From the Molecular Virology Section, Laboratory of
Molecular Microbiology, National Institutes of Allergy and Infectious
Diseases, NIH, Bethesda, Maryland 20892-0460 and
§ Laboratoire de Virologie Moléculaire et Transfert de
Gène, Institut de Génétique Humaine, CNRS Unité
Propre de Recherche 1142, 34296 Montpellier, France
Received for publication, September 6, 2002
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ABSTRACT |
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P-TEFb, cyclin T1 + CDK9, is needed for the
expression of cellular promoters and primate lentiviral long terminal
repeats (LTRs). Curiously, cellular and lentiviral promoters differ
dramatically in the requirements for positive transcriptional
elongation factor (P-TEF) b activity. Lentiviral LTRs, but not cellular
promoters, need an RNA-associated P-TEFb/Tat/TAR
(trans-activation-responsive) RNA ternary complex. Ternary complex
defective murine cycT1 is apparently inactive for lentiviral
transcription. Why P-TEFb requires Tat/TAR for LTRs but not for
cellular promoters remains unknown. To explore this question, we sought
to determine whether DNA targeting of murine and human cyclin T1 can
reconstitute a Tat/TAR-independent activity to the HIV-1 LTR. In the
absence of Tat and TAR, we found that both HuCycT1 and MuCycT1 can
robustly activate the HIV-1 LTR. We further showed that Sp1 is
necessary and sufficient for this DNA-targeted activity. Thus, like
cellular promoters, HIV-1 LTR can use P-TEFb function without a Tat/TAR
RNA complex. This activity could explain recent findings of robust
HIV-1 replication in rat cells that cannot form a P-TEFb/Tat/TAR moiety.
Human immunodeficiency virus 1 (HIV-1)1 transcription is an
excellent paradigm for studying RNA polymerase II (RNAP II) elongation (1). HIV-1 encodes a transactivator protein, Tat, that stimulates transcription elongation through interaction with a
trans-activation-responsive element (TAR) located at the 5' end of the
nascent transcripts (2). Tat has been suggested to recruit an RNAP II
C-terminal domain (CTD) kinase to the HIV-1 promoter (3-6).
Hyperphosphorylation of RNAP II CTD by this kinase correlates closely
with productive transcriptional elongation (5, 7, 8). The
Tat-associated CTD-kinase has been shown to comprise cdc2-related
kinase (PITALRE/CDK9) (9, 10) and cyclin (cyclin T1) (11, 12). CDK9 and
cyclin T1 (cycT1) are components of previously identified positive
transcriptional elongation factor (P-TEFb) in Drosophila
melanogaster and mammals (10, 13, 14). Our current understanding
is that P-TEFb forms a ternary complex with Tat and TAR to mediate
transcriptional elongation from the HIV-1 LTR. Hence, mouse cycT1,
which differs from human cycT1 by a cysteine at position 261, cannot
form a P-TEFb/Tat/TAR complex; this defect is presumed to explain the long-observed defect in HIV-1 transcription in rodent cells (12, 15-17).
Recent findings suggest that P-TEFb also plays an essential role in the
expression of mammalian and D. melanogaster genes (10, 13,
14, 18, 19). In these settings, P-TEFb must engage promoter-DNA
independently of TAR RNA and must promote processive transcription
without Tat. Indeed, it is a mystery why the cellular-promoter
operative activity of P-TEFb does not apparently apply to the HIV-1
LTR. Here, we sought to determine whether DNA targeting of murine or
human cycT1 to the HIV-1 LTR can bypass a requirement for Tat/TAR. We
show that both murine and human cycT1 can interact with the Sp1 "A"
domain to reconstitute Tat/TAR independent transcriptional activity and
that Sp1 is necessary and sufficient to recruit cycT1 to the HIV-1 LTR.
These results support the finding of P-TEFb in HIV-1 preinitiation
complexes before the synthesis of TAR RNA (20) and help to explain the normal expression of HIV-1 LTR in rat cells that cannot form an RNA-bound P-TEFb (21, 22). Thus, we propose that lentiviral LTRs, like
cellular promoters, can use P-TEFb despite absence of Tat/TAR. This
proposition concurs with recent suggestions that successful promoter
recruitment of cycT1 is fully sufficient for HIV-1 LTR transcription
(23).
Plasmids--
pCMV-HuCycT1 and pCMV-MuCycT1 (1-708) were kindly
provided by Drs. David Price and Kathy Jones, respectively (11, 24). Full-length MuCycT1 and HuCycT1 were reconstructed by PCR. pCMV-HA (Clontech) and pSG424 (Gal4 binding domain) vectors
were used for cyclin T1 fusion vectors. p( Cell Culture, Transfection, and Reporter Assays--
Cell
propagation, transfection, and reporter assays were as described
previously (28). All transfections were repeated three or more times
and were normalized to Antibodies--
Mouse monoclonal anti-HA (Sigma Chemical); mouse
monoclonal anti-Gal4BD (Santa Cruz Biotechnology, Santa Cruz, CA)
rabbit polyclonal anti-Sp1 and anti-HA antibody (Upstate Biotechnology, Lake Placid, NY) were used as described.
Western Blotting, Immunoprecipitation, and Confocal
Imaging--
Western blotting and immunoprecipitation were performed
as described previously (28). For confocal microscopy, HeLa cells were
cultured on 25-mm coverslips (Thomas Scientific) and transfected with
plasmid DNA. One day later, cells were fixed with 3.7% formaldehyde, permeabilized with PBS containing 0.1% Triton X-100, and incubated with monoclonal anti-HA antibody followed with anti-mouse conjugated to
Alexa Fluor 488 (Molecular Probes, Eugene, OR). Nuclei were stained
with 4,6-diamidino-2-phenylindole (Molecular Probes). Coverslips were
mounted onto glass slides with ProLong antifade kit (Molecular Probes)
and examined with a Leica laser-scanning microscope.
Robust Activation of Primate Lentiviral LTRs by cycT1 in the
Absence of Tat--
The great preponderance of HIV-related P-TEFb
studies have focused on transcriptional elongation in the context of a
ternary P-TEFb/Tat/TAR RNA complex (20, 24, 29). Recent findings, however, demonstrate that P-TEFb activates numerous cellular promoters in Tat/TAR independent settings (18, 30). Moreover, P-TEFb is in HIV-1
preinitiation complexes before the synthesis of TAR RNA (20, 31),
suggesting a role distinct from its Tat/TAR RNA-bound form. Prompted by
these observations, we sought to investigate the impact of P-TEFb on
lentiviral LTR-expression in the absence of Tat/TAR.
Human and subhuman primate lentiviral LTRs conserve biological function
(32) without preserving sequence identity. For example, although three
or more copies of Sp1 binding motifs are found in all primate
lentiviral LTRs, NF- Requirement for Sp1 in cycT1-mediated LTR Activation--
The
HIV-1 LTR contains several transcription factor binding sites,
including AP-1, NFAT, NF-
To better understand the Sp1 requirement, we explored the possibility
that Sp1 and cycT1 directly interact. We transfected HA-tagged HuCycT1
or MuCycT1 into HeLa cells and immunoprecipitated mock- (lanes
1 and 5), HuCycT1- (lanes 1, 2,
6, 7), or MuCycT1- (lanes 4 and
8) transfected cells with either anti-HA (lanes
1-4) or an irrelevant isotype-matched control serum
(lanes 5-8). Immunoprecipitates were then
analyzed by Western blotting for corecovery of HeLa cell-endogenous
Sp1. Consistent with their similar functional activities (Fig.
2A), both human and murine CycT1 bound Sp1.
The Sp1 A Domain Suffices for Functional and Physical Interaction
with cycT1--
Sp1 is a 95- to 105-kDa transcription factor that
binds DNA through its C-terminal zinc fingers (33, 34). Previously, it
was shown that Sp1 contributes importantly to the expression of the
HIV-1 LTR (35) and that its serine/threonine- and glutamine-rich "A" activation domain suffices for functional interaction with Tat
(27, 36). These observations together with above results (Fig. 2) lead
us to ask whether the functional interaction between cycT1 and Sp1 is
entirely circumscribed in the A domain. To address this, we transfected
HeLa cells with a chloramphenicol acetyltransferase reporter
(G3(
To confirm whether Sp1A-alone was sufficient to bind CycT1 (Fig.
2B), we checked coimmunoprecipitations of Gal4Sp1A and
CycT1, cyclin H, or cyclin K (Fig. 3C). HeLa cells were
transfected with Gal4Sp1A and HA-tagged MuCyCT1 (lane 1),
MuCycT1 (1-631; lane 2), Hu cyclin H (lane 3),
or Hu cyclin K (lane 4). Gal4Sp1A was captured with
anti-Gal4BD serum, and coassociated proteins were probed by Western
blotting with anti-HA. We found that wild-type MuCycT1 (closely
migrating as a doublet with a background band; lane 1),
MuCycT1 (1-631; lane 2), and cyclin K (lane 4)
bound Sp1A. Cyclin H (lane 3) did not. These results agree
with cyclin K providing transcriptional roles partially redundant with
cyclin T1 (30, 37) and cyclin H serving functions distinct from cyclin T1 (38).
The C Terminus of Cyclin T1 Interacts with Sp1A to Functionally
Activate Expression--
Human cyclin T1 is a 726-amino acid protein.
Its N-terminal 1-290 amino acids contain a "cyclin homology box."
This "box" binds cdk9 (39-41) and mediates physical interaction
with HIV-1 Tat. Indeed, a single cysteine at position 261 of HuCycT1 is
critical for forming a human P-TEFb/Tat/TAR complex (12, 17). Above, our results indicate a cycT1-Sp1 activity distinct from that of P-TEFb/Tat/TAR. We next examined whether the former uses a region of
cycT1 different from that of the latter.
We generated several HA-tagged HuCycT1 and MuCycT1 deletion mutants
(Fig. 4). Each was challenged in
expression assays with cotransfected Gal4BD or Gal4Sp1A (Fig. 4). All
deletion mutants expressed comparably in HeLa cells (Fig.
4B), but not all supported activated expression. Thus,
HuCycT1-wild type, HuCycT1 (1-633), HuCycT1 (240-726), and HuCycT1
(300-726) produced high (
To verify that the C-terminal region of cycT1 is discretely sufficient
for transcription, we fused a sub-portion, amino acids 419 to 726, to
Gal4BD. Gal4HuCycT1 (419-726) when targeted to promoter upstream
Gal4-binding sites was sufficient for activating promoter expression
(Fig. 5). Surprisingly, in parallel
assays, Gal4HuCycT1 (1-444) and Gal4HuCycT1 (1-426), previously shown by others to be competent for cdk9-binding and transcriptional elongation when bound to Tat/TAR RNA, were inactive when targeted to
DNA via Gal4-binding sites (Fig. 5, left). Because all
Gal4HuCycT1 chimeras were expressed comparably (Fig. 5,
right), one interpretation of these results is that simple
recruitment of cdk9 insufficiently supports transcription from the
HIV-1 promoter.
Sp1A-active HuCycT1s Localize to Nuclear Speckles--
Fig. 4
defined forms of HuCycT1 that are highly active, moderately active, and
inactive for cooperation with Sp1A. Previously, transcriptionally
intact full-length HuCycT1 was found in nuclear dots/speckles (42). To
check how Sp1A-competent forms of HuCycT1 might behave, we visualized
using confocal microscopy to identify the subcellular location of seven
HA-tagged mutants. Intriguingly, both highly active, (HuCycT1 (1-726),
HuCycT1 (1-633), HuCycT1(240-726, not shown), and HuCycT1 (300-726))
and moderately active (HuCycT1 (419-726) and HuCycT1 (500-726))
cyclin T1s stained identically in nuclear speckles (Fig.
6). On the other hand, Sp1A-inactive HuCycT1 (1-444) and HuCycT1 (1-419), shown elsewhere to support P-TEFb/Tat/TAR function (24, 39), were not in speckles and stained
predominantly cytoplasmic. To the extent that nuclear speckling might
be a requisite for transcriptional activity, the divergent patterns
suggest that Sp1A-active portion of cycT1 dictates such physical
localization.
HIV-1 expression occurs in two phases. After integration of the
provirus into cellular chromosome, basal transcription from the LTR
must initially drive the synthesis, however inefficiently, of a small
amount of full-length 9.6-kb mRNA that provides for the synthesis
of Tat. Tat then activates the LTR through TAR RNA (2) to further
increase processive transcription. Over the past few years, P-TEFb
(reviewed in Ref. 43) has emerged as an important cofactor for
Tat/TAR-activated phase of LTR transcription. Thus, we understand the
P-TEFb/Tat/TAR ternary complex modulates transcriptional elongation by
hyperphosphorylation of RNAP II CTD (12, 14). Interestingly, although
P-TEFb is present in elongation complexes, before the synthesis of TAR
RNA, it is also found in HIV-1 LTR-engaged preinitiation complexes
(20). Currently, despite much learned about Tat/TAR interactions with
elongation-associated P-TEFb, little is known regarding how P-TEFb
enters preinitiation complexes and the role that it might play in such moieties.
Here, we present evidence that P-TEFb may be recruited into
preinitiation complexes through physical association of its cycT1 subunit with DNA-bound Sp1. Sp1 is a glutamine-rich promoter-proximal activator that interacts with TFIID to stabilize preinitiation complexes (44). We found that the 179-amino acid Sp1A domain that
contains its glutamine-rich sequence is sufficient for physical (Fig.
3C) and functional (Figs. 3B and 4A)
interaction with cycT1. Surprisingly, the portion of cycT1 delineated
to cooperate with Sp1A mapped to a short, ~419-633-amino acid
C-terminal stretch (Figs. 4A and 6). This Sp1A-active region
resides outside of the cycT1 cyclin homology box (i.e. amino
acids 1-290) and associates with neither cdk9 nor Tat. Thus, unlike
TAR RNA-bound P-TEFb, DNA-bound cycT1-Sp1A activity apparently does not
use cdk9. Although provocative, this unexpected conclusion is
consistent, in part, with findings that Sp1-driven transcription can
occur through a CTD-kinase independent mechanism (45, 46) and with
evidence that productive and processive transcriptional elongation from the HIV-1 LTR can proceed without cdk9 (47). Currently, we do not rule
out that in the absence of cdk9, other kinases such as CDK2 (48) may be involved.
It has been suggested elsewhere that cycT1 might be recruited to the
HIV-1 preinitiation complexes through binding to NF- In addition to binding Sp1, the C terminus of cycT1 has another
important activity. It is well known that transcription factors and
actively transcribing RNAP II physically localize into nuclear foci
with speckled patterns (52, 53). Accordingly, a prerequisite for
P-TEFb-activity is its entry into nuclear speckles (42, 54). A
priori, one might suppose that P-TEFb is directed to such a locale
through the N-terminal Tat/TAR associative domain of its cycT1
component. Surprisingly, our data show that the Sp1A-interactive C
terminus (500-726 amino acids) of cycT1 dictates its
localization into nuclear dots (Fig. 6). The N terminus
(e.g. 1-444 amino acids) of cycT1, which binds cdk9, has no
such function (Fig. 6). Hence, the first step of P-TEFb function, its
constitutive (or Sp1-associated) migration to nuclear speckles, seems
to be determined through its C terminus.
DNA recruitment of cycT1 by Sp1 serves also to address the
long-standing observation that Tat function and HIV-1 transcription are
apparently defective in rodent cells (29, 55, 56). Two recent studies
have unexpectedly shown that HIV-1 is both transcriptionally intact and
replication competent in rat cells (21, 22), despite the fact that rat
cycT1 lacks the human specific cysteine 261 residue (29, 55-57)
necessary for forming a P-TEFb/Tat/TAR complex. Accordingly, in rat
cells, the prototypic elongation-associated P-TEFb/Tat/TAR complex
cannot form. How then can HIV-1 transcription occur successfully in rat
cells? Although we have not examined rat cycT1 directly, we find in
human cells that MuCycT1 and HuCycT1 are equally active in their
Sp1-dependent activity. Thus, DNA-targeted cycT1 does not
need cysteine 261 for LTR expression. Intriguingly in rat cells, we
have observed that Tat-independent activity of MuCycT1 is actually
considerably higher than that of
HuCycT1.2 Conceivably, a
rat-specific cell factor may enhance DNA-based Sp1-cycT1 interaction to
sufficiently bypass the need for RNA-tethered P-TEFb/Tat/TAR. Indeed
active P-TEFb at cellular promoters functions effectively without Tat
or TAR (43). We have recently observed that, consistent with the idea
that Tat/TAR may not be absolutely necessary for lentiviral LTR and
consistent with the proposal of Bieniasz and Cullen (58),
overexpression of either HuCycT1 or MuCycT1 fully activates the
transcription and synthesis of viral proteins from a Tat(
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)43 chloramphenicol
acetyltransferase (25), HIV-1 LTR luciferase plasmids (26),
Gal4Sp1A (27), and G5-83HIV-Luc (21) have been described previously.
SIVsykes and SIVmac LTR-luciferase plasmids were cloned into pGL3-basic (Promega, Madison, WI). HIV-1 linker scanning mutants were from AIDS
reference reagent program donated by Dr. Steven Zeichner (26).
-galactosidase activity expressed from a
cotransfected pCMV-
(Clontech). Average
values ± S.D. are shown. In all experiments, we have assayed both
the HuCycT1 and the equivalent MuCycT1 plasmids. Because of space limitations, only one or the other set of results is shown.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-binding sites are sporadically reduced in
number or absent altogether in simian immunodeficiency (SIV) viral LTRs
(Fig. 1A). Using three
different primate LTRs, HIV-1 LTR (HXB2), and SIV LTRs (SIVmac and
SIVsykes), we asked how each responds to trans-expression of either
human cyclinT1 (HuCycT1) or murine cyclinT1 (MuCycT1). When the
appropriate LTR-luciferase reporters were cotransfected into HeLa cells
with either HuCycT1-alone (Fig. 1B, lanes 2,
5, 8) or MuCycT1-alone (Fig. 1B,
lanes 3, 6, and 9), we observed strong
activation of all LTRs. Previously, the salient difference between
HuCycT1 and MuCycT1 was that the latter cannot form a functional
P-TEFb/Tat/TAR ternary complex (20, 24, 29). Interestingly, the current
Tat-independent assays revealed equivalent activities for both cyclin
T1s. Dose-dependent titrations further confirmed that
MuCycT1 activates expression effectively from NF-
B-containing and
NF-
B-absent LTRs (Fig. 1C).
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Fig. 1.
HuCycT1 and MuCycT1 activate lentiviral LTRs
in the absence of Tat. A, schematic representations of human
(HIV-1 HXB2) and simian (SIVmac and SIVsykes) LTR-luciferase reporters.
HIV-1, SIVmac, and SIVsykes LTRs have two, one, and zero NF- B
binding sites, respectively. B, HeLa cells were transfected
with 200 ng of HIV-1 HXB2 LTR (lanes 1-3), SIVmac LTR
(lanes 4-6) or SIVsykes (lanes 7-9)
LTR-luciferase plasmid with 2.0 µg of either HuCycT1 or MuCycT1.
Transfections were normalized to cotransfected pCMV-
-galactosidase
plasmid. C, dose-titrated activity of MuCycT1. HeLa cells
were cotransfected with 200 ng of either HIV-1 HXB2 or SIVsykes
LTR-luciferase plasmid with increasing amounts of MuCycT1. All
transfections were normalized to cotransfected pCMV-
. All
values are averages ± S.D. from three or more independent
experiments.
B, Sp1, and TAR. Results in Fig. 1A suggest that NF-
B is not needed for cycT1-activation.
To address the other motifs in the HIV-1 LTR, we employed several,
otherwise isogenic, linker-scanning LTR-mutants (Fig.
2A). In parallel assays, we
found that the Sp1 motifs were strictly required for cycT1 activity,
whereas AP-1, NFAT, NF-
B, or TAR sequences were dispensable (Fig.
2A). Hence, although Tat-dependent activity of
cycT1 requires TAR, the Tat-independent activity does not.
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Fig. 2.
Requirement for Sp1 binding sites in cycT1
mediated LTR-activation. A, HIV-1 LTR linker scanning
mutants with selective mutation in AP1 (lane 1-3), NF-AT
(lanes 4-6), TAR (lanes 7-9), NF- B
(lanes 10-12), or Sp1 (lanes 13-15), were
transfected with either HuCycT1 or MuCycT1. B, HeLa cells
transfected with HA-tagged HuCycT1 and MuCycT1 were lysed and
immunoprecipitated with mouse monoclonal anti-HA antibody (lanes
1-4) or an irrelevant isotype-matched control serum (lanes
5-9). Immunoprecipitates (IP) were solubilized and
probed in Western blotting (WB) with rabbit anti-Sp1
antibody. Control represents transfection with pCMV-HA. Lane
9 shows HeLa cell extract.
)43CAT) composed of three Gal4 binding sites positioned upstream
of a TATA box with a downstream chloramphenicol acetyltransferase cDNA (Fig. 3A). To this
reporter, we separately added Gal4BD-, Gal4Sp1A-, or Gal4E1a-plasmid
expressing respectively the Gal4-DNA-binding-domain, a
binding-domain-Sp1A-fusion, or a binding-domain adenovirus E1a fusion.
These DNA mixes were cotransfected with or without a MuCycT1 expression
vector (Fig. 3B). We found that neither Gal4BD (Fig. 2B, lanes 1 and 2) nor Gal4E1a (Fig.
2B, lanes 5 and 6) supported MuCycT1-activated expression over basal. By contrast Gal4Sp1A plus
MuCycT1 mediated a >70-fold increase in expression (Fig. 2B, lanes 3 and 4), consistent with
the Sp1A segment being wholly sufficient for function.
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Fig. 3.
Sp1A domain sufficiently mediates cycT1
activation. A, schematic representations of G3( )43CAT,
Gal4BD, Gal4Sp1A, Gal4E1A, and Hu/MuCycT1 vectors. B, HeLa
cells were transfected with G3(
)43CAT and Gal4BD Gal4Sp1A, or Gal4E1A
with or without MuCycT1. C, Gal4Sp1A was transfected into
HeLa cells separately with HA-tagged MuCycT1 (wild-type or 1-631
mutant), HA-tagged human cyclin H, or HA-tagged human cyclin K
expression vectors. Cells were immunoprecipitated with monoclonal
anti-Gal4BD antibody followed by Western blotting (WB) with
rabbit anti-HA. Arrows, expected positions for MuCycT1s;
asterisks, expected positions for cycH and cycK.
IP, immunoprecipitates.
30-fold increase) activity; HuCycT1
(419-726) and HuCycT1 (500-726) provided moderate (7-10-fold
increase) activity; and HuCycT1 (1-444) and HuCycT1 (1-426) were
inactive (Fig. 4A). From the results, we mapped a Sp1A
cooperative domain to the C-terminal 300 amino acids of cycT1. This
C-terminal region contrasts with its N-terminal cyclin homology box,
which serves cdk9-binding and P-TEFb/Tat/TAR function (24, 39).
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Fig. 4.
The C terminus of cycT1 cooperates with Sp1A.
A, HeLa cells were cotransfected with G3( )43CAT plus
either Gal4Sp1A or Gal4BD with the indicated full-length or deletion
mutant of HuCycT1. B, Western blot verification of the
expression of transfected cyclin T1s.
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Fig. 5.
CycT1 C-terminal amino acids 419 to 726 are
sufficient for promoter activation. Left, HeLa cells were
transfected with G5-83HIV-Luc and the indicated wild type and deletion
mutants of HuCycT1 fused to Gal4BD. Luciferase was assayed 48 h
later. Right, Western blot verification of comparable
expression of transfected DNAs.
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Fig. 6.
Localization of Sp1A-active cycT1 to nuclear
speckles. HeLa cells were transfected with the indicated HA-tagged
HuCycT1. Cells were incubated with anti-HA and stained with anti-mouse
conjugated to Alexa Fluor 488 (Molecular Probes). Nuclei were stained
with 4,6-diamidino-2-phenylindole (Molecular Probes). Light field
(DIC (differential interference contrast)) is left;
4,6-diamidino-2-phenylindole (DAPI) is middle; anti-HA is
right.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and contact
with RNAP II CTD (49, 50). Although we have not directly examined
interactions between NF-
B and cycT1, several observations seemingly
disfavor this interpretation. First, the natural phylogeny of primate
lentiviral LTRs is such that many simian LTRs have either single or no
NF-
B binding sites (Fig. 1A). Thus, recruitment of cycT1
by NF-
B cannot be a generally conserved mechanism for all primate
lentiviruses. Second, our linker-scanning analyses (Fig. 2A)
showed that Sp1 is required, whereas NF-
B is not, for DNA-targeted
cycT1 activity. Other studies (40, 51) have also independently
suggested this Sp1-requirement. Hence, although we do not exclude that
NF-
B might recruit cycT1 to HIV-1 DNA, our current results show that
NF-
B is unnecessary, whereas Sp1 is necessary and sufficient.
Intriguingly, in the two studies that proposed NF-
B recruitment of
cycT1, presence of Sp1-binding sites in the reporter constructs were
required for such activity (49, 50). Because NF-
B binds the
N-terminal cyclin homology domain of cycT1 (49) and Sp1 binds the C
terminus of cycT1, it is possible, however, that in the natural context of the HIV-1 LTR, both factors cooperate in high affinity capture of
cycT1. DNA-tethered cycT1 may then subsequently contact RNAP II CTD
directly (50).
) HIV-1
provirus (data not shown).
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Bldg. 4, Rm. 306, 9000 Rockville Pike, Bethesda, MD 20892-0460. Tel.: 301-496-6680; Fax: 301-480-3686; E-mail: kj7e@nih.gov.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M209162200
2 V. S. R. K. Yedavalli, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
HIV-1, human
immunodeficiency virus type 1;
RNAP II, RNA polymerase II;
TAR, trans-activation-responsive element;
CTD, C-terminal domain;
P-TEFb, positive transcriptional elongation factor;
cycT1, cyclin T1;
CMV, cytomegalovirus;
HA, hemagglutinin;
NF-B, nuclear factor-
B;
SIV, simian immunodeficiency virus;
MuCycT1, murine cyclin T1;
HuCycT1, human cyclin T1;
AP-1, activator protein 1;
NFAT, nuclear factor of
activated T cells;
Gal4BD, Gal4 DNA binding domain.
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