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Journal of Virology, January 2000, p. 652-660, Vol. 74, No. 2
0022-538X/00/$04.00+0
Cell Cycle-Regulated Transcription by the Human
Immunodeficiency Virus Type 1 Tat Transactivator
Fatah
Kashanchi,1
Emmanuel T.
Agbottah,1
Cynthia A.
Pise-Masison,1
Renaud
Mahieux,1
Janet
Duvall,1
Ajit
Kumar,2 and
John N.
Brady1,*
Virus Tumor Biology Section, Laboratory of
Receptor Biology and Gene Expression, National Cancer Institute,
National Institutes of Health, Bethesda,
Maryland,1 and Department of
Biochemistry, George Washington University, Washington,
D.C.2
Received 10 February 1999/Accepted 8 October 1999
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ABSTRACT |
Cyclin-dependent kinases are required for the Tat-dependent
transition from abortive to productive elongation. Further, the human
immunodeficiency virus type 1 (HIV-1) Vpr protein prevents proliferation of infected cells by arresting them in the G2
phase of the cell cycle. These findings suggest that the life cycle of
the virus may be integrally related to the cell cycle. We now demonstrate by in vitro transcription analysis that Tat-dependent transcription takes place in a cell cycle-dependent manner. Remarkably, Tat activates gene expression in two distinct stages of the cell cycle.
Tat-dependent long terminal repeat activation is observed in
G1. This activation is TAR dependent and requires a
functional Sp1 binding site. A second phase of transactivation by Tat
is observed in G2 and is TAR independent. This later phase
of transcription is enhanced by a natural cell cycle blocker of HIV-1,
vpr, which arrests infected cells at the G2/M
boundary. These studies link the HIV-1 Tat protein to cell
cycle-specific biological functions.
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INTRODUCTION |
Progression through the cell cycle
requires an ordered array of biochemical interactions, illustrated by
the cyclin-Cdk complexes and their Cdk-inhibitory regulators (45,
56, 65). There is increasing evidence that regulation of basal
(26, 81, 82, 86) and upstream activator transcription
factors (1, 11, 33, 79) also plays an important role in the
coordinated cascade of the cell cycle. For example, the activity of the
cellular transcription factor E2F is tightly regulated by the Rb
protein. In early G1, the interaction of Rb with E2F
inactivates the transcription factor. In late G1, the
cyclin D-Cdk complex phosphorylates Rb, triggering its release from the
complex. Activated E2F then increases the transcription of genes which
are critical for entry into the S phase, including the family of E2F
genes (17, 53, 55, 85).
The human immunodeficiency virus type 1 (HIV-1) Tat protein is required
for viral replication and is a potent stimulator of viral transcription
(41). Following activation of quiescent viral promoters by
mitogenic stimulation, presumably resulting in an increase in
transcription factors such as NF-
B, Tat stimulates viral
transcription through a unique TAR RNA enhancer (5-8, 14, 16, 18,
19, 22, 27, 29, 36, 41-44, 47, 50, 51, 58, 74, 75, 83, 90). In
addition, Tat has been reported to activate a number of cellular
promoters (9, 10, 62, 64, 77, 80). In carrying out its
transcriptional activation, Tat interacts with a number of cellular
transcription factors, including TFIID (TBP) (39, 40, 78),
TFIID-associated TAFs (13), TFIIH (57),
Tat-associated protein (TAP) (87), Tat-associated kinase
(TAK/pTEFb) (20, 21, 30-32, 48, 89, 92), TTK
(54), Sp1 (25), and SF-1 (91), as well
as TAR RNA (84). It has also been reported that Tat
regulates the binding of RNA polymerase II to the TAR RNA
(84).
Interaction of Tat with TFIID and TFIIH, both of which may be involved
in cell cycle regulation (2, 69-72, 76, 86), prompted us to
ask whether Tat transactivation is regulated during the cell cycle. The
hypothesis that Tat's physical and functional interaction with a
number of basal and upstream factors which may be cell cycle regulated
is of further interest in view of the fact that HIV-1 encodes a
conserved gene, vpr, that blocks infected cells at the
G2/M boundary by inhibiting p34cdc2 activation. This block
may be important for efficient HIV-1 replication, since vpr
mutants slow viral replication (4, 37, 59, 60). In this
report, we present evidence that Tat activates transcription at two
distinct phases of the cell cycle, G1 and G2.
Moreover, G1 Tat transactivation is TAR dependent, whereas
G2 Tat transactivation is TAR independent. We hypothesize
that G1 transcription is important for viral mRNA and
genomic RNA synthesis, whereas G2 transcription may
activate some of the cellular genes (coding for cytokines) important
for activation of neighboring cells subsequent to virus infection.
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MATERIALS AND METHODS |
In vitro transcription.
HeLa whole-cell extract (25 to 50 µg total) (49) was added to reaction mixtures containing
either long terminal repeat (LTR)-TAR+ template,
LTR-TAT
template, or a construct with Sp1 deleted (41, 66)
linearized with EcoRI (100 ng). In vitro transcription
reactions were incubated for 1 h at 30°C and contained the
nucleoside triphosphates ATP, GTP, and CTP at a final concentration of
50 µM and [32P]UTP (20 µCi; 400 Ci/mmol; Amersham) in
buffer D (10 mM HEPES [pH 7.9], 50 mM KCl, 0.5 mM EDTA, 1.5 mM
dithiothreitol, 6.25 mM MgCl2, and 8.5% glycerol).
Transcription reactions were terminated by the addition of 20 mM
Tris-HCl (pH 7.8), 150 mM NaCl, and 0.2% sodium dodecyl sulfate (SDS).
The quenched reactions were extracted with equal volumes of
phenol-chloroform and precipitated with 2.5 volumes of ethanol and 0.1 volume of 3.0 M sodium acetate. Following centrifugation, the RNA
pellets were resuspended in 12 µl of formamide denaturation mix
containing xylene cyanol and bromophenol blue, heated at 90°C for 3 min, and electrophoresed at 400 V in a 4% polyacrylamide (19:1
acrylamide-bisacrylamide) gel containing 7 M urea (prerun at 200 V for
30 min) in 1× Tris-borate-EDTA. The gels were analyzed with the
Molecular Dynamics PhosphorImager screen. Radioactivity was quantitated
with the ImageQuant program.
Generation of epitope-tagged Tat-expressing cell line.
HeLa
CD4+ cells (a generous gift of Bruce Chesebro, National
Institutes of Health, Rocky Mountain Laboratories, Hamilton, Mont.) were used for transfection with either an epitope-tagged (the influenza
epitope at the C terminus of Tat 1-86) plasmid or the parental vector
pCEP4. Following transfection, cells were selected under 200 µg of
hygromycin/µl. Hygromycin-resistant lines established from
single-cell clones were maintained for up to 6 months with continuous
passage and used to make extracts for in vitro transcription analysis.
Transfections and CAT assays.
Wild-type and TAR mutant
constructs were electroporated into pCEP4 and etat cells as described
previously (38). Extracts were prepared 18 h later for
chloramphenicol acutyltransferase (CAT) assay. The cells were
harvested, washed once with phosphate-buffered saline (PBS) without
Ca2+ and Mg2+, pelleted, and resuspended in 150 µl of 0.25 M Tris (pH 7.8). The cells were freeze-thawed three times
with vortexing and then incubated for 5 min at 68°C followed by
centrifugation. The supernatants were transferred to 1.5-ml Eppendorf
tubes. After one final spin, the supernatant was again transferred to
1.5-ml Eppendorf tubes, and the protein concentration was determined.
CAT assays were performed with 2 µg of protein according to the
method of Gorman et al. (25).
For vpr experiments, both etat and control cells (3 × 107/time point) were transfected with
HIV-1-LTR-vpr (15 µg) (Mike Emerman, Fred Hutchinson
Cancer Research Center) and pSV2-neo (5 µg). Zero-hour samples were
processed immediately after electroporation for cell sorting and in
vitro transcription analysis. Twenty-four-hour samples were
electroporated with vpr and neo plasmids,
followed by selection with hygromycin (200 µg/ml) and G418 (100 µg/ml). Two sets of templates (100 ng each),
EcoRI-linearized HIV-1 wild type and TM26 (66)
and HIV-1 wild type and Sp1
(41) were used for
in vitro transcription assays (50 µg of total cellular protein).
Immunoprecipitation assays.
Immunoprecipitations were
performed as described previously (39). Cellular protein
(100 µg) was mixed with monoclonal antibody (2.5 µg) for 2 h
at 4°C. Protein A + G agarose beads (5 µl; Calbiochem, Inc.)
were added and incubated at 4°C for another 2 h. The
immunoprecipitated complex was then spun down and washed with buffer D
containing 500 mM KCl (three times; 1 ml each). Where indicated,
immunoprecipitated proteins were eluted with the influenza virus tag
peptide (88). The supernatant was retained for in vitro
transcription analysis.
Cell cycle analysis.
The etat or control cells were either
blocked with hydroxyurea for 18 h or blocked with hydroxyurea (2 mM final concentration), washed, and released for 1 h, followed by
addition of nocodazole (50 ng/ml) for 14 h. Following the block,
the cells were washed with PBS (2×) and released with complete medium.
Samples were collected every 3 h, and the cells were used to make
whole-cell extracts (5 × 107 cells/time point) for in
vitro transcription or Western blot analysis or processed for
fluorescence-activated cell sorter (FACS) sorting. Single-color flow
cytometric analysis of DNA content was performed on both etat and
control cell lines. The cells were washed with PBS, and approximately
2 × 106 were fixed by the addition of 500 µl of
70% ethanol. The cell pellets were washed with PBS (three times; 10 ml
each time) and incubated in 1 ml of PBS containing 150 µg of RNase A
(Sigma)/ml and 20 µg of propidium iodide (Sigma)/ml at 37°C for 30 min. The stained cells were analyzed for red fluorescence (FL2) on a
FACScan (Becton Dickinson), and the distribution of cells in the
G1, S, and G2/M phases of the cell cycle was
calculated from the resulting DNA histogram with Cell FIT software,
based on a rectangular S-phase model (FAST Systems, Inc., Gaithersburg,
Md.).
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RESULTS |
Construction of epitope-tagged Tat cell line.
HeLa cells
(12) were transfected with either the backbone control
plasmid (PCEP4) or a hemagglutinin epitope-tagged Tat plasmid
(etat/PCEP4) at the C terminus. HeLa cells were chosen for these
studies because Tat transactivation can be reproduced in vitro and in
vivo with these cells. T-lymphocyte cell lines, which were also
considered, could not be developed, presumably due to the apoptotic
effect of Tat in T cells (52, 63, 67). Two HeLa cell lines,
containing either the control or etat plasmid, were successfully
selected by single-cell dilution. Both cell types were selected and
maintained under 200 µg of hygromycin/ml. The control PCEP4 HeLa line
is designated the "control" cell line, and the etat/PCEP4 HeLa line
is designated "etat" throughout the text.
To determine if the endogenous Tat was active, etat and control cells
were transfected with the wild-type HIV-1 LTR CAT construct
(pLTR-CAT)
or a TAR mutant, pLTR TM26-CAT (
66). As a further
control
for transfection efficiency, the cells were also transfected
with
pSV2-CAT. At 18 h posttransfection, cell extracts were prepared
and assayed for CAT activity with 2 µg of protein. The results
of
this study demonstrate that the endogenous Tat protein is functional
in
the etat cell line. The pLTR-CAT, but not the pLTR TM26-CAT,
promoter
is activated in the etat cell line (Fig.
1A, lanes 2,
3, 5, and 6). A 25- to
50-fold increase in HIV-1 LTR promoter
activity is routinely observed
in the etat cell line. Importantly,
transcription from the simian virus
40 promoter as assayed by
pSV2-CAT activity was similar in both cell
lines (Fig.
1A, lanes
1 and 4). These studies demonstrated that the
endogenous Tat protein
was able to transactivate the wild-type HIV-1
LTR in the etat
cells. It is important to point out that five
independent clones
each of the PCEP4 and etat cell lines were tested.
All PCEP4 and
etat clones had similar growth properties, Tat
expression, and
Tat activities. One clone of PCEP4 and etat were
selected for
the subsequent experiments.
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FIG. 1.
Functional assay of HIV-1 etat and control cell lines.
(A) Transient assays. Five micrograms of wild-type HIV-1 LTR
(LTR-TAR+) (LTR-CAT), a TAR mutant (LTR-TAR)
(LTR TM26-CAT), or control pSV2-CAT was electroporated into each cell
type (38), and CAT assays were performed with extracts
prepared 24 h posttransfection. (B) In vitro transcription.
Whole-cell extracts from the control or etat cell line were used for in
vitro transcription assay of either HIV-1 LTR-TAT+ (WTLTR)
or HIV-1 LTR-TAT (TM26) templates linearized with
EcoRI (40). Twenty-five micrograms of extract was
used. Arrow, RNA transcript. (C) (Top) Immunodepletion of etat cell
extracts with 12CA5 or Tab172 antibody. Pellets from the
immunoprecipitates (lanes 1 and 2) or depleted extracts (lanes 3 and 4)
were analyzed by Western blotting with polyclonal immunoglobulin G
purified  -Tat antibody. (Bottom) Supernatants from extracts (25 µg) immunodepleted with either 12CA5 (lanes 1 and 3) or Tab172 (lanes
2 and 4) antibody (2.5 µg) were used for in vitro transcription of
the HIV-1 LTR-TAR+ template. (D) Supernatants from extracts
(25 µg) immunodepleted with either 12CA5 or Tab172 antibody (2.5 µg) were used for in vitro transcription of the AdML template.
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As further controls for the experiment, we selected transfected cells
by using an affinity antibody to interleukin-2R. The
number of cells
expressing interleukin-2R were similar in the
etat and pCEP cell lines,
suggesting a similar transfection efficiency.
Moreover, the CAT assay
results presented in Fig.
1A were reproduced.
The wild-type HIV LTR,
but not the TM26 mutant, was specifically
transactivated in the eTat
cell
line.
In vitro transcription from epitope-tagged cell line extracts.
We next prepared whole-cell extracts from control and etat cells for in
vitro transcription assays. pLTR-CAT and pLTR TM26-CAT reporter
plasmids were digested with the restriction enzyme EcoRI to
generate a template which would yield an RNA runoff transcript of 330 bases (40). Equivalent amounts of the templates were added
to in vitro transcription reaction mixtures. Extracts were carefully
titrated to optimize for LTR transcription. Similar to the results
obtained in vivo, the wild-type HIV-1 LTR, but not the
TAR template, was transactivated in the in vitro
transcription assay with etat extracts (Fig. 1B, lanes 1 and 2). The
level of transcription from the wild-type LTR template was
approximately 10-fold above that observed with the TM26 TAR mutant
template. In contrast, the levels of transcription from the two
templates were equivalent in the control cell extracts (Fig. 1B, lanes
3 and 4). Moreover, the levels of transcription observed in the
"nontransactivated" samples were equivalent, demonstrating that the
basal levels of transcription were similar in the two extracts (Fig.
1B, compare lanes 1, 3, and 4). Similar results were obtained with HIV
wild-type, TAR mutant, and AdML G-free cassette templates (data not
shown). Control transcription reactions which included a Pol II
(-amanitin) or Pol III (tagetoxin) inhibitor demonstrate that the
transcription is Pol II dependent.
To demonstrate that the increase in HIV-1 LTR transcription was in fact
due to Tat, etat extracts were cleared by using the
monoclonal
antibodies 12CA5 (anti-epitope-tagged Tat) and Tab172
(anti-Tax
control). Western blot analysis of the immunoprecipitates
from the
clearing experiments demonstrated that Tat was specifically
immunoprecipitated with the anti-epitope antibody (12CA5) but
not the
control antibody (Fig.
1C, top, lanes 1 and 2). Further,
Western blot
analysis of the extracts from the clearing experiments
demonstrated
that Tat was, in fact, depleted from the extract
(Fig.
1C, lanes 3 and
4). When the extracts were used for transcription
assays with the HIV
template, the 12CA5 antibody cleared transactivation
activity whereas
the control monoclonal antibody failed to inhibit
transcription (Fig.
1C, bottom, lanes 1 and 2). These results
suggest that transactivation
is a direct effect of Tat, since
depletion of Tat from the extract
abolishes transcription. It
was important to demonstrate that the
decrease in transcription
was not due to clearance of important
transcription factors from
the extract. A control experiment, utilizing
the AdML template,
is presented in Fig.
1D. The results of this
experiment demonstrate
that while immunoprecipitation of Tat from the
extract decreases
HIV transcription, the level of AdML transcription is
unchanged.
These experiments demonstrated that the Tat protein
expressed
in the etat cells was active and that extracts prepared from
dividing
cell populations transactivate the HIV-1 template. Similar to
in vivo studies, in vitro transactivation is dependent upon the
HIV-1
TAR RNA enhancer. These studies demonstrate that our transcription
extracts correctly reflect in vivo Tat
transactivation.
TAR-dependent Tat transactivation occurs during the G1
phase of the cell cycle.
To examine the profile of HIV
transcription and Tat transactivation during the cell cycle, we
performed experiments with the reversible G1/S cell cycle
blocker hydroxyurea to synchronize the cells (34, 61).
Following an 18-h block, the cells were washed and placed in fresh
medium, and samples were collected every 3 h. An aliquot of the
cells was analyzed by flow cytometry (Fig.
2A). The results of this analysis
demonstrate that hydroxyurea effectively blocked etat and control cells
at the G1/S border (Fig. 2A, zero-hour profile). Upon
release, there was a synchronous progression of the cells through S
(Fig. 2A, 3-h profile), G2/M (Fig. 2A, 6- and 9-h
profiles), and into G1 (Fig. 2A, 12- to 24-h profiles). A
graphic presentation of the cell cycle block and progression is
presented in Fig. 2B. The percentages of cells in G1, S,
and G2/M are plotted at each time point.
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FIG. 2.
Cell cycle analysis and in vitro transcription of etat
and control cell lines following hydroxyurea or hydroxyurea-nocodazole
block. Log-phase growing cells were blocked with hydroxyurea for
18 h (2 mM) and released by removing the inhibitor and adding
fresh medium. Samples were collected every 3 h, and the cells were
used to make whole-cell extracts for in vitro transcription or
processed for FACS analysis. (A) Cell cycle analysis of HIV-1
transcription. The cells were removed from the medium at each time
point, washed with PBS without Mg2+ or Ca2+,
fixed with 70% ethanol, and stained with propidium iodide followed by
cell-sorting analysis on a Coulter EPICS cell analyzer. The FACS
profile for each time point is presented. Un, untreated. (B) (Top)
Graphic presentation of the cell cycle block and progression. The
percentage of cells in G1, S, or G2/M is
plotted as percent total at each time point. (Middle) Whole-cell
extracts were made from 5 × 107 cells/time point, and
25 µg of total protein was used for in vitro transcription from both
the LTR-TAT+ and LTR-TAT (100 ng) templates.
(Bottom) Cell extract (100 µg) from each time point was
immunoprecipitated and separated on an SDS-polyacrylamide gel
electrophoresis gel, and Western blot analysis was performed with
anti-Tat antibody. (C) Cell cycle analysis of AdML transcription. The
AdML template (250 µg) was incubated with 25 µg of the same cell
cycle extracts described above. Following a 1-h incubation, labeled
transcripts were purified and analyzed as described above. (D) Cell
cycle analysis of HIV-1 transcription following hydroxyurea-nocodazole
treatment. Cells were blocked with hydroxyurea for 18 h, washed,
and released for 1 h followed by addition of nocodazole (50 ng/ml)
for 14 h. Following the block, the cells were washed and released.
Samples were collected at each time point and processed for in vitro
transcription analysis.
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Whole-cell extracts were made from 5 × 10
7 cells/time
point and assayed for activity by in vitro transcription with both
wild-type
and TAR mutant templates. Transcription from control cell
extracts
showed a low level of transcription from both the wild-type
and
the mutant templates throughout the S and G
2/M phases
of the cell
cycle. At approximately 18 h after release,
corresponding roughly
to early to mid-G
1 phase of the cell
cycle, a transient but significant
10-fold increase in TAR-independent
basal HIV-1 transcription
was observed (Fig.
2B,
right).
When in vitro transcription was performed with the etat extracts, a
distinct profile was observed. We consistently observed
that cells at
late G
1/early S (21 h) showed 10-fold-higher levels
of
transcriptional activity on the wild-type LTR template
(LTR-TAR
+) than on the TAR mutant template
(LTR-TAR
) (Fig.
2B, left). Of interest, the timing of the
Tat-dependent
transcription was routinely observed at a slightly later
phase
of G
1 (18 versus 21 h). The fact that the timing
of basal (in
control cells) and Tat-transactivated (in etat cells)
transcription
are distinct may suggest that Tat switches the HIV-1
transcription
phase. This point is under investigation. Unexpectedly,
we also
observed a distinct peak of Tat-dependent activation of both
the
LTR-TAT
+ and LTR-TAT
templates in the 6-h
G
2 extracts. The Tat-dependent G
2 transcription
was increased 10-fold over
background.
Western blot analysis of Tat protein demonstrated that the protein was
relatively constant throughout the cell cycle. Quantitative
analysis of
the Western blot determined that there was less than
a twofold
variation in Tat protein. These results suggest that
the
transcriptional levels in 6- and 21-h extracts were not simply
due to
changes in Tat protein levels (Fig.
2B, Tat immunoprecipitation/Western
blot [IP/WB]). Moreover, the peaks in HIV-1 transcription are
not due
to variation in the overall transcription activity. Utilizing
the same
extracts, transcription assays with the AdML promoter
were performed.
The results of these transcription assays demonstrated
that there was a
fairly consistent level of transcription throughout
the cell cycle
(Fig.
2C). Quantitative analysis of the transcription
assays
demonstrated that there was less than a twofold difference
in AdML
transcription across the cell
cycle.
In a separate series of experiments, we have tested the transcriptional
activity of extracts from unselected cells synchronized
at different
stages of the cell cycle, supplemented with exogenous
Tat. As reported
by ourselves and numerous other groups, the addition
of Tat to
unsynchronized G
1 cell extracts results in an increase
in
transcription (
8,
40,
42,
44,
48,
50,
51,
92).
This
transactivation, which is TAR dependent, is reproduced in
the etat
extracts from synchronized G
1 cells. In contrast, the
addition of exogenous Tat to extracts from cells synchronized
in
G
2 does not result in activation of transcription. The
potential
for in vivo protein-protein interactions and
posttranslational
modifications that are necessary for Tat
transactivation are evidently
not totally reproduced when Tat is simply
added to the G
2 extracts.
The 21-h extract showed specific Tat activation on the
LTR-TAR
+ promoter, suggesting that Tat transactivation
occurred during
the G
1 phase of the cell cycle. To confirm
this observation, hydroxyurea-synchronized
cells were released and
treated with the mitotic blocker nocodazole,
followed by release at
various time points. Extracts were made
from the control and etat cell
lines at 0, 2, 4, 6, and 8 h post-nocodazole
release for in vitro
transcription. The results of such an experiment
are shown in Fig.
2D.
Low levels of HIV-1 transcription were observed
during the early
G
1 phase (Fig.
2D, 0-, and 2-h samples). Tat
transactivation of the LTR-TAR
+, but not the
LTR-TAR
, increased transcription 9- to 10-fold at 4 h after nocodozole
release. The level of Tat transactivation decreased
to fivefold
in the later stages of G
1/early S (Fig.
2D, 6 and 8
h).
TAR-independent Tat transactivation occurs at the G2
phase of the cell cycle.
FACS analysis measures the DNA content of
cells as determined by propidium iodide incorporation and scores cells
of G2 or M as a mixed G2/M population. We were
interested in determining if G2- or M-phase cells were
responsible for the TAR-independent transcription effect seen in the
etat line (Fig. 2A, 6 h). To distinguish these cell populations,
the cells were blocked with hydroxyurea and released for 6 h to
obtain primarily G2 extracts (73, 81). To obtain
M-phase extracts, hydroxyurea-blocked cells were released and
subsequently blocked with nocodazole for 18 h. G2
extracts from etat cells showed high transcriptional activity compared
to M-phase or control cell extracts (Fig.
3A, lanes 1 to 4). Consistent with the
results presented in Fig. 2A, Tat transactivation (10- to 20-fold) at
the G2 phase was TAR independent (Fig. 3A, lane 1, upper
and lower gels). Antibody clearing experiments demonstrated that the
G2/M transcription activity was Tat dependent in the etat
cell line (Figure 3B). The epitope-directed 12CA5, but not the control
Tab172, antibody decreased transcriptional activity approximately
11-fold (Fig. 3B, lanes 1 and 2). We further show that the
G2-phase transcription is Pol II transcription, since it is
sensitive to low levels of -amanitin (Fig. 3B, lane 3). The low
transcriptional activity of the M-phase extracts is in agreement with
published reports showing the downregulation of Pol II transcription
during the M phase (68).
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FIG. 3.
Analysis of HIV-1 transcription at G2 and M
phases of cell cycle. (A) etat or control cells were blocked with
hydroxyurea and released for 6 h to obtain G2-phase
cells or double-blocked with hydroxyurea and nocodazole to obtain
M-phase cells. The cells were then processed for in vitro transcription
analysis. (B) In vitro transcription following immunodepletion of Tat
protein from etat G2 extracts. G2-phase extract
was treated with either 12CA5 (anti-epitope) or Tab172 (anti-Tax
control) antibody followed by in vitro transcription from the
supernatant. -amanitin was used at 1.0 µg/ml to inhibit Pol
II-specific transcription.
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G1/S Tat transactivation, but not G2
transactivation, is Sp1 dependent.
We next compared in vitro
transcription of HIV-1 wild-type and Sp1 deletion mutant
templates (73). Consistent with the results presented above,
the level of HIV-1 transcription was significantly higher in the
extracts containing Tat (Fig. 4, compare
lanes 2 and 6 and lanes 4 and 8). Interestingly, the requirement for
Sp1 was dependent upon the stage of the cell cycle (Fig. 4, lanes 1 and
3). G1 activation (21-h extracts) required Sp1 binding
sites (Fig. 4, lanes 1 and 2), whereas G2 extracts (6-h)
support activated transcription in the absence of Sp1 sites (Fig. 4,
lanes 3 and 4). Western blot analysis of the pCEP and etat extracts
demonstrated that the levels of Sp1 were similar for an asynchronous
population of cells which are primarily in G1 (21-h) (Fig.
4B, lanes 1 and 5). In the etat cells, a slight decrease in the level
of Sp1 was observed as the cells progressed from G1/S (0 h)
through S (3 h) and into G2 (6 h) (Fig. 4B). The change in
Sp1 protein levels (twofold) was, however, less dramatic than the
change in Sp1-dependent transcription activity, suggesting a
fundamental change in the transcription program by Tat. It will be of
interest to determine which upstream factors regulate HIV-1
transcription during the G2 phase of the cell cycle. Of
interest, the Sp1-del-LTR plasmid has all three Sp1 sites deleted,
moving the NF-B sites adjacent to the TATA box. The basal activity
of the promoter in the PCEP4 cells correlates with in vivo transfection
assays (41).
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FIG. 4.
In vitro transcription of HIV-1 LTR Sp1 deletion
mutants. (A) The etat or control cells were blocked with hydroxyurea
for 18 h, washed, and released for 6 or 21 h for
G2 and G1/S extracts, respectively. Following
the block, the cells were washed twice with PBS and released with
complete medium. Whole-cell extracts were made from 5 × 107 cells/time point. One hundred nanograms of HIV-1 LTR
(Wt) or HIV-1 LTR Sp1 deletion mutant (Sp1 del) template linearized
with EcoRI (41) was added to the in vitro
transcription reaction mixture containing 25 µg of extract. (B)
Western blot analysis of Sp1 in pCEP and etat cell extracts. Log-phase
growing cells were blocked with hydroxyurea for 18 h (2 mM) and
released by removing the inhibitor and adding fresh medium. Samples
were collected every 3 h, and the cells were used to make
whole-cell extracts for in vitro transcription or Western blot
analysis. One hundred micrograms of cell extract from each time point
was separated on an SDS-polyacrylamide gel electrophoresis gel, and
Western blot analysis was performed with anti-Sp1 anti-TBP antibody. A,
asynchronous cell extract, primarily G1; 0, 3, 6, time (in
hours) after release from hydroxyurea block.
|
|
vpr cell cycle block induces Tat-dependent and
TAR-independent transcription.
It was important to relate these
observations on Tat transactivation to events which occur during the
normal viral infection. The HIV-1 vpr protein prevents Cdc2
activation, thereby delaying or preventing replication of infected
cells at the G2/M boundary (4, 37, 60). It was
of interest, therefore, to examine if vpr-blocked cells
exhibited a high level of Tat-dependent and TAR-independent
transactivation activity as observed in cells progressing through
G2 (Fig. 2). Control and etat cells were cotransfected with
a HIV-1 LTR-driven vpr expression vector (4) and
a plasmid carrying the gene for neomycin resistance. Cells were
selected by addition of 100 µg of G418/ml to the media. Transfected
cells were harvested at zero hour, immediately after transfection, or at 24 h and analyzed by FACS or used for preparation of in vitro transcription extracts. FACS analysis demonstrated that control or etat
cells collected immediately after transfection were primarily in
G1 (Fig. 5A, zero-hour etat
and control FACS profiles). In contrast, etat cells harvested 24 h
after transfection with HIV-1 LTR-driven Vpr expression vector were
primarily in G2 (Fig. 5A, 24-h etat FACS profile). The 24-h
sample from control cells transfected with HIV-1 LTR-driven Vpr
expression vector did not accumulate in G2, since there was
no activator to increase Vpr expression (Fig. 4, 24-h control FACS
profile). Extracts made from 0- and 24-h control or etat transfected
cells were used for in vitro transcription of the wild-type HIV-1
promoter, TAR mutant TM26, or an Sp1 deletion mutant. Extracts from the
zero-hour etat cells (vpr), which were
primarily in G1, exhibited a requirement for both Sp1 and
TAR (Fig. 5B and 5C, compare lanes 1 and 2). Mutations within either
the TAR or Sp1 regulatory domain decreased transcription 10- to
14-fold. In contrast, vpr-blocked cells at G2
support efficient Tat-dependent transcription from the Sp1 and TAR
mutant TM26 templates (Fig. 5B and C, compare lanes 3 and 4). These
results demonstrate that the G2-blocked cells support a
qualitatively different mode of activation than the G1
population. The fact that control cells, which do not express high
levels of vpr because of the absence of the Tat
transactivator to drive LTR-vpr expression, did not lock at
G2/M indicates that vpr is responsible for the
observed results.
View larger version (51K):
[in this window]
[in a new window]
|
FIG. 5.
vpr block at G2 increases
Tat-dependent and TAR-independent transcription. etat and control cells
(3 × 107 cells/time point) were transfected with 15 µg of the HIV-1-LTR-vpr expression vector and 5 µg of
pSV2-neo. Zero-hour samples for cell sorting and in vitro transcription
analysis were processed after electroporation. Twenty-four-hour samples
were selected with hygromycin (200 µg/ml) and G418 (100 µg/ml) for
24 h. Two sets of templates (100 ng each),
EcoRI-linearized HIV-1 wild type (Wt) and TM26
(66) and HIV-1 wild type and Sp1
(41), were used for in vitro transcription assays (25 µg
of total cellular protein). IRF, integral red fluorescence.
|
|
| |
DISCUSSION |
The experiments presented here provide important and novel insight
into the nature of HIV-1 Tat transactivation and its regulation during
the cell cycle. In particular, the data demonstrate that Tat
transactivation through the TAR RNA enhancer element (5, 6,
14-16, 18, 19, 22, 27, 29, 35, 36, 42, 44, 47, 74, 83, 84, 91)
occurs distinctly during mid- to late G1. Tat
transactivation during this phase of the cell cycle likely represents a
window in which critical transcription factors required for Tat
transactivation are present. A second and distinct phase of Tat
transactivation, which is not dependent upon the TAR RNA enhancer or
the Sp1 binding site, is observed during the G2 phase of
the cell cycle. Tat transactivation at this stage may represent the
transactivation of cellular genes which are critical for virus
replication (data not shown) (10, 62, 64, 77, 80). Because
viral activators are absolutely essential for the virus life cycle, it
is plausible to predict that they carry multiple functions to
manipulate the host machinery at different stages of the cell cycle. It
will be interesting to find out if this dual function of Tat is
accomplished by interacting with different transcription factors or
kinases at various stages of the cell cycle.
It is likely that Tat transactivation at the G2 phase of
the cell cycle plays an important role in virus replication. The HIV-1
protein Vpr inhibits cell growth by inducing a cell cycle arrest at the
G2/M boundary (4, 37, 59, 60). This arrest correlates with inactivation of the cellular Cdc2 kinase. All primate
lentiviruses encode Vpr, and recent studies by Planelles et al.
(60) suggest that the cell cycle arrest induced by
vpr is a property shared by HIV-1 strains with diverse
biological phenotypes. Further, deletion studies of HIV-1
vpr result in viruses with slower replication kinetics in
lymphoid cells and peripheral blood mononuclear cells and severely
attenuated virus replication in macrophages (3, 28). Goh et
al. (24) have recently reported that Vpr increases viral
gene expression by arresting cells in G2, which the authors
find is an optimal phase for viral transcription. Our results support
the observation that cells in G2 have a high level of
Tat-dependent LTR transcription. Importantly, we find that this peak of
viral transcription is TAR independent. We think it is also possible
that the vpr block accentuates the Tat-dependent and
TAR-independent transactivation of cellular genes which are critical
for successful virus replication. Along these lines, it has been
demonstrated that HIV-1 infection requires a "mitogenically activated" state. We would postulate that one of the targets of Tat
is cellular cytokine genes whose products facilitate virus spread to
neighboring cells by creating an environment which favors viral
infection and replication.
It is becoming increasingly apparent that transcription factors are
regulated in a cell cycle-dependent manner. For example, phosphorylation of the carboxy-terminal domain (CTD) of Pol II by
cyclin-dependent kinases can result in activation or inactivation of
the polymerase. CTD phosphorylation by TFIIH-associated Cdk7, the
catalytic subunit of cyclin-activating kinase, results in activation of
the elongation phase of Pol II transcription (46). In
contrast, phosphorylation of the CTD by Cdc2 inhibited
promoter-dependent transcription (23). It is also relevant
that TFIIH phosphorylates Cdc2 and Cdk2 (72), suggesting a
link between transcription and the cell cycle machinery.
TFIID-associated TAFs are also apparently regulated during the cell
cycle. It has been reported that a hamster cell line bearing a mutation
in CCG1 (TAF 250) was defective in G1 progression. TAF 250 is a major component of the holo-TFIID complex, acting as a scaffold
for other TAFs and transcriptional activators. Since TFIID and TFIIH
both play critical roles in HIV-1 Tat transactivation, it will be
important to determine how cell cycle regulation of these transcription
factors influences interaction with Tat and Tat transactivation.
Finally, it has recently been reported that the cell cycle regulator
E2F mediates repression of HIV-1 transcription through NF-B
(38). Interestingly, the other component of the E2F complex,
Rb, also inhibits HIV-1 transcription through NF-B (71).
Thus, repression of HIV-1 transcription during the S phase may follow
dissociation of the E2F-Rb complex at the G1/S border.
The mechanism of Tat transactivation is unique among the viral
activators in that Tat requires an RNA enhancer. From a virus replication standpoint, the use of an RNA enhancer likely provides an
advantage for the virus in specifically activating viral transcription. It is possible that Pol II elongation factors are diminished during the
G1 window of HIV-1 transcription. Tat's ability to
stimulate transcription elongation may overcome this inhibition. It is
also possible that specific kinases important for Tat transactivation are active during the G1 phase. Our experiments suggest
that the key to understanding these RNA enhancer questions will lie in analyzing transcription activation during the G1 phase of
the cell cycle. In addition, our experiments demonstrate that there is
a discrete Tat transactivation that occurs during the G2
phase of the cell cycle. This activation window has been largely missed by previous analysis, since the majority of dividing cells used in
either transient assays or preparation of in vitro transcription extracts are derived from populations of cells that are 60 to 80%
G1/S. Our studies, therefore, point to novel and unexpected pathways linking Tat transactivation to the cell cycle. Finally, the
concept that a retroviral activator may work at different stages of the
cell cycle opens the possibility of a similar phenomenon among all DNA
and RNA virus activators.
| |
ACKNOWLEDGMENT |
Fatah Kashanchi and Emmanuel T. Agbottah contributed equally to
this study.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Virus Tumor
Biology Section, LRBGE, Building 41, Room B201, National Cancer
Institute, National Institutes of Health, Bethesda, MD 20892. Phone:
(301) 496-0986. Fax: (301) 496-4951. E-mail:
bradyj{at}exchange.nih.gov.
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Journal of Virology, January 2000, p. 652-660, Vol. 74, No. 2
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Abstract
Introduction
