal chemistry optimization cycles, based on hit compounds
identified in a high-throughput screening of purified recombinant
CDK9/cyclin T1 complex against a kinase-biased screening collection.
The characteristics of R22 indicate a high CDK9 potency, cellular
CDK9 activity and selectivity within the kinase family. Moreover,
several pharmacological properties, such as solubility, bioavailability
and metabolic stability, appear very promising. Compounds were
prepared in DMSO and aliquots were stored at 220 or at 280 uC (for
periods longer than 3 months).
Selectivity of CDK inhibitor R22. For selectivity panels of
R22, various cellular protein kinases were assayed for activity by a
[33P-c]ATP-based in vitro kinase assay (Herget et al., 2004; Mett et al.,
2005). Assays were adequate when the Z prime value was .0.5.
Reactions were performed in the linear range for reaction time and
enzyme concentration as well as an ATP concentration according to
the Km of the respective enzyme for comparison of the inhibitory
effects of the compounds, as described previously (Mett et al., 2005).
In addition to the radioactive in vitro kinase assays, substrate
phosphorylation was determined in an alternative assay format by an
immobilized metal assay of phosphorylation (IMAP, Molecular
Devices; Sportsman et al., 2004). IMAP technology is based on the
covalent-coordinate, high-affinity interaction of trivalent metalcontaining nanoparticles with phospho-groups linked to serines,
threonines or tyrosines. In brief, fluorescence-labelled peptides were
phosphorylated in a kinase reaction in a microtitre plate-based
format. The reaction was stopped by addition of the IMAP reagent
(Molecular Devices), which contained IMAP beads binding to
phosphorylated substrate peptides, thereby leading to an increase
in fluorescence polarization upon phosphorylation. Signals were
detected by fluorescence polarization measurement in a Victor Wallac
reader (Perkin Elmer).
Cell culture, HCMV infections and plasmid transfection. 293T
cells were cultivated in Dulbecco’s modified Eagle’s medium
containing 10 % FBS. HFFs were cultivated in minimal essential
medium containing 7.5 % FBS. HCMV laboratory strain AD169 was
propagated in HFFs. Titres of virus stocks were determined using
standard plaque titration. HFFs (2.56105 cells per well in a six-well
plate) were infected as reported previously (Marschall et al., 2000).
Transfection of 293T cells was performed using polyethylenimine
reagent (Sigma) as described previously (Schregel et al., 2007).
Generation of recombinant UL69-deleted HCMV HB5DUL69.
The recombinant AD169-derived HCMV HB5DUL69 was generated,
isolated and propagated as described previously (Tavalai et al., 2008).
Briefly, BAC mutagenesis was performed via homologous recombination in Escherichia coli using a linear recombination cassette and
the parental BAC pHB5 (Borst et al., 1999). The recombination
cassette consisted of a kanamycin resistance gene framed by 50 nt
with homology to the 59- and 39-flanking regions of UL69. The
1528
respective fragment was generated by PCR amplification using the
primer pair F-P1UL69-3 (59-AACGGGATAAGGGACAGCAATCATCACGCACAACACCCTTCACTCTCTTTGTGTAGGCTGGAGCTGCTTC-39) and R-P4UL69-5 (59-CTATATATACATCAGCGTGCCCGAACGTGACCTTCCTAGCGACGGCGGCCATTCCGGGGATCCGTCGACC-39) together with plasmid pKD13 as template, which codes
for the kanamycin resistance marker (Datsenko & Wanner, 2000). In
the next step, homologous recombination in E. coli was performed as
described previously (Tavalai et al., 2008). Thereafter, the structural
integrity of the recombinant BAC was confirmed via restriction
enzyme digestion and nucleotide sequence analysis of the recombined
gene region. BAC DNA of positive recombinants was isolated using the
Nucleobond AX 100 kit according to the manufacturer’s instructions
(Machery Nagel). For the subsequent reconstitution of recombinant
viruses, HFFs were seeded into six-well dishes at a density of 3.56105
cells per well and transfected with 1 mg of the respective BAC DNA
together with an expression plasmid for pp71 using the transfection
reagent Fugene HD according to the manufacturer’s protocol (Roche).
One week after transfection, the cells were transferred into 25 cm2
flasks and incubated until an almost complete cytopathic effect
occurred so that the supernatant could be used to infect fresh HFF
cultures for the preparation of virus stocks.
HCMV GFP-based antiviral assay. HFFs cultivated in 12-well
plates were used for a GFP-based antiviral infection assay as described
previously (Marschall et al., 2000). In brief, HFFs were infected with a
recombinant GFP-expressing HCMV, AD169–GFP, at an m.o.i. of
GFP of 0.25 (i.e. 25 % GFP-forming infectious dose at 7 days p.i.).
Infections were performed in duplicate. CDK inhibitors were added
after virus adsorption, and at 7 days p.i. cells were lysed. Lysates from
each well were divided into two samples before processing and
subjected to automated GFP quantification in a Victor 1420
Multilabel Counter (Perkin-Elmer Wallac).
Indirect immunofluorescence assay and confocal microscopy.
HFFs were grown on coverslips in six-well plates (36105 cells per
well) and infected with AD169 at an m.o.i. of 1 or with HCMV
HB5DUL69. For infection with HB5DUL69, HFFs were infected with
16104 p.f.u. and incubated for 7 days until infection of cells was
microscopically observable. CDK inhibitors were added to the culture
medium at 24 h p.i. for HCMV AD169 or at 7 days p.i. for
HB5DUL69, followed by a change of the culture media including
inhibitors every 24 h. Cells were fixed after 2 days of CDK inhibitor
treatment using 4 % paraformaldehyde (10 min, room temperature)
and permeabilized using 0.2 % Triton X-100 in PBS (20 min, 4 uC).
Cells were incubated with the following primary antibodies for
60 min at 37 uC: mAb-UL69 (clone 69-66, kindly provided by W.
Britt, University of Birmingham, AL, USA), mAb-UL44 (clone BS
510; kindly provided by B. Plachter, University of Mainz, Germany),
mAb-CDK9 (clone D-7; Santa Cruz Biotechnology), mAb-RNAPII
(clone CTD4H8; Covance), mAb-RNAPII-0 (clone 8WG16;
Covance), mAb-RNAPII-5P (clone H14; Covance) and mAbRNAPII-2P (clone H5; Covance); and polyclonal antibodies pAbUL69 (Winkler et al., 1994), pAb-IE2 (anti-pHM178; Hofmann et al.,
2000), pAb-UL97 (kindly provided by D. Michels, University of Ulm,
Germany), goat pAb-cycT1 (Santa Cruz Biotechnology) and pAbcycB1 (Santa Cruz Biotechnology). Secondary antibodies used for
triple staining in green (Alexa Fluor 488-conjugated antibodies;
Molecular Probes), red (Alexa Fluor 555-conjugated) and far-red
fluorescence (Alexa Fluor 647-conjugated) were incubated for 30 min
at 37 uC (nuclear counterstaining with DAPI Vectashield mounting
medium; Vector Laboratories). Immunofluorescence data were
collected by confocal laser-scanning microscopy with a TCS SP5
microscope (Leica).
Co-immunoprecipitation assay. 293T cells were transfected with
expression plasmids in 10 cm dishes via a polyethylenimine transfec-
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Journal of General Virology 92
Inter-regulation between HCMV pUL69 and cyclin T1/CDK9
tion technique as described previously (Schregel et al., 2007) and lysed
2 days post-transfection. For interaction studies with proteins from
infected cells, HCMV AD169-infected HFFs (m.o.i. of 1) were treated
with CDK inhibitors (as described for indirect immunofluorescence
analysis) and lysed at 72 h p.i. Both transfected 293T cells and HCMVinfected HFFs were lysed in 500 ml CoIP buffer [50 mM Tris/HCl
(pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5 % NP-40, 1 mM PMSF,
2 mg aprotinin ml21, 2 mg leupeptin ml21 and 2 mg pepstatin ml21] and
used for CoIP with 1–5 ml antibody as indicated for 2 h at 4 uC under
rotation in the presence of protein A–Sepharose beads (2.5 mg;
Amersham Pharmacia Biotech). The precipitates were then pelleted in a
microfuge (10 000 r.p.m., 1 min) and washed five times (800 ml each)
with CoIP buffer before the samples were subjected to a standard
Western blot analysis using tag- or protein-specific antibodies for the
detection of co-immunoprecipitates and protein expression (ECL
staining; New England Biolabs) as follows: mAb-FLAG (clone M2;
Sigma), mAb-Myc (clone 1-9E10.2; ATCC), mAb-HA (clone 12CA5;
Roche), mAb-UL69 (clone 69-66) and mAb-CDK9, pAb-UL69
(Winkler & Stamminger, 1996), rabbit pAb-cycT1 (Santa Cruz
Biotechnology) and goat pAb-cycT1 (Santa Cruz Biotechnology).
Yeast two-hybrid analysis. Protein interactions were analysed using
GAL4 fusion proteins in a yeast two-hybrid system (Fields & Song,
1989). Saccharomyces cerevisiae strain Y153 expressing viral or cellular
proteins (fused with GAL4-AD or GAL-BD; vector pGAD424 or
pGBT9, respectively; Clontech) was used for interaction analysis
(Durfee et al., 1993). Selection for the presence of bait and interactor
plasmids was achieved by cultivation on medium that restricted
growth to combined tryptophan/leucine prototrophy. Transformants
were analysed for b-galactosidase activity by filter lift assays.
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ACKNOWLEDGEMENTS
The authors wish to thank Gillian Scott and William Rawlinson
(Virology Division, University of NSW, Sydney, Australia) for providing
clinical HCMV isolates; Hartmut Hengel and Albert Zimmermann
(Institute of Virology, University of Düsseldorf, Germany) for providing
HCMV TB40/EDUL27; Peter Hemmerich (Institute of Molecular
Biotechnology, Jena, Germany) for support in microscopy techniques;
Marco Thomas, Sabrina Auerochs and Jens Milbradt (Institute of
Clinical and Molecular Virology, University of Erlangen-Nuremberg,
Germany) for very helpful methodical advice and scientific cooperation;
and Axel Choidas (Lead Discovery Center GmbH, Dortmund,
Germany) for co-work in CDK inhibitor characterization. This work
was supported by Deutsche Forschungsgemeinschaft SFB 796 (C3
and B3), DFG MA 1289/6-1 and Bayerische Forschungsstiftung (grant
4SC-2010).
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