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Previous Article | Next Article 
Journal of Virology, May 1999, p. 4341-4349, Vol. 73, No. 5
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
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.
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.
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 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.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Polyvalent Rev Decoys Act as Artificial
Rev-Responsive Elements
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
References
-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.).
TABLE 1.
Oligonucleotides used
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.
-galactosidase activity detected in each extract.
The -galactosidase levels were determined by incubating 15 µl of
extract with 0.1 mM MgCl2, 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
NaH2PO4-Na2HPO4. 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.
| |
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).
|
|
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.
|
|
|
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).
|
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.
|
|
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.
|
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 |
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* 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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REFERENCES |
|---|
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Top
Abstract
Introduction
Materials and Methods
References
|
|---|
| 1. | Ausubel, F., R. Brent, R. Kingston, D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1997. Short protocols in molecular biology, 3rd ed. John Wiley & Sons, Inc., New York, N.Y. |
| 2. | Bartel, D. P., M. L. Zapp, M. R. Green, and J. W. Szostak. 1991. HIV-1 Rev regulation involves recognition of non-Watson-Crick base pairs in viral RNA. Cell 67:529-536[Medline]. |
| 3. | Bogerd, H. P., R. A. Fridell, S. Madore, and B. R. Cullen. 1995. Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins. Cell 82:485-494[Medline]. |
| 4. |
Cook, K. S.,
G. J. Fisk,
J. Hauber,
N. Usman,
T. J. Daly, and J. R. Rusche.
1991.
Characterization of HIV-1 Rev protein: binding stoichiometry and minimal RNA substrate.
Nucleic Acids Res.
19:1577-1583 |
| 5. | Cullen, B. R. 1991. Regulation of HIV-1 gene expression. FASEB J. 5:2361-2367[Abstract]. |
| 6. | Daly, T. J., R. C. Doten, P. Rennert, M. Auer, H. Jaksche, A. Donner, G. Fisk, and J. R. Rusche. 1993. Biochemical characterization of binding of multiple HIV-1 Rev monomeric proteins to the Rev responsive element. Biochemistry 32:10497-10505[Medline]. |
| 7. | Fischer, U., S. Meyer, M. Teufel, C. Heckel, R. Luhrmann, and G. Rautmann. 1994. Evidence that HIV-1 Rev directly promotes the nuclear export of unspliced RNA. EMBO J. 13:4105-4112[Medline]. |
| 8. | Fischer, U., J. Huber, W. C. Boelens, I. W. Mattaj, and R. Luhrmann. 1995. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82:475-483[Medline]. |
| 9. | Fornerod, M., M. Ohno, M. Yoshida, and I. W. Mattaj. 1997. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90:1051-1060[Medline]. |
| 10. | Fritz, C. C., M. L. Zapp, and M. R. Green. 1995. A human nucleoporin-like protein that specifically interacts with HIV Rev. Nature 376:530-533[Medline]. |
| 11. | Gerace, L. 1995. Nuclear export signals and the fast track to the cytoplasm. Cell 82:341-344[Medline]. |
| 12. |
Giver, L.,
D. Bartel,
M. Zapp,
A. Pawul,
M. Green, and A. D. Ellington.
1993.
Selective optimization of the Rev-binding element of HIV-1.
Nucleic Acids Res.
21:5509-5516 |
| 13. | Good, P. D., A. J. Krikos, S. X. L. Li, E. Bertrand, N. S. Lee, L. Giver, A. D. Ellington, J. A. Zaia, J. J. Rossi, and D. R. Engelke. 1997. Expression of small, therapeutic RNAs in human cell nuclei. Gene Ther. 4:45-54[Medline]. |
| 14. |
Hadzopoulou-Cladaras, M.,
B. Felber,
C. Cladaras,
A. Athanassopoulos,
A. Tse, and G. N. Pavlakis.
1989.
The Rev (Trs/Art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the Env region.
J. Virol.
63:1265-1274 |
| 15. |
Heaphy, S.,
J. T. Finch,
M. J. Gait,
J. Karn, and M. Singh.
1991.
Human immunodeficiency virus type 1 regulator of virion expression, rev, forms nucleoprotein filaments after binding to a purine-rich "bubble" located with the rev response region of viral mRNAs.
Proc. Natl. Acad. Sci. USA
88:7366-7370 |
| 16. |
Holland, S. M.,
M. Chavez,
S. Gerstberger, and S. Venkatesan.
1992.
A specific sequence with a bulged guanosine residue(s) in a stem-bulge-stem structure of Rev-responsive element RNA is required for trans activation by human immunodeficiency virus type 1 Rev.
J. Virol.
66:3699-3706 |
| 17. |
Hope, T. J.,
D. McDonald,
X. Huang,
J. Low, and T. G. Parslow.
1990.
Mutational analysis of the human immunodeficiency virus type 1 Rev transactivator: essential residues near the amino terminus.
J. Virol.
64:5360-5366 |
| 18. |
Hope, T. J.,
X. Huang,
D. McDonald, and T. G. Parslow.
1990.
Steroid-receptor fusion of the human immunodeficiency virus type 1 Rev transactivator: mapping cryptic functions of the arginine-rich motif.
Proc. Natl. Acad. Sci. USA
87:7787-7791 |
| 19. |
Hope, T. J.,
N. P. Klein,
M. E. Elder, and T. G. Parslow.
1992.
Trans-dominant inhibition of human immunodeficiency virus type 1 Rev occurs through formation of inactive protein complexes.
J. Virol.
66:1849-1855 |
| 20. | Hope, T. J. 1997. Viral RNA export. Chem. Biol. 4:335-344[Medline]. |
| 21. |
Huang, X.,
T. J. Hope,
B. L. Bond,
D. McDonald,
K. Gahl, and T. G. Parslow.
1991.
Minimal Rev-response element for type 1 human immunodeficiency virus.
J. Virol.
65:2131-2134 |
| 22. | Inouye, R. T., B. Du, D. Boldt-Houle, A. Ferrante, I.-W. Park, S. M. Hammer, L. Duan, J. E. Groopman, R. J. Pomerantz, and E. F. Terwilliger. 1997. Potent inhibition of human immunodeficiency virus type 1 in primary T cells and alveolar macrophages by a combination anti-Rev strategy delivered in an adeno-associated virus vector. J. Virol. 71:4071-4078[Abstract]. |
| 23. |
Iwai, S.,
C. Pritchard,
D. A. Mann,
J. Karn, and M. J. Gait.
1992.
Recognition of the high affinity binding site in rev-response element RNA by the human immunodeficiency virus type 1 rev protein.
Nucleic Acids Res.
20:6465-6472 |
| 24. |
Jaeger, J. A.,
D. H. Turner, and M. Zuker.
1989.
Improved predictions of secondary structures for RNA.
Proc. Natl. Acad. Sci. USA
86:7706-7710 |
| 25. | Jaeger, J. A., D. H. Turner, and M. Zuker. 1990. Predicting optimal and suboptimal secondary structure for RNA. Methods Enzymol. 183:281-306[Medline]. |
| 26. | Junker, U., K. Rittner, M. Homann, D. Bevec, E. Bohnlein, and G. Sczakiel. 1994. Reduction in replication of the human immunodeficiency virus type 1 in human T cell lines by polymerase III-drived transcription of chimeric tRNA-antisense RNA genes. Antisense Res. Dev. 4:165-172[Medline]. |
| 27. | Kjems, J., A. D. Frankel, and P. A. Sharp. 1991. Specific regulation of mRNA splicing in vitro by a peptide from HIV-1. Cell 67:169-178[Medline]. |
| 28. | Kjems, J., B. J. Calnan, A. D. Frankel, and P. A. Sharp. 1992. Specific binding of basic peptide from HIV-1 Rev. EMBO J. 11:1119-1129[Medline]. |
| 29. |
Kjems, J., and P. A. Sharp.
1993.
The basic domain of Rev from human immunodeficiency virus type 1 specifically blocks the entry of U4/U6-U5 small nuclear ribonucleoprotein in spliceosome assembly.
J. Virol.
67:4769-4776 |
| 30. |
Lee, S.-W.,
H. F. Gallardo,
E. Gilboa, and C. Smith.
1994.
Inhibition of human immunodeficiency virus type 1 in human T cells by a potent Rev response element decoy consisting of the 13-nucleotide minimal Rev-binding domain.
J. Virol.
68:8254-8264 |
| 31. | Lee, T. C., B. A. Sullenger, H. F. Gallardo, G. E. Ungers, and E. Gilboa. 1992. Overexpression of RRE-derived sequences inhibits HIV-1 replication in CEM cells. New Biol. 4:66-74[Medline]. |
| 32. | Lin, Y. S., and M. R. Green. 1989. Similarities between prokaryotic and eukaryotic cyclic AMP-responsive promoter elements. Nature 340:656-659[Medline]. |
| 33. | Malim, M. H., S. Bohnlein, J. Hauber, and B. R. Cullen. 1989. Functional dissection of the HIV-1 Rev trans-activator: derivation of a trans-dominant repressor of Rev function. Cell 58:205-214[Medline]. |
| 34. | Malim, M. H., J. Hauber, S.-Y. Le, J. V. Maizel, and B. R. Cullen. 1989. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 338:254-257[Medline]. |
| 35. | Malim, M. H., L. S. Tiley, D. F. McCarn, J. R. Rusche, J. Hauber, and B. R. Cullen. 1990. HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence. Cell 60:675-683[Medline]. |
| 36. | Malim, M. H., and B. R. Cullen. 1991. HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency. Cell 65:241-248[Medline]. |
| 37. |
Malim, M. H.,
D. F. McCarn,
L. S. Tiley, and B. R. Cullen.
1991.
Mutational definition of the human immunodeficiency virus type 1 Rev activation domain.
J. Virol.
65:4248-4254 |
| 38. | Mann, D. A., I. Mikaelian, R. W. Zemmel, S. M. Green, A. D. Lowe, T. Kimura, M. Singh, J. G. Butler, M. J. Gait, and J. Karn. 1994. Co-operative rev binding to stem I of the rev-response element modulates human immunodeficiency virus type 1 late gene expression. J. Mol. Biol. 241:193-207[Medline]. |
| 39. |
McDonald, D.,
T. J. Hope, and T. G. Parslow.
1992.
Posttranscriptional regulation by the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type 1 Rex proteins through a heterologous RNA binding site.
J. Virol.
66:7232-7238 |
| 40. |
Meyer, B. E., and M. H. Malim.
1994.
The HIV-1 Rev trans-activator shuttles between the nucleus and cytoplasm.
Genes Dev.
8:1538-1547 |
| 41. |
Molinaro, M., and I. J. Tinoco.
1995.
Use of ultra stable UNCG tetraloop hairpins to fold RNA structures: thermodynamic and spectroscopic applications.
Nucleic Acids Res.
23:3056-3063 |
| 42. |
Olsen, H. S.,
A. W. Cochrane,
P. J. Dillon,
C. M. Nalin, and C. A. Rosen.
1990.
Interaction of the human immunodeficiency virus type 1 Rev protein with a structured region in env mRNA is dependent on multimer formation mediated through a basic stretch of amino acids.
Genes Dev.
4:1357-1364 |
| 43. | Parslow, T. G. 1993. Post-transcriptional regulation of human retroviral gene expression, p. 101-136. In B. R. Cullen (ed.), Human retroviruses. Oxford University Press, New York, N.Y. |
| 44. |
Peterlin, B. M.,
P. A. Luciw,
P. J. Barr, and M. D. Walker.
1986.
Elevated levels of mRNA can account for the trans-activation of human immunodeficiency virus.
Proc. Natl. Acad. Sci. USA
83:9734-9738 |
| 45. | Richard, N., S. Iacampo, and A. Cochrane. 1994. HIV-1 Rev is capable of shuttling between the nucleus and cytoplasm. Virology 204:123-131[Medline]. |
| 46. |
Ruhl, M.,
M. Himmelspach,
G. M. Bahr,
F. Hammerschmid,
H. Jaksche,
B. Wolff,
H. Aschauer,
G. K. Farrington,
H. Probst,
D. Bevec, and J. Hauber.
1993.
Eukaryotic initiation factor 5A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating transactivation.
J. Cell Biol.
123:1309-1320 |
| 47. |
Schatz, O.,
M. Oft,
C. Dascher,
M. Schebesta,
O. Rosorius,
H. Jaksche,
M. Dobrovnik,
D. Bevec, and J. Hauber.
1998.
Interaction of the HIV-1 Rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5.
Proc. Natl. Acad. Sci. USA
95:1607-1612 |
| 48. |
Sczakiel, G.,
M. Oppenlander,
K. Rittner, and M. Pawlita.
1992.
Tat- and Rev-directed antisense RNA expression inhibits and abolishes replication of human immunodeficiency virus type 1: a temporal analysis.
J. Virol.
66:5576-5581 |
| 49. | Stutz, F., E. Izaurralde, I. W. Mattaj, and M. Rosbash. 1996. A role for nucleoporin FG repeat domains in export of human immunodeficiency virus type 1 Rev protein and RNA from the nucleus. Mol. Cell. Biol. 16:7144-7150[Abstract]. |
| 50. | Symensma, T. L., L. Giver, M. Zapp, G. B. Takle, and A. D. Ellington. 1996. RNA aptamers selected to bind human immunodeficiency virus type 1 Rev in vitro are Rev responsive in vivo. J. Virol. 70:179-187[Abstract]. |
| 51. | Tuerk, C., and S. MacDougal-Waugh. 1993. In vitro evolution of functional nucleic acids: high-affinity RNA ligands of HIV-1 proteins. Gene 137:33-39[Medline]. |
| 52. |
Venkatesan, S.,
S. M. Gerstberger,
H. Park,
S. M. Holland, and Y. S. Nam.
1992.
Human immunodeficiency virus type 1 Rev activation can be achieved without Rev responsive element RNA if Rev is directed to the target as a Rev/MS2 fusion protein which tethers the MS2 operator RNA.
J. Virol.
66:7469-7480 |
| 53. | Wen, W., J. L. Meinkoth, R. Y. Tsien, and S. S. Taylor. 1995. Identification of a signal for rapid export of proteins from the nucleus. Cell 82:463-473[Medline]. |
| 54. | Yamada, O., G. Kraus, L. Luznik, M. Yu, and F. Wong-Staal. 1996. A chimeric human immunodeficiency virus type 1 (HIV-1) minimal Rev response element-ribozyme molecule exhibits dual antiviral function and inhibits cell-cell transmission of HIV-1. J. Virol. 70:1596-1601[Abstract]. |
| 55. |
Yuyama, N.,
J. Ohkawa,
T. Koguma,
M. Shirai, and K. Taira.
1994.
A multifunctional expression vector for an anti-HIV-1 ribozyme that produces a 5'- and 3'-trimmed trans-acting ribozyme, targeted against HIV-1 RNA, and cis-acting ribozymes that are designed to bind to and thereby sequester trans-activator proteins such as Tat and Rev.
Nucleic Acids Res.
22:5060-5067 |
| 56. | Zapp, M. L., and M. R. Green. 1989. Sequence-specific RNA binding by the HIV-1 Rev protein. Nature 342:714-716[Medline]. |
| 57. |
Zapp, M. L.,
T. J. Hope,
T. G. Parslow, and M. R. Green.
1991.
Oligomerization and RNA binding domains of the type 1 human immunodeficiency virus Rev protein: a dual function for an arginine-rich binding motif.
Proc. Natl. Acad. Sci. USA
88:7734-7738 |
| 58. | Zemmel, R. W., A. C. Kelley, J. Karn, and P. J. G. Butler. 1996. Flexible regions of RNA structure facilitate co-operative rev assembly on the rev-response element. Mol. Biol. 258:763-777. |
| 59. | Zhou, C., I. C. Bahner, G. P. Larson, J. A. Zaia, J. J. Rossi, and D. B. Kohn. 1994. Inhibition of HIV-1 in human T-lymphocytes by retrovirally transduced anti-tat and rev hammerhead ribozymes. Gene 149:33-39[Medline]. |
| 60. |
Zuker, M.
1989.
On finding all suboptimal foldings of an RNA molecule.
Science
244:48-52 |
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