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Journal of Virology, May 1999, p. 4341-4349, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Polyvalent Rev Decoys Act as Artificial
Rev-Responsive Elements
Tonia L.
Symensma,1
Scott
Baskerville,1
Amy
Yan,2 and
Andrew
D.
Ellington2,*
Department of Microbiology, Indiana
University, Bloomington, Indiana 47405,1 and
Department of Chemistry, University of Texas at Austin, Austin,
Texas 787122
Received 9 October 1998/Accepted 18 January 1999
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ABSTRACT |
Interactions between Rev and the Rev-responsive element (RRE)
control the order, rate, and extent of gene expression in human immunodeficiency virus type 1. Rev decoys may therefore prove to be
useful RNA therapeutics for the treatment of AIDS. To improve upon the current generation of Rev decoys that bind single Rev molecules, it would be useful to generate polyvalent Rev decoys that
could bind multiple Rev molecules. J. Kjems and P. A. Sharp (J. Virol. 67:4769-4776, 1993) originally constructed functional polyvalent Rev decoys, but the structural context of these polyvalent decoys remains unclear, and it has been argued that the individual decoys were either structurally discrete (Kjems and Sharp, J. Virol. 67:4769-4776, 1993) or were part of an extended helix (R. W. Zemmel et al., Mol. Biol. 258:763-777, 1996). To resolve the differences between these models, we have designed and synthesized concatemers of Rev-binding elements (RBEs) that fold to form
multiple, discrete, high-affinity Rev-binding sites. We find that the
concatenated RBEs can facilitate the cytoplasmic transport of
viral mRNAs and therefore likely bind multiple Rev molecules. These
artificial RREs may simultaneously sequester Rev and hinder access to
the cellular transport machinery.
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INTRODUCTION |
The Rev protein of human
immunodeficiency virus type 1 (HIV-1) interacts with a Rev-responsive
element (RRE) on retroviral mRNAs to regulate the expression of viral
proteins (reviewed in references 5 and
43). Early in the HIV-1 life cycle, fully spliced
(~2-kb) viral messages accumulate in the cytoplasm and produce
regulatory proteins such as Rev, Tat, and Nef. Rev then enters the
nucleus and facilitates the nucleocytoplasmic transport of incompletely
spliced (~4-kb) and unspliced (~9-kb) viral messages that encode
viral structural proteins (33; reviewed in reference 20).
The mechanism of Rev-dependent mRNA transport has been elucidated in
some detail. An N-terminal arginine-rich motif (ARM) within a single
Rev protein first interacts with a 30-nucleotide Rev-binding element
(RBE) (4, 23, 28) on a 234-nucleotide RRE. The RRE is in
turn located within an intron that spans the env gene
(14, 34, 56). An oligomerization domain that flanks and
overlaps the ARM guides the formation of Rev tetramers and higher-order
Rev oligomers (42, 57). After Rev binds to the RBE,
additional Rev molecules accumulate on the RRE (6, 36, 57).
The specific RNA-protein complex interacts with the nuclear export machinery to promote the transport of incompletely spliced mRNAs that contain the RRE (7, 9, 37, 49). A C-terminal leucine-rich activation domain has been shown to be critical for nucleocytoplasmic transport (33, 36), to act as a nuclear export signal (8, 53; reviewed in reference
11), and to bind to proteins involved in the nuclear
pore complex (3, 10). The Rev activation domain redirects
RRE-containing mRNAs to the non-mRNA export pathway used by 5S rRNA and
U snRNAs (8, 46, 47; reviewed in reference
20). The Rev ARM includes a nuclear localization
signal that allows Rev to reenter the nucleus and transport additional
mRNA molecules (18). Rev thus shuttles between the nucleus
and cytoplasm and regulates the timing and level of structural protein
expression by kinetically competing with the splicing machinery
(40, 45).
Rev and the RBE have both been targets for the development of antiviral
therapies. Mutants of the Rev protein that inhibit oligomerization can
transdominantly disrupt Rev function and inhibit viral replication
(19, 37, 42, 57). Antisense oligonucleotides (26,
48) and ribozymes (59) directed against the RRE have also been used to interrupt the viral life cycle. Rev decoys based on
the RBE have been shown to inhibit RNA-protein interactions and viral
replication (30, 31, 54, 55). Similarly, aptamers that can
bind tightly and selectively to the Rev protein have been selected from
random sequence nucleic acid population (2, 12, 51). These
anti-Rev aptamers have also been shown to bind Rev in vivo
(50) and to inhibit viral replication (13, 22).
Just as single Rev molecules bind to the RBE whereas multiple Rev
molecules accumulate on the RRE, the efficacies of individual Rev
decoys can potentially be augmented by building polyvalent Rev decoys
in which multiple Rev-binding sites are present on a single RNA
transcript. However, it is unclear how polyvalent Rev decoys should be
constructed. Kjems and Sharp have suggested that simple concatenation
of multiple high-affinity sites may successfully sequester multiple Rev
molecules (29). In contrast, Zemmel and coworkers have
suggested that Rev accumulates on extended helices adjacent to a single
high-affinity site (38, 58). Although these models are not
necessarily mutually exclusive, Zemmel et al. (58) have
argued that the constructs originally designed by Kjems and Sharp
(29) did not in fact fold to form discrete high-affinity
sites but instead formed an extended helix. To clearly distinguish
between these different structural models, we designed a polyvalent Rev
decoy in which multiple, high-affinity Rev-binding sites were presented
in a nonhelical structural context. The polyvalent decoy functions as
an artificial RRE and efficiently supports mRNA transport in tissue
culture cells. These findings have important implications not only for
the design of antiviral therapies but also for understanding the
mechanism of Rev-dependent viral mRNA transport.
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MATERIALS AND METHODS |
Materials.
Plasmids pDM128 (17, 18), pDM138
(21), and pRSVRev (17) were generous gifts from
T. Hope. Plasmids pDM128 and pDM138 contain the HIV-1 intron that
splits the rev gene and flanking exonic sequences; a
chloramphenicol acetyltransferase (CAT) reporter gene has been inserted
into the intron. The HIV-1 intron and CAT gene are under the control of
the simian virus 40 promoter and enhancer (44). In pDM138, a
unique ClaI restriction site was introduced into pDM128 in
place of the RRE. Plasmid pRSVbgal (2), generously supplied
by M. Zapp, contains the 
-galactosidase gene under the control of
the strong Rous sarcoma virus (RSV) promoter. Plasmids to be used in
cellular transfection experiments were purified by using Qiagen
(Chatsworth, Calif.) columns. Media, reduced-serum media, supplements,
phosphate-buffered saline, and Lipofectamine were purchased from Gibco
BRL (Gaithersburg, Md.). Fetal bovine serum was purchased from Summit
Biotechnologies (Fort Collins, Colo.).
Cell lines.
African green monkey kidney (CV-1) cells were
grown according to standard procedures in high-glucose Dulbecco's
modified Eagle medium supplemented with fetal bovine serum (11%),
penicillin (2.5 U/ml), streptomycin (2.5 mg/ml), and
L-glutamine (2 mM) in 24-well cell culture plates (Costar,
Cambridge, Mass.).
Construction of RBE concatemers.
A modular synthesis scheme
was devised for the construction of RBE concatemers. Three overlapping
pairs of oligonucleotides encoded a 5' T7 RNA polymerase promoter, an
RBE-linker monomer, and a 3' cap, respectively. The paired
oligonucleotides corresponding to the RBE-linker monomer contained
overhangs that facilitated their oligomerization. These overhangs also
facilitated addition of a T7 RNA polymerase promoter to the 5' end of
RBE concatemers and a constant sequence cap to the 3' end. Both
constant sequences also contained ClaI restriction sites to
facilitate cloning. The sequences of the oligonucleotides are shown in
Table 1.
All oligonucleotides were phosphorylated by using T4 polynucleotide
kinase (New England Biolabs, Beverly, Mass.) for 30 min
at room
temperature. The oligonucleotides encoding the RBE linker
monomers were
denatured for 1 min at 95°C, annealed for 3 min
at 45°C, and
incubated at room temperature for at least 10 min.
The oligonucleotide
concatemers that were formed were ligated
with T4 DNA ligase (New
England Biolabs). The T7 promoter and
3' cap were then ligated to the
RBE concatemers. PCR amplification
yielded a mixture of products which
served as templates for Ampliscribe
in vitro transcription reactions
(Epicentre Technologies, Madison,
Wis.). Transcribed RNAs were treated
with DNase I and gel purified
on a 6% polyacrylamide denaturing gel.
Individual bands were eluted
from the gel, ethanol precipitated, and
reverse transcribed by
using avian myeloblastosis virus reverse
transcriptase (Seikagaku
America, Ijamsville, Md.) for 1 h at
42°C. The reverse transcription
reaction products corresponding to
individual concatemers were
then PCR amplified and cloned into the
pCRII vector from the TA
cloning kit (Invitrogen, San Diego, Calif.).
Individual colonies
were screened for the presence of RBE-linker
inserts. Clones containing
concatemers corresponding to one to five
tandem copies of the
RBE (T1 to T5) were identified, and the sequences
of the concatemers
were confirmed by standard dideoxy sequencing
methods. The RBE
concatemers were PCR amplified from the pCRII vector,
digested
with
ClaI, and cloned into
ClaI-digested
pDM138 to generate plasmids
pDM138-T1 to pDM138-T5. The sequences and
orientations of the
concatemers in pDM138 were verified by dideoxy
sequencing. The
insert in plasmid pDM138-T5 contained point mutations
in two of
the linker regions (one linker lacked a U, while the second
had
an additional
C).
Nuclease mapping.
A DNA template corresponding to T5 was
transcribed in vitro by using an Ampliscribe transcription kit
according to the manufacturer's directions. The transcribed RNA was
purified on a 6% denaturing polyacrylamide gel, ethanol precipitated,
and dephosphorylated with alkaline phosphatase (Boehringer Mannheim,
Indianapolis, Ind.). The dephosphorylated RNA was phenol-chloroform
extracted and treated with T4 polynucleotide kinase (New England
Biolabs) in the presence of 5 fmol of [
-32P]ATP (7,000 Ci/mmol; ICN, Costa Mesa, Calif.). The radiolabeled RNA was again
purified on a 6% polyacrylamide gel and precipitated. Radiolabeled T5
was digested with various amounts of RNase A (1, 0.1, 0.01, 0.001, and
0.0001 U) or RNase T1 (10, 5, 2.5, 1.25, 0.63, and 0.31 U)
in 6 µl of Hanks balanced salt solution (1) at 37°C for
5 min. The digestion reactions were quenched with 2 µg of yeast tRNA
(Gibco Life Technologies, Gaithersburg, Md.) in 4 µl of 0.5 M EDTA,
immediately phenol-chloroform extracted, and precipitated. To determine
the spacings between hydrolysis products, 0.5 µg of radiolabeled T5
RNA was hydrolyzed in the presence of 25 mM sodium bicarbonate, 1 mM
EDTA, and 10 µg of yeast tRNA. This alkaline hydrolysis reaction
mixture was heated to 90°C for 10 min, neutralized with 2 µl of 0.5 M acetic acid, and ethanol precipitated. Hydrolysis products were
separated on a 6% denaturing polyacrylamide gel, and the digestion
patterns were analyzed with a PhosphorImager (Molecular Dynamics,
Sunnyvale, Calif.).
Cellular assays of RBE concatemers.
To determine if RBE
concatemers supported Rev function, the reporter plasmids were
cotransfected with a Rev expression plasmid, pRSVRev, and CAT
activities were assessed. One day prior to transfection, CV-1 cells
were counted with a Coulter (Hialeah, Fla.) cell counter, and 55,000 cells were seeded into each well of a 24-well cell culture plate. The
next day, the cells (at 70 to 80% confluency) were transiently
transfected in duplicate with either a Rev-responsive reporter plasmid
(pDM128; 0.2 µg) or derivatives of pDM138 containing RBE
concatemers (0.2 µg), pRSVbgal (0.5 µg) as a control
for transfection efficiency, and a Rev expression plasmid (pRSVRev).
The amount of pRSVRev used in each transfection was varied
between limiting pRSVRev (0.01 µg), saturating pRSVRev (0.2 µg), or
a titration of pRSVRev (0.002 µg to 0.2 µg). Cells were prepared
for transfection by preincubation for 30 min in 1 ml of OptiMEM
reduced-serum medium. Plasmid DNAs (1.2 µg in total, including
various amounts of a carrier plasmid, pUC118) in 100 µl of OptiMEM
were mixed with 1.5 µl of Lipofectamine in 100 µl of OptiMEM. Lipid
amalgams (200 µl) were incubated at ambient temperature for 30 min to
allow complex formation to occur; the mixture was added to cells in a
total volume of 0.5 ml of OptiMEM (7.2 ng of liposome:2.4 ng of DNA per
ml [final concentration]) and incubated for an additional 4.5 to
5 h at 37°C. The transfection medium was removed from the wells
and replaced with complete medium. Forty-eight hours posttransfection, the cells were washed with 1 ml of phosphate-buffered saline and lysed
in 0.25 M Tris-Cl (pH 7.6)-0.5% Triton X-100 (150 µl). Harvested cellular extracts (100 µl) were centrifuged for 5 min, and the supernatant was used for CAT assays and -galactosidase assays.
CAT activity was measured according to standard procedures
(
32). Cellular extracts (40 µl) were mixed with acetyl
coenzyme
A (0.45 mM [final concentration]; Pharmacia, Piscataway,
N.J.),
glycerol (1.8% final), and
14C-labeled
chloramphenicol (0.1 µCi; 50 mCi/mmol; DuPont NEN, Boston,
Mass.) in
176 µl of 0.14 M Tris-Cl (pH 7.6). The reaction mix
was incubated at
37°C for 1.5 h; control experiments indicated
that this was
within the linear range of the assay. Chloramphenicol
and less polar
acetylated products were ethyl acetate extracted
and separated by
thin-layer chromatography in 5% methanol-95%
chloroform on silica
gel IB2 sheets (J. T. Baker, Phillipsburg,
N.J.). The labeled
chloramphenicol products were quantitated with
a Phosphorimager
(Molecular Dynamics). The amount of CAT activity
present in each
extract was defined as the percentage of total
chloramphenicol that had
been converted to acetylated products.
CAT activities were normalized
to the amount of
-galactosidase
activity detected in each extract.
The
-galactosidase levels
were determined by incubating 15 µl of
extract with 0.1 mM MgCl
2,
0.35%
-mercaptoethanol
(Sigma, St. Louis, Mo.), and 0.88 mg of
o-nitrophenyl-
-
D-galactopyranoside (Sigma)
per ml in a final
volume of 150 µl of 0.1 M
NaH
2PO
4-Na
2HPO
4. After
incubation at
37°C for 45 min,
-galactosidase activities were
determined with
a microtiter plate reader (Cambridge Technology,
Cambridge, Mass.)
fitted with a 450-nm
filter.
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RESULTS AND DISCUSSION |
The structural context of polyvalent Rev decoys.
Rev decoys
have been developed as potential therapeutics to combat HIV infection.
The RBE mounted on a retroviral vector sequesters Rev and inhibits
viral replication (30, 31, 54, 55). Anti-Rev aptamers that
have higher affinities for Rev than the wild-type RBE have been
selected from random-sequence populations (2, 12, 51) and
have been shown to be efficient Rev decoys both in vitro and in vivo
(13, 22, 50).
To further improve the efficacies of Rev decoys, we wished to design
RNA molecules that would capture multiple Rev molecules.
The fact that
Rev responsiveness is potentiated by the accumulation
of multiple Rev
molecules on the RRE (
33,
36) suggested that
polyvalent
decoys might prove to be especially effective at blocking
viral
replication. Biochemical analyses have previously revealed
that 7 to 8 Rev molecules form a stable complex with the RRE in
vitro
(
6), and up to 11 to 12 Rev molecules may contribute
to full
Rev responsiveness in vivo (
38). Moreover, the accumulation
of Rev on the RRE is assisted by the tetramerization or oligomerization
of Rev monomers (
15,
36,
42,
57), and mutations in the
multimerization domain of Rev inhibit in vivo Rev function (
36,
57).
The simplest model for the design of polyvalent Rev decoys would be the
concatenation of multiple high-affinity Rev-binding
sites. In support
of this model, fusion proteins between Rev and
the bacteriophage MS2
coat protein directed transport of intron-containing
RNAs when multiple
MS2 phage operator sites were present (
39,
52). These
studies found that three to four MS2 operator sites
were required for
efficient Rev-dependent mRNA transport (
39).
Similarly,
Kjems and Sharp (
29) designed constructs that contained
one,
three, or six copies of RBEs. RNAs containing only one RBE
fostered no
Rev-dependent mRNA transport, while RNAs expressing
three copies of the
RBE had an intermediate Rev response (55%
of the wild-type RRE level),
and six copies produced an almost
full Rev response (74% of the
wild-type RRE level). A hexamer
RBE has also been shown to mediate
Rev-dependent suppression of
RNA splicing in vitro (
27).
However, a competing model suggested that the simple concatenation of
high-affinity Rev-binding sites might not direct the
accumulation of
multiple Rev molecules (
38,
58). In this model,
the
high-affinity RBE within stem IIB of the RRE binds the first
Rev
molecule, while the adjacent stem I, a long and imperfect
RNA duplex,
mediates the subsequent accumulation of additional
Rev molecules
(
23,
38,
58) (Fig.
1).
Nuclease protection
studies and gel shift analyses have shown that
initial Rev binding
to the high-affinity RBE is required for additional
binding to
adjacent, lower-affinity RNA duplexes (
38,
58).
Deletion analyses
originally revealed that the presence of stem I was
important
for Rev responsiveness (
35) and that truncation of
stem I reduces
the number of Rev molecules bound in vitro and Rev
responsiveness
in vivo (
38). Taken together, these results
support the notion
that coupled high- and low-affinity sites on the RRE
function
as a molecular rheostat that is sensitive to the concentration
of Rev in a cell (
38).
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FIG. 1.
The molecular rheostat model for the accumulation of Rev
on the RRE. One model for Rev function suggests that Rev initially
binds to the RBE whereas subsequent Revs oligomerize along an adjacent,
imperfectly paired duplex in stem I (38, 58). In this model,
the overall architecture of the RRE complex plays a critical role in
sequestering multiple Rev molecules. The RRE is drawn and numbered
according to Mann et al. (38). Rev protein molecules are
represented as oblongs; the actual number and alignment of Revs on the
RRE is unknown.
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While these two models are not of necessity mutually exclusive, Zemmel
et al. (
58) have argued that the RBE concatemers
constructed
by Kjems and Sharp (
29) (Fig.
2a) could be folded
into
an alternative structure in which one or more RBEs was adjacent
to a
long and imperfect RNA duplex (Fig.
2b). An energetic analysis
(
60) of the Kjems and Sharp (
29) RBE concatemers
suggests
that the alternative conformation predicted by Zemmel et al.
(
58)
was in fact the more favorable conformation. In this
view, increasing
numbers of RBEs promoted greater levels of mRNA
transport not
because multiple, high-affinity Rev-binding sites were
sequentially
introduced into a transcript but rather because a
proportionately
longer, albeit unplanned, stem structure was formed.
Before design
strategies for polyvalent RBEs could be established it
was first
necessary to resolve these disparate interpretations.
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FIG. 2.
Alternative structures of RBE concatemers. (a) Design of
an RBE hexamer by Kjems and Sharp (29). The desired
structure is shown. Rev protein molecules interacting with the internal
loop of the RBE are again represented as oblongs. (b) Predicted
secondary structure of the RBE hexamer. When the secondary structure of
the RBE hexamer shown in panel a is modeled with the program Mulfold
(24, 25, 60), an elongated RNA originally predicted by
Zemmel et al. (58) is observed.
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Concatenation of discrete RBEs into polyvalent Rev decoys.
A
minimal, 30-nucleotide, stem-internal loop-stem RBE has previously been
shown to interact with Rev monomers both in vitro (23, 28)
and in vivo (16, 35). We therefore attempted to link
together several RBEs to form polyvalent Rev decoys (Fig. 3a). The RBEs were topped with stable
tetraloops (UUCG [41]) that should have guided the
formation of discrete stem-internal loop-stem structures within the
cell, and they were flanked by pyrimidine spacers that should not have
allowed the formation of an extended helix similar to that shown in
Fig. 2b. An energetic analysis of the RBE concatemers suggested that
the structure shown in Fig. 3b was likely the most favorable
conformation.
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FIG. 3.
Design and construction RBE concatemers. (a) Design of
RBE concatemers. Individual RBEs were capped with a stable UUCG
tetraloop sequence and separated by nine-nucleotide pyrimidine linkers
(CUCUUCUCU). The tetramer T4 is shown. (b) Predicted
secondary structure of a RBE pentamer. When the secondary structure of
the RBE concatemer T5 (this report) is modeled with the program
Mulfold, a discrete series of unit-length RBE structures is observed.
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The length (>200 nucleotides) of the larger RBE concatemers prevented
their direct synthesis as a single oligonucleotide,
and a modular
synthetic scheme was instead devised. Individual
oligonucleotides
encoding RBE-linker monomers were synthesized.
The RBE monomers were
ligated to form concatemers, which were
then capped with
oligonucleotides that allowed PCR amplification;
one of the capping
oligonucleotides also contained a T7 RNA polymerase
promoter to further
biochemical analysis. Following amplification,
a ladder of bands
corresponding to concatemers of different lengths
was observed on an
agarose gel. The concatemer pool was cloned,
and concatemers T1, T2,
T3, T4, and T5 were recovered and sequenced.
To further buttress our
contention that the concatemers folded
into discrete Rev-binding sites,
the secondary structure of the
T5 concatemer was mapped in vitro (Fig.
4). The nuclease sensitivity
of T5 is
consistent with the formation of unit-length RBEs and
inconsistent with
the formation of an extended secondary structure.
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FIG. 4.
Mapping the structure of T5. The secondary structure of
T5 was mapped with RNase T1 and RNase A as described in
Materials and Methods. Lanes 1 to 5, digestion of T5 with decreasing
concentrations of RNase A; lanes 7 to 12, digestion of T5 with
decreasing concentrations of RNase T1; lanes 6 and 13, alkaline hydrolysis ladder of T5. A repeating pattern is observed with
both nucleases; elements of this pattern are indicated by arrows
stretching between the proposed secondary structure of the RBE
concatemer and the digests. The sizes and spacings of the digestion
products that make up this pattern are consistent with the formation of
unit-length RBEs that contain single-stranded pyrimidines (in the case
of RNase A) or single-stranded guanosines (in the case of RNase
T1). The digestion patterns are inconsistent with the
formation of a long, extended helix.
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The RBE concatemers were subcloned into pDM138, a well-characterized
Rev-dependent CAT reporter for use with tissue culture
cells (
17,
18,
21). Plasmid pDM138 is derived from pDM128
(Fig.
5a), which contains the RRE and a CAT
reporter gene within
an HIV-1 intron. In the absence of Rev, RNAs
transcribed from
pDM128 are spliced and negligible CAT activity is
observed. When
Rev is present and interacts with the RRE, Rev-mediated
mRNA transport
competes with splicing and CAT activity is detected.
Plasmid pDM138
contains a unique
ClaI site in place of the
RRE (Fig.
5b). By
placing the RBE concatemers into the pDM138 reporter,
constructs
that were superficially similar to pDM128 were formed. If
the
RBE concatemers can in fact bind multiple Rev molecules and thereby
be transported from the nucleus, then higher amounts of CAT activity
should be present in transfected cellular extracts in the presence
of
Rev than in its absence.
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FIG. 5.
Reporter plasmids. (a) Plasmid pDM128 (17,
18), which serves as a positive control and contains the
wild-type RRE within an HIV-1 intron adjacent to a CAT reporter gene.
SD, splice donor; SA, splice acceptor; LTR, long terminal repeat.
(b) Insertion of RBE concatemers into pDM138. Plasmid pDM138
(21), a derivative of pDM128, contains a unique
ClaI site in place of the RRE. RBE concatemers T1, T2,
T3, T4, and T5 were inserted into the ClaI site of pDM138.
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Optimization of assay conditions.
To determine how much
reporter plasmid should be transfected into cells to obtain a robust
Rev response, we first carried out a series of ranging experiments.
Plasmid pRSVRev (driver) expresses the Rev protein under the control of
the strong RSV promoter. Various amounts of the pRSVRev driver were
transfected into CV-1 African green monkey kidney cells along with the
parental reporter plasmid, pDM128. A plasmid that contained the
-galactosidase gene was transfected in parallel with the reporter
and driver plasmids as a control for transfection efficiencies
(2). At 48 h posttransfection, the cells were harvested
and levels of CAT activity were determined and normalized to levels of
-galactosidase activity. As shown in Fig.
6, when 0.2 µg of pDM128 is transfected into tissue culture cells the cotransfection of small amounts (e.g.,
0.01 µg) of pRSVRev leads to the production of small amounts of CAT,
while the cotransfection of large amounts of pRSVRev (e.g., 0.2 µg)
saturates the Rev-dependent CAT signal. These results are similar to
those previously observed in our lab with pDM128 and mutant derivatives
of pDM128 (50).
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FIG. 6.
Rev responsiveness as a function of pRSVRev
concentration. The relative activities of the wild-type RRE in pDM128
are shown as a function of the amount of pRSVRev included in
transfection experiments. CV-1 cells were transiently transfected with
constant amounts of pDM128 (0.2 µg) and constant amounts of pRSVbgal
(0.5 µg). The amounts of pRSVRev included in each transfection varied
(0.002, 0.01, 0.02, 0.05, and 0.2 µg). CAT activities (arbitrary
units) were determined and normalized to -galactosidase levels
(-gal) (arbitrary units). The results of eight separate
determinations (four repetitions in duplicate) were averaged. Bars
indicate standard deviations of the data.
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Polyvalent Rev decoys function as artificial RREs.
RBE
concatemers in pDM138 were cotransfected with pRSVRev, and CAT signals
were assayed at 48 h posttransfection. Since the Rev-dependent CAT
signal from the pDM128 reporter was readily observed and reproducible
in the presence of both limiting and saturating amounts of
cotransfected pRSVRev driver, the RBE concatemer reporter constructs
were assayed under both conditions. A representative sample of the data
is shown in Fig. 7; a graphical summary
of all of the data is shown in Fig. 8.
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FIG. 7.
RBE concatemers are Rev responsive. A representative
sample of results of CAT assays carried out with the RBE concatemer
series is shown. Lane 1, no-plasmid control; lanes 2 to 4, pDM128 plus
pRSVREV (positive control; triplicate determinations); lanes 5, 6, and
22, pDM138 plus pRSVRev (negative control); lanes 7 to 11, RBE
concatemers pDM138-T1 through pDM138-T5, respectively; lanes 12 to 21, RBE concatemers pDM138-T1 through pDM138-T5 plus pRSVRev, respectively
(duplicate determinations).
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FIG. 8.
Rev responsiveness of RBE concatemers at limiting and
saturating concentrations of pRSVRev. Graphical summaries of the data
from Fig. 4 and other determinations. CV-1 cells were transiently
transfected with constant amounts of RBE concatemer (0.2 µg),
pRSVbgal (0.5 µg), and limiting (0.01 µg) (a) or saturating (0.2 µg) (b) levels of pRSVRev. The CAT activities of RBE concatemers were
normalized to -galactosidase (gal) activities and to the CAT
activity of the positive control (pDM128 plus pRSVRev). Results of six
separate determinations (three repetitions in duplicate) were averaged.
Bars indicate standard deviations of the data.
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In the absence of Rev, the RBE concatemer reporters (pDM138-T1, -T2,
-T3, -T4, and -T5) produced only negligible levels of
CAT activity,
comparable to results for a no-transfection control
and to pDM138 in
the presence of Rev. However, when the Rev expression
plasmid was
included in the transfection experiment, substantially
greater amounts
of CAT activity were observed. The levels of Rev
responsiveness
increased in a graded fashion; each RBE that was
added to a concatemer
led to the production of additional CAT
activity. At limiting
concentrations of the driver plasmid, the
amount of Rev-dependent CAT
activity observed ranged from 1.3%
of wild-type activity (pDM128) for
pDM138-T1 to 51.5% of wild-type
activity for pDM138-T5 (background
activity subtracted relative
to Fig.
8). At saturating concentrations
of the driver plasmid,
the values ranged from 4.2% above background
for pDM138-T1 to
25.2% for pDM138-T5.
Rev responsiveness of artificial RREs as a function of Rev
concentration.
The experiments at limiting and saturating
concentrations of the Rev expression plasmid gave some indication of
how the artificial RREs differed from their natural counterparts. To
derive a fuller understanding of how the Rev responses of the
artificial and natural elements differed or overlapped, Rev titration
experiments similar to those originally carried out with pDM128 were
also carried out with pDM138-T2 and -T5 (Fig.
9). The RBE concatemer reporter pDM138-T2
was chosen rather than pDM138-T1 because the extremely small amounts of
signal produced by pDM138-T1 would have been difficult to follow at low
concentrations of pRSVRev. The amounts of RBE concatemer reporter
plasmids used for transfection were held constant (0.2 µg), while the
amounts of pRSVRev were varied from 0.002 to 0.2 µg.
View larger version (13K):
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|
FIG. 9.
Rev responsiveness of artificial RREs as a function of
pRSVRev concentration. The relative CAT activities of pDM138-T2 and
pDM138-T5 are shown as a function of Rev concentration. CV-1 cells were
transiently transfected with constant amounts of T2, T5, or pDM128 (0.2 µg), constant amounts of pRSVbgal (0.5 µg), and various amounts of
pRSVRev (0.002, 0.01, 0.02, 0.05, and 0.2 µg). CAT activities were
normalized to -galactosidase (-gal) levels. Results of eight
separate determinations (four repetitions in duplicate) were
averaged.
|
|
In consonance with the results shown in Fig.
8, the RBE concatemer
reporter pDM138-T5 was again more Rev responsive than pDM138-T2
and
elicited higher levels of Rev-dependent CAT activity. Interestingly,
the level of Rev-responsive mRNA transport exhibited by pDM138-T5
at
saturating concentrations of the driver plasmid was roughly
2.5 times
the level exhibited by pDM138-T2 at saturating concentrations
of the
driver plasmid. This result is consistent with a model
in which the
number of Rev-binding sites on a multivalent decoy
is the sole
determinant of the level of mRNA transport. However,
both
pDM138-T2 and pDM138-T5 reached maximal levels of Rev responsiveness
when approximately 0.01 µg of pRSVRev was added, while the RRE
did
not reach maximal levels of Rev responsiveness until approximately
0.05 µg of pRSVRev had been added (Fig.
6). The results presented
in Fig.
6,
8, and
9 indicate that there is not only a quantitative
difference
between the natural and artificial RREs but a qualitative
one as well.
While concatenated Rev-binding sites allow a transcript
to be
transported in a Rev-dependent fashion, they do not necessarily
respond
to Rev or accumulate Rev in the same manner as the natural
RRE
does.
Toward design principles for polyvalent decoys and artificial
RREs.
By eliminating heterologous (i.e., MS2) components (39,
52) and by clarifying the structures of the RNA substrates
(29), our studies are the first to fully delineate and
delimit Rev-RNA interactions in a functional (albeit artificial) RRE.
The graded increase in Rev-dependent mRNA transport activity observed
with increasing numbers of RBEs confirms the findings of Kjems and Sharp (29) and is consistent with the model in which
multiple, discrete Rev-RNA complexes can mediate Rev responsiveness.
The use of discrete Rev-binding sites greatly simplifies the analysis of where and how many Rev molecules are bound by a RNA molecule. For
example, Huang et al. (21) demonstrated that a single copy of an 88-nucleotide RRE fragment encompassing the RBE supported a
partial Rev response (26% of the wild-type RRE response), but it was
unclear how many Rev-binding sites may have been present on this
fragment. Based on our results, a similar level of Rev-dependent mRNA
transport would have required approximately five independent, high-affinity Rev-binding sites (see also Fig. 8). By simplifying the
structural context for the presentation of high-affinity Rev-binding sites, it should now be possible to directly compare the efficacies of
polyvalent Rev decoys in which the number, spacing, and affinities of
the binding sites are varied.
However, it should be noted that our results are also in accord with
the rheostat model for the accumulation of Rev on the
RRE, since
artificial RREs with either two or five RBEs saturated
at roughly the
same concentration of the driver plasmid, while
the natural RRE
saturated at a higher concentration. Thus, it
is likely that the
discrete-binding-site and rheostat models may
be simultaneously
correct. To accumulate sufficient Rev molecules
to mediate mRNA
transport, it is sufficient either to have multiple,
independent
high-affinity Rev-binding sites or to have multiple,
interdependent
high- and low-affinity Rev-binding sites. Thus,
in addition to varying
the placement and type of Rev decoys in
a polyvalent decoy, it would
likely be fruitful to explore whether
the inclusion of additional
secondary structural elements can
drive the cooperative accumulation of
even more Rev
molecules.
Overall, our results support the simple design principle that we
originally set out to assess, that simple concatenation of
Rev-binding sites can be used to generate polyvalent Rev decoys.
Most importantly, since polyvalent Rev decoys can function as
artificial RREs, they may be able to inhibit viral replication
by
additional mechanisms, such as binding cytoplasmic as well
as nuclear
Rev or competing for nuclear export
pathways.
| |
ACKNOWLEDGMENT |
This work was supported by Department of Health and Human
Services grant AI-36083 from the National Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Chemistry, ICMB A4800, University of Texas at Austin, Austin, TX 78712. Phone: (512) 471-6445. Fax: (512) 471-7014. E-mail:
andy.ellington{at}mail.utexas.edu.
| |
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Top
Abstract
Introduction
