From the Institute of Applied Biochemistry,
Center for Tsukuba Advanced Research Alliance, University of
Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8577, the
§ Division of Human Retroviruses, Center for Chronic Viral
Diseases, Faculty of Medicine, Kagoshima University, 8-35-1,
Sakuragaoka, Kagoshima 890-8520, and ** PRESTO, Japan Science and
Technology Corporation,
4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan
Received for publication, August 1, 2000, and in revised form, October 18, 2000
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ABSTRACT |
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RNA helicase A (RHA) has two double-stranded (ds)
RNA-binding domains (dsRBD1 and dsRBD2). These domains are conserved
with the cis-acting transactivation response element
(TAR)-binding protein (TRBP) and dsRNA-activated protein kinase (PKR).
TRBP and PKR are involved in the regulation of HIV-1 gene expression through their binding to TAR RNA. This study shows that RHA also plays
an important role in TAR-mediated HIV-1 gene expression. Wild-type RHA
preferably bound to TAR RNA in vitro and in
vivo. Overexpression of wild type RHA strongly enhanced viral
mRNA synthesis and virion production as well as HIV-1 long terminal
repeat-directed reporter (luciferase) gene expression. Substitution of
lysine for glutamate at residue 236 in dsRBD2 (RHAK236E)
reduced its affinity for TAR RNA and impaired HIV-1 transcriptional
activity. These results indicate that TAR RNA is a preferred target of
RHA dsRBDs and that RHA enhances HIV-1 transcription in
vivo in part through the TAR-binding of RHA.
RNA helicase A (RHA)1
catalyzes the unwinding of duplex RNA and DNA in a process coupled with
the hydrolysis of NTPs (1, 2). We have previously shown that RHA
mediates association of the CREB-binding protein (CBP) with RNA
polymerase II (pol II) (3) and that RHA links breast cancer-specific
tumor suppressor protein (BRCA1) to pol II (4). RHA consists of two
type A double-stranded RNA-binding domains (dsRBDs) (5, 6), a classical
Walker type NTP-binding site, a DEAH/D helicase domain, and a
single-stranded nucleic acid-binding domain characterized by
Arg-Gly-Gly (RGG) repeats (7). Trypsin-digested RHA, lacking both
dsRBDs and the RGG repeat sequence, has reduced helicase activity,
implicating these domains in the unwinding function of RHA (7). Amino
acids 1-262 and 255-664 of RHA have proved to be CBP- and pol
II-binding sites, respectively (3). RHA shuttles between nucleus and
cytoplasm with a cis-acting constitutive transport element
in simian retroviruses (8). It has been proposed that RHA is necessary
for releasing both constitutive transport element- and HIV-1 Rev
response element (RRE)-containing RNA from spliceosomes prior to the
completion of splicing (9).
After integration of HIV-1 into the host genome, the nuclear
factor- TAR, a nascent viral leader RNA transcribed from the R region of the
LTR, plays an important role in HIV-1 gene expression and forms a
unique stem and loop structure (17, 18). Tat function is mediated by
the TAR RNA and requires the recruitment of a complex consisting of Tat
and cyclin T1 component of positive transcription elongation factor b
(P-TEFb) bound to TAR (19). A defect of Tat-induced transactivation in
murine cells was attributed to the lack of a functional cyclin T1 (20,
21). Alternatively, the defect was linked to the reduced abundance of
p300 and p300/CBP-associated factor (22). In addition, several cellular
cofactors play crucial roles in HIV-1 gene expression through binding
to the TAR RNA. For instance, TRBP consists of two type A dsRBDs and
another type B dsRBD and activates the HIV-1 LTR (6, 23) through
binding of the second type A dsRBD (dsRBD2) to the TAR RNA (24). PKR contains two type A dsRBDs and functions as a cellular antiviral factor
by inhibiting eukaryotic initiation factor 2 via phosphorylation of its
Cell Culture--
Human embryonic kidney (HEK) 293 (27) and HeLa
cells were maintained in Dulbecco's modified Eagle's medium (Sigma)
supplemented with 10% fetal bovine serum (Sigma), 100 units/ml
penicillin, 100 µg/ml streptomycin, and 2 mM glutamine.
The cells were grown at 37 °C in a humidified 95% air, 5%
CO2 atmosphere.
Plasmids--
Plasmid pHyg LTR-Luc encodes a chimeric gene
consisting of the HIV-1 LTR containing two intact
A series of RHA polypeptides (1-262, 255-664, 649-1077, and
1064-1270) fused to glutathione S-transferase (GST) was
described previously (3). RHA-(1-90), RHA-(76-174), RHA-(160-262),
RHA-(1-262/ Transient Transfection and Luciferase Assays--
HEK 293 and
HeLa cells were transiently transfected with 100 ng of pHyg LTR-Luc or
mNF TAR Binding Assays--
Total cellular extract from
Escherichia coli BL21, expressing a series of RHA
polypeptides or full-length TRBP fused to GST protein, was
electrophoresed on an SDS gel and transferred to a polyvinylidene
difluoride membrane (Immobilon P, Millipore). TAR binding assays were
performed as reported previously (23). Filters were incubated in
binding buffer (20 mM HEPES, pH 7.3, 40 mM KCl,
1.5 mM MgCl2, and 1 mM
dithiothreitol) with 10 µg/ml yeast RNA, 10 µg/ml calf thymus DNA,
and 200 fmol/ml gel-purified 32P-labeled TAR RNA probe.
Filters were washed with binding buffer and then exposed to x-ray film.
Comparable expression of GST-fused protein was determined by Western
blotting with an anti-GST antibody (Amersham Pharmacia Biotech) and
Coomassie Brilliant Blue staining.
Wild-type TAR, Immunoprecipitation and Slot Blot Assays--
HEK 293 cells were
transfected with 3 µg of pHyg LTR-Luc or G5b-Luc (32), 150 ng of
pCD-SR p24 Assays--
HEK 293 cells were transfected with 10 ng of
pNL4-3, 20 ng of PGV-C control plasmid containing the SV40-luciferase
gene (Toyo-inki, Tokyo Japan), and various amounts of RHA construct. To
ensure an equal amount of DNA, empty plasmid was added in each
transfection. After 24 and 48 h of transfection, culture
supernatants were collected and tested for HIV-1 p24 antigen levels
using a p24 antigen detection kit (Retro-tek, Zepto Metrix Corporation,
Buffalo, NY). Equivalent transfection efficiency was verified by
luciferase activity derived from the cotransfected PGV-C control
plasmid. All experiments were performed in triplicate.
Northern Blot Assays--
HEK 293 cells were transfected with
500 ng of pNL4-3, 1 µg of PGV-C control plasmid, and 5 µg of each
RHA construct. After 24 h of transfection, polyadenylated RNA was
extracted from the transfected cells using an mRNA purification kit
(Amersham Pharmacia Biotech), run on a 0.8% agarose gel, and blotted
onto GeneScreen Plus membrane. The membrane was hybridized with a
32P-labeled HIV-1 LTR probe and then exposed to x-ray film.
The probe was amplified by PCR using the primers
5'-AGTGCTCAAAGTAGTGTGTG-3' and 5'-GATCTCCTCTGGCTTTACTTT-3' and pNL4-3
as a template. Equivalent transfection efficiency was determined by
measuring the amount of luciferase mRNA derived from the
cotransfected PGV-C control plasmid.
The Effects of RHA on HIV-1 LTR-directed Gene Expression--
To
assess the regulatory effects of RHA on HIV-1 LTR-directed gene
expression, several human cell lines were transfected with the pHyg
LTR-Luc reporter plasmid. In HEK 293 cells, wt RHA markedly enhanced
Tat-induced reporter activity in a dose-dependent manner (Fig. 1A). In contrast,
overexpression of wt RHA did not affect HIV-1 LTR-directed luciferase
activity in HeLa cells (Fig. 1B) as described previously
(9).
Determination of the RHA Regions Required for TAR
Binding--
Northwestern assays were conducted to determine whether
RHA binds to the TAR RNA. First, we divided full-length RHA into four fragments (Fig. 2A) and
examined each fragment for their TAR RNA binding activity. Only
RHA-(1-262), which contains both dsRBD1 and dsRBD2, could bind to the
TAR RNA (Fig. 2B, left panel). Second, we constructed three
deletion mutants of RHA-(1-262) (Fig. 2A). Unexpectedly,
only RHA-(160-262) (containing dsRBD2) and not RHA-(1-90) (containing
dsRBD1) bound to the TAR RNA (Fig. 2B, middle panel). Amino
acids 235-249 in the RHA dsRBD2 are well conserved among the
dsRNA-binding protein family (Fig. 2C). In particular, the lysine at residue 211 (Lys-211) in the TRBP dsRBD2 is critical for
binding to the TAR RNA (34). Therefore, we constructed two mutants
(RHA-(1-262/ RHA dsRBD Preferentially Binds the Stem of TAR RNA in
Vitro--
HIV Tat and cyclin T1 recognize the bulge and loop of TAR
RNA, respectively (20). Similarly, it is important to define the region
of TAR that interacts with the RHA dsRBD and to confirm its relative
specificity. Presumably, RHA dsRBD binds to the stem region of TAR RNA,
since dsRBD binds to dsRNA. The Association of RHA with TAR in Vivo--
To test for in
vivo interaction of the TAR RNA with the RHA complex,
coimmunoprecipitation assays were performed on whole cell lysates from
HEK 293 cells cotransfected with pHyg LTR-Luc (to express
TAR-containing luciferase mRNA) or G5b-Luc (to express TAR-negative
luciferase mRNA) and each RHA construct. Except for "no-transfectant," a comparable amount of TAR-containing and
TAR-negative mRNA was detected in each cell lysate (Fig.
4, middle panel). Equal
amounts of wt RHA and RHAK236E were precipitated with
anti-HA antibody (Fig. 4, lower panel). The TAR-containing
mRNA was present in the wt RHA precipitant (Fig. 4, upper
panel). However, although significantly less mRNA was
coprecipitated with RHAK236E. No precipitant was identified
in the negative controls (no-transfectant and "mock-transfectant"). Furthermore, the association of wt RHA with the TAR-negative luciferase mRNA was as weak as that of RHAK236E and the
TAR-containing luciferase mRNA. These results indicate that the
TAR-containing mRNA existed in the RHA complex in vivo
and that the complex was formed primarily via the binding of RHA dsRBD2
to the TAR RNA. Although RHA-(1-262/K236E) could not bind to the TAR
RNA in vitro (Fig. 2B), the TAR-containing mRNA was weakly associated with RHAK236E complex
in vivo. Thus, it cannot be excluded that RHA also binds to
mRNA in a TAR-independent fashion, as in the case of RHA binding to
ssRNA through the RGG motif (7).
Enhancement of TAR-dependent Gene Expression by
RHA--
The finding that RHA binds to the TAR RNA in vivo
prompted a study of the role of RHA in TAR-mediated gene expression.
Transient transfections of the pHyg LTR-Luc reporter plasmid were
conducted to analyze the effect of RHAK236E on HIV-1
LTR-directed gene expression. RHAK236E, but neither
RHAmATP nor RHAW339A, enhanced the reporter activity (Fig. 5, lanes
7-12), although the effect of RHAK236E was
significantly lower than that of wt RHA (~40%). This reduced effect
of RHAK236E may be due to the lack of TAR-binding ability. Alternatively, RHA may be functionally bound to the
CBP·NF- The Effects of RHA on HIV-1 Production--
To elucidate the role
of TAR-binding ability of RHA in HIV-1 viral replication, HEK 293 cells
were cotransfected with pNL4-3 and different amounts of wt RHA or
RHAK236E (Fig.
6A). After 24 h of
transfection, HIV-1 p24 production was enhanced ~5-fold in the wt RHA
transfectants in a dose-dependent manner (Fig. 6A, left panel), as previously demonstrated in HeLa cells (9). The
effect of RHAK236E on HIV-1 p24 production was
significantly less than that of wt RHA (~30%), which was consistent
with the results in the reporter gene assays (Fig. 5). Similar results were also obtained after 48 h of transfection, except in the cells expressing for higher p24 antigen levels (Fig. 6A,
right panel). These results suggest that the association of
RHA with the TAR RNA is partly required for the RHA-enhanced HIV-1 gene
expression.
Finally, to elucidate the role of RHA in transcriptional regulation of
HIV-1, HEK 293 cells were transfected with pNL4-3 and each RHA
construct. Fig. 6B demonstrates that three different lengths
of HIV-1 mRNA (~2-, 4-, and 9-kb transcripts) were detected by
Northern blot assay. Equivalent transfection efficiency was confirmed
by the amount of luciferase mRNA derived from each cotransfected PGV-C control plasmid (data not shown). The amounts of all HIV-1 mRNA transcripts were increased by wt RHA coexpression (Fig.
6B). No other RHA mutants affected HIV-1 mRNA synthesis
or p24 production. These results strongly support a functional role of
RHA in the transcriptional activation of HIV-1.
RHA belongs to the dsRNA-binding protein family, which includes
TRBP and PKR. These proteins display variable RNA-binding characteristics. For instance, the PKR dsRBD1 displays higher affinity
than the PKR dsRBD2 for several kinds of RNA, including the TAR RNA
(37, 38). The TRBP dsRBD2 alone can bind to TAR RNA independent of
other dsRBDs (39). Two polypeptides containing each of the RHA dsRBDs
bind to poly(rI·rC) dsRNA with similar affinity, and it has been
considered that the two RHA dsRBDs cooperate to interact with dsRNA,
such as with poly(rI·rC) (7). The results presented here show that
only dsRBD2, and not dsRBD 1, could bind to TAR RNA in vitro
(Fig. 2B). This RNA-binding property of RHA is similar to
that of TRBP. Gel mobility shift assays with polypeptides containing
each or both RHA dsRBD were used to confirm the contribution of dsRBD1
in binding to the TAR RNA. This approach was unsuccessful due to
aggregation of purified dsRBD polypeptide (data not shown).
The dsRBDs interact with highly structured RNA and dsRNA, generally
without obvious RNA sequence specificity (40). In fact, RHA could bind
to various RNA species, including ssRNA in vitro. Like TRBP
(24), RHA bound to the TAR RNA with higher affinity than to ssRNA or
yeast tRNA and preferably bound to TAR-containing mRNA rather than
TAR-negative mRNA. Thus, it appears that TAR RNA is one of the
binding targets of RHA dsRBDs. These findings further demonstrate that
the conserved amino acids 235-249 in the RHA dsRBD2 are essential for
TAR binding. It was previously reported that the Lys-211 residue of
TRBP dsRBD2 is important for interaction with TAR RNA (34). It was
confirmed by x-ray crystallography that the corresponding residue of
Xlrbpa, the Xenopus homologue of TRBP, directly interacts
with dsRNA (41). These residues correspond to the Lys-236 residue of
RHA dsRBD2, and the importance of Lys-236 was demonstrated here the
using a RHA-(1-262/K236E) mutant. Together, the results confer in that the characteristics of RHA for TAR-binding are similar to those of
TRBP.
It is known that transcription factor levels differ between cell lines.
For instance, murine NIH3T3 cells express a small amount of p300
compared with HeLa cells. Thus, the failure of HIV-1 LTR-directed
transactivation by Tat in the murine cells could be attributed to the
small amount of p300 (22). Western blot analysis showed that the amount
of RHA in HEK 293 cells was less than 50% that in HeLa cells, although
an equivalent amount of CBP or CREB was identified in both cell
lines.3 The HIV-1
LTR-directed luciferase activity was enhanced by exogenous RHA in HEK
293 cells but not in HeLa cells. HEK 293 cells are widely used in
transfection experiments with HIV-1 proviral DNA clones (29, 42). Taken
together, the results indicate HEK 293 cells to be a favorable host
cell system to assess the functions of RHA in HIV-1 gene expression by
transient transfection. RHA was reported to bind weakly to HIV-1 RRE
and be involved in post-transcriptional regulation of HIV-1 (9).
However, the reporter gene constructs used in this study do not contain
RRE, and the results in the reporter gene assays accorded well with
those in the p24 assays, suggesting that the reduced activity of
RHAK236E in the p24 assays was due primarily to the lack of
the TAR-binding ability of RHA in HEK 293 cells. This discrepancy may
be due to either the different cell types used or to the distinct roles
of RHA in transcription and post-transcriptional regulation in HIV-1
gene expression.
We have previously suggested that both the ATPase/helicase activity and
pol II binding ability of RHA contribute to the
CREB-dependent transcription (3). It can be proposed from
the current studies that these functions of RHA may also be important
for HIV-1 transcription. Moreover, the data demonstrate that RHA
enhances HIV-1 LTR-directed gene expression in a
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (NF-
B) binds to enhancer elements in the HIV-1 long terminal repeat (LTR) and stimulates the expression of the viral genome
in a signal-dependent manner (10, 11). HIV-1 expression can
be divided into two phases (early and late). In the early phase, the
majority of viral mRNA is multiply spliced to produce 2-kb
transcripts that encode the regulatory proteins, including Tat and Rev,
necessary to activate HIV-1 expression. A transition subsequently
occurs to accumulate singly spliced (4-kb) and full-length (9-kb)
transcripts encoding the viral structural proteins and providing the
genomic RNA (12-14). Regulation of the viral RNA-splicing transition
mechanism is reported to involve the protection and transport of
full-length HIV-1 RNA by Rev (15, 16).
-subunit (25). A similar function of PKR is mediated by an
interaction with several kinds of RNA, leading to PKR
autophosphorylation (26). Like TRBP and PKR, RHA possesses dsRBDs and
may act as a cellular transcriptional regulator. Of particular interest
is whether RHA binds to the TAR RNA and influences HIV-1 gene
expression. This study shows that RHA acts as a novel TAR-binding
cellular cofactor and enhances HIV-1 LTR-directed gene expression and
viral production in vivo.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B elements and a
luciferase gene (28). The mNF
B LTR-Luc plasmid was constructed by
inserting both of the
B element-mutated LTR fragments (14) into the
SmaI/HindIII site of PGV-B containing a
luciferase gene without a promoter (Toyo-Inki). The pCD-SR
/tat
plasmid, a mammalian expression vector for HIV-1 Tat, was constructed
as described previously (28). The HIV-1 pNL4-3 proviral DNA clone (30)
was obtained from the AIDS Research and Reference Reagent Program
(Division of AIDS, NIAID, National Institutes of Health) and was
contributed by Malcolm Martin. Wild-type (wt) RHA and
RHAmATP constructs were prepared as described previously
(3). The RHAmATP and
RHAW339A2
constructs contain single amino acid substitutions leading to defects
in ATPase/helicase activity and pol II binding ability of RHA,
respectively. RHAK236E containing the lysine to glutamate substitution at residue 236 was generated by PCR. The wt RHA construct and three RHA mutants were tagged by hemagglutinin (HA) for Western blot assays and immunoprecipitation studies. To obtain the RNA probes
by in vitro transcription, the pcTAR and
loopTAR plasmids were constructed by inserting PCR-generated cDNA fragments
(5'-GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCC-3' and 5'-GGGTCTCTCTGGTTAGACCAGATCTGAGCGCTCTCTGGCTAACTAGGGAACCC-3') with
HindIII linker into the HindIII site of
pcDNA3 (Invitrogen). The plasmids as described above were purified
using cesium chloride gradients.
235-249), RHA-(1-262/K236E), and full-length TRBP were
created by PCR. These fragments were cloned into pGEX-5X-1 (Amersham
Pharmacia Biotech), and all of the PCR-generated plasmids were
confirmed by sequence analysis.
B-Luc reporter, 100 ng of RSV-
-gal control plasmid, 2 or 10 ng
pCD-SR
/tat, and various amounts of RHA, using the calcium phosphate
method as described previously (31). To ensure an equal amount of DNA,
"empty plasmids" were added in each transfection. In the reporter
gene assays, luciferase activity derived from expression of the pHyg
LTR-Luc and mNF
B LTR-Luc reporter plasmids was normalized to
-galactosidase activity from cotransfected Rous sarcoma virus
(RSV)-expression plasmid containing the
-galactosidase gene
(RSV-
-gal) (3). Luciferase activity was measured with AutoLumat
(Berthold). All experiments were performed in triplicate, and all
results were obtained from at least three separate experiments.
Equivalent expression of RHA constructs was verified by Western blot
analysis using the anti-HA antibody 12CA5 (Roche Molecular Biochemicals).
loop TAR RNA, and ssRNA were obtained by in
vitro transcription using BamHI-cleaved pcTAR or
loopTAR plasmid and XhoI-cleaved pcDNA3,
respectively. GST and RHA-(1-262) polypeptide blots were incubated in
binding buffer with 10 µg/ml calf thymus DNA, 1 pmol/ml
32P-labeled wt TAR,
loop TAR RNA or ssRNA probe, and 300 pmol/ml unlabeled RNA or yeast tRNA (Sigma) as a competitor. The
filters were washed with binding buffer and exposed to x-ray film.
/tat or 300 ng Gal4-VP16 (33), and 3 µg of each RHA
construct. After 24 h of transfection, the cells were lysed in 1 ml of 0.65% Nonidet P-40 lysis buffer (20 mM HEPES, pH
7.3, 150 mM KCl, 1.5 mM MgCl2,
0.65% Nonidet P-40). Cell lysates were pre-cleared with 5 µg of
normal mouse IgG (Santa Cruz Biotechnology) conjugated to protein
G-Sepharose beads (Amersham Pharmacia Biotech) for 1 h at 4 °C.
After a brief centrifugation, the lysates were mixed with 5 µg of
normal mouse IgG or 5 µg of anti-HA antibody conjugated to protein
G-Sepharose beads. After an overnight incubation at 4 °C, the beads
were washed three times with lysis buffer. RNA was extracted from the
beads with ISOGEN (Nippon Gene) and then blotted onto GeneScreen Plus
membrane (PerkinElmer Life Sciences) using a slot blotting apparatus
(HYBRI-SLOT MANIFOLD, Life Technologies, Inc.). The membrane was
hybridized with a 32P-labeled luciferase gene probe
generated by PCR using the primer pair 5'-GGATGGAACCGCTGGAGAG-3' and
5'-GTTTCATAGCTTCTGCCAACCG-3' and exposed to x-ray film. Equivalency for
immunoprecipitation of HA-tagged RHA was verified by Western blot analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The effects of RHA on HIV-1 LTR-directed gene
expression. HEK 293 (A) and HeLa cells (B)
were transfected with 100 ng of pHyg LTR-Luc reporter, and 2 ng (for
HEK 293 cells) or 10 ng (for HeLa cells) of pCD-SR /tat (tat) or
"empty vector" (
tat), together with various amounts of the wt RHA
expression vector as indicated. To ensure an equal amount of DNA, the
control plasmid was added in each transfection. Luciferase activity was
normalized to transfection efficiency via cotransfection with 100 ng of
RSV-
-gal. All experiments were performed in triplicate, and results
were taken from three separate experiments. *, lanes 5 and
6 versus 4, p < 0.05, by multiple comparisons using the t test.
235-249) and RHA-(1-262/K236E)) that contain the
intact dsRBD1 and the mutated dsRBD2. RHA-(1-262/
235-249) is a
deletion mutant missing amino acids 235-249 from RHA-(1-262). RHA-(1-262/K236E) is also an RHA-(1-262) mutant with lysine at residue 236 (Lys-236) substituted with glutamate. The Lys-236 of RHA
corresponds to the Lys-211 of TRBP. Both RHA-(1-262) mutants failed to
interact with the TAR RNA (Fig. 2B, right panel), indicating that amino acids 235-249 and in particular residue Lys-236 are essential for TAR binding in vitro.
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Fig. 2.
Determination of the RHA region required for
TAR binding in vitro. A, schematic
representation of RHA (top line) and its functional domains
(dsRBD, dsRNA binding domain; pol II, RNA
polymerase II-binding site; ATP, ATP-binding site;
DEAH/D, helicase domain; RGG, Arg-Gly-Gly repeat;
CBP, CBP-binding site). Bottom lines represent
the portions of RHA deletion mutants used to make the GST fusion
proteins. TAR binding activity of each mutant is indicated at
right. B, binding of RHA mutants to TAR RNA.
Total cellular extract was electrophoresed on an SDS gel and then
transferred to polyvinylidene difluoride membrane for Northwestern
analysis (TAR binding). The same membrane was used for
Western blotting with an anti-GST antibody ( -GST
Western), and Coomassie Brilliant Blue (CBB)
staining. The clones used are indicated at the top of each
lane and correspond to the protein described in A. Negative
(GST) and positive (GST-TRBP) controls are the
cell lysate from E. coli BL21 cells containing the GST and
GST-TRBP fusion proteins, respectively. The arrowheads
indicate GST fusion proteins in the panels of CBB and
-GST Western. C, comparison of
amino acids sequences between RHA and TRBP dsRBD2.
loop TAR RNA probe illustrated in
Fig. 3A was therefore
constructed. GST-fused RHA-(1-262) and GST alone (negative control)
were blotted onto filters, and the filters were incubated with an
equivalent amount of 32P-labeled wt TAR,
loop TAR RNA,
or ssRNA. Interestingly, the binding affinity of
loop TAR RNA to
RHA-(1-262) was significantly stronger than that of wt TAR RNA. ssRNA
bound weakly to RHA-(1-262). None of the tested probes bound to GST
(Fig. 3B). In competition assay (Fig. 3C), the
binding of wt TAR RNA to RHA-(1-262) was almost completely inhibited
by wt TAR or
loop TAR RNA competitor at a concentration of 300-fold
higher than that of the 32P-labeled probe. In contrast, the
ssRNA competitor and yeast tRNA slightly reduced the probe binding.
These results suggest that the RHA dsRBD preferably recognizes the stem
of the TAR RNA.
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Fig. 3.
The feature of RHA dsRBD binding to TAR
RNA. A, primary sequence and RNA secondary structures
of wild-type TAR (wt TAR) and mutant TAR construct
( loop TAR). B, the element in TAR
that binds to RHA dsRBD. Filters transferred with various amounts of
RHA-(1-262) as indicated and 100 pmol of GST proteins were incubated
with 1 pmol/ml 32P-labeled wt TAR,
loop TAR RNA, or
ssRNA probe (see "Experimental Procedures"). C, relative
specificity for RHA binding to TAR RNA. The same filters as indicated
above were incubated with 1 pmol/ml 32P-labeled wt TAR RNA
alone (no competitor) or together with 300 pmol/ml of either
unlabeled wt TAR,
loop TAR, ssRNA, or yeast tRNA
(competitor).
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Fig. 4.
Detection of in vivo
interaction of RHA with TAR-containing mRNA. Lysates
from HEK 293 cells transfected with 3 µg of pHyg LTR-Luc or 3 µg of
G5b-Luc, 150 ng of pCD-SR /tat or 300 ng of Gal4-VP16, and 3 µg of
wt RHA (HA-wt RHA), RHAK236E
(HA-RHAK236E), or empty vector (mock)
were immunoprecipitated with normal mouse IgG (IgG) or
anti-HA antibodies (
-HA). The extracted RNA
from each immunoprecipitated complex (Co-IP RNA) was
subjected to slot blot assays with luciferase gene-specific probe.
Comparable amounts of TAR-containing or TAR-negative luciferase
mRNA from each lysate were determined (10% input).
Equivalency for immunoprecipitation of HA-tagged RHA with anti-HA
antibodies was verified by Western blot analysis
(
-HA IP-Western). No-transfectant (No
Tf), mock-transfectant (mock), and normal mouse IgG
(IgG) were used as negative controls.
B complex on the
B elements of HIV-1 LTR (35,
36). To exclude this possibility, transient transfection assays with
the mNF
B LTR-Luc reporter plasmid that contained two mutated
B
elements were also performed. Mutation of the
B elements strongly
reduced luciferase activity (Fig. 5, lane 4 versus 16), as described previously (11).
Wild-type RHA markedly enhanced the Tat-induced reporter activity in a
dose-dependent manner (Fig. 5, lanes 16-18),
and although both RHAmATP and RHAW339A slightly
increased the luciferase activity, RHAK236E had no effect
(Fig. 5, lanes 19-24). These results indicate that the
association of RHA with the TAR RNA is required for the RHA-induced
transactivation in the mutated
B LTR. Conversely, the higher
activity of RHAK236E in intact
B LTR compared with
mutated
B LTR suggests that RHA can also interact with HIV-1 LTR
through the intact
B elements in a TAR-independent fashion.
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Fig. 5.
The effect of RHA on HIV-1 LTR-directed gene
expression in B element-dependent
and -independent fashions. HEK 293 cells were transfected with 100 ng of pHyg LTR-Luc or mNF
B LTR-Luc reporter and 2 ng of
pCD-SR
/tat (tat) or empty vector (
tat),
together with various amounts of each RHA construct as indicated. To
ensure an equal amount of DNA, the control plasmid was added in each
transfection. Luciferase activity was normalized to transfection
efficiency via cotransfection with 100 ng of RSV-
-gal. All
experiments were performed in triplicate, and results were taken from
at least three separate experiments. (*1, lane 8 versus 6, p = 0.0006;
*2, lanes 17 and 18 and
21-24 versus 16, p < 0.03; *3, lanes 22 and 24 versus 18, p < 0.008, by
multiple comparisons using the t test).
View larger version (34K):
[in a new window]
Fig. 6.
The effect of RHA on HIV-1 production.
A, implication of TAR-binding of RHA in HIV-1 replication.
HEK 293 cells were transfected with 10 ng of pNL4-3, 20 ng of PGV-C
plasmid, and various amounts of each RHA construct as indicated. To
ensure an equal amount of DNA, the control plasmid was added in each
transfection. After 24 and 48 h of transfection, culture
supernatants were tested for p24 antigen levels. Equivalent
transfection efficiency was verified by the luciferase activity derived
from cotransfected PGV-C plasmid. All experiments were performed in
triplicate. (*1, lanes 2 and 3 versus 1, p < 0.04;
*2, lane 5 versus 3,
p = 0.0006 by multiple comparisons using the
t test). B, the effect of RHA on HIV-1 mRNA
synthesis. HEK 293 cells were transfected with 500 ng of pNL4-3, 1 µg
of PGV-C control plasmid, and 5 µg of each RHA construct as
indicated. After 24 h of transfection, polyadenylated RNA was
extracted, electrophoresed on an agarose gel, and blotted onto
GeneScreen Plus membrane. The membrane was hybridized with the
32P-labeled HIV-1 LTR probe. To compare the
amount of RNA in each lane, the replica membrane was hybridized with
a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA
probe. Equivalent transfection efficiency was verified by measuring the
amount of luciferase mRNA derived from each cotransfected PGV-C
control plasmid. The p24 antigen levels in culture supernatants of the
transfected cells were determined.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
element-dependent and -independent fashion. NF-
B
functionally binds to the
B elements in the HIV-1 LTR (10, 11) and
associates with CBP (35, 36). Furthermore, RHA was shown to mediate the
association of CBP with pol II (3). The RHAK236E mutant was
capable of binding to CBP in GST pull-down assays (data not shown).
Therefore, the certain activity of RHAK236E in the reporter
assays using intact
B elements but not in the assays using the
mutated
B elements may indicate that RHA also mediates the
association of CBP with pol II on the
B-dependent HIV-1
preinitiation complex. Conversely, it was shown that RHA enhances HIV-1
gene expression, at least in part, through its TAR binding. TAR RNA is
required for the recruitment of a complex consisting of Tat and the
cyclin T1 component of P-TEFb (19-21). It is possible that both the
ATPase/helicase activity and pol II binding ability of RHA may be
required for the unwinding of highly structured RNA, such as TAR RNA,
and following HIV-1 transcription.
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ACKNOWLEDGEMENTS |
---|
We thank Yukiko Okada and Megumi Fujita for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Japanese Ministry of Education, Science, Culture, and Sports (10480196 and 10177230), Japanese Ministry of Health and Welfare, Japan Science and Technology Corporation (PRESTO), Human Health Science Foundation Medicitial Welfide Foundation, and by funds from Memorial Yamanouchi Foundation, Kaken Pharmaceutical Co. Ltd., and Santen Pharmaceutical Co. Ltd.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.
¶ Present address: Dept. of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-8656, Japan.
To whom correspondence should be addressed: Department
of
Genome Science, Institute of Medical Science, St.
Marianna University of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki
Kanagawa 216-8512, Japan. Tel.: 81-44-977-8111 (ext. 4113); Fax:
81-44-975-4599; E-mail: nakashit@marianna-u-ac.jp.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M006892200
2 S. Aratani, R. Fujii, T. Oishi, H. Fujita, T. Amano, T. Ohshima, M. Hagiwara, A. Fukamizu, and T. Nakajima, manuscript in preparation.
3 S. Aratani and T. Nakajima, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
RHA, RNA helicase A;
CREB, cAMP-responsive element-binding protein;
CBP, CREB-binding
protein;
pol II, RNA polymerase II;
dsRBD, double-stranded RNA-binding
domains;
HIV-1, human immunodeficiency virus, type 1;
LTR, long
terminal repeat;
TAR, cis-acting transactivation response
element;
RRE, Rev response element;
HEK, human embryonic kidney;
HA, hemagglutinin;
GST, glutathione S-transferase;
P-TEFb, positive transcription elongation factor b;
kb, kilobase pair;
wt, wild
type;
TRBP, TAR-binding protein;
PCR, polymerase chain reaction;
ssRNA, single-stranded RNA;
PKR, dsRNA-activated protein kinase;
RGG, Arg-Gly-Gly;
NF-B, nuclear factor-
B;
RSV, Rous sarcoma
virus.
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