1. ABSTRACT The viral transactivator Rev is essential for HIV replication, since it allows the nuclear export of unspliced and partially spliced viral mRNAs that encode the structural proteins. Rev is an RNA binding protein that interacts with a highly structured RNA element, the RRE, found within the envelope sequences. This viral protein also interacts with cellular proteins, termed nucleoporins, and acts as an adaptor between the viral mRNAs and the cellular nuclear export machinery. Both interactions are specific, and required for Rev function. Because of its crucial role in the HIV replication cycle, and its novel mechanism of action, Rev represents an ideal target for therapeutic intervention. This review describes the efforts towards Rev inhibition. Gene therapy approaches, including the expression of trans-dominant mutants and RNA decoys, as well as antisense therapies and small molecule inhibitors of Rev-RRE binding or Rev interaction with the cellular machinery will be discussed.
2. INTRODUCTION HIV-1, the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS), displays a complex regulation of __________________________________________Received 6/2/97 Accepted 6/4/97
1 To whom correspondence should be addressed at: Oncogene Science, Inc., 106 Charles Lindbergh Blvd., Uniondale, NY 11553 Tel: 516-222-0023 Fax: 516-222-0114 E-mail: email@example.com
viral gene expression during its life cycle. Unlike many “simple” retroviruses (i.e. avian and murine leukemia viruses), which express only three viral genes, the genome of HIV-1 encodes nine genes whose expression patterns are tightly regulated during the HIV-1 replication cycle (Figure 1, for reviews see 1, 2, 3). In the infected host cell, HIV expresses over 20 distinct mRNA species (reviewed in 4). The early stage of regulation of the HIV-1 life cycle is marked by the appearance of the viral regulatory molecules Tat, Rev, and Nef, encoded by the fully spliced 2 kb class of viral mRNAs. The late viral life cycle gene expression is characterized by the cytoplasmic appearance of the 4 kb class of single spliced and 9.2 kb unspliced mRNAs, that encode the proteins required for the assembly of infectious virions. The viral transactivator Rev allows this transition into the late cycle (5, 6), and is therefore essential for viral replication. In effect, proviral mutants that do not express Rev fail to produce structural proteins and therefore cannot form new infectious viral particles (5, 7).
Because of its essential role in HIV replication, Rev constitutes an excellent target for therapeutic intervention. Its mode of action and specific interactions with its target RNA and cellular proteins have been extensively studied and elegantly elucidated, and this body of knowledge adds to the attractiveness of Rev as a target. The purpose of this article is to briefly review the latest developments on Rev, and how this knowledge can be used for development of anti-viral strategies, as well as t
Figure 1. Schematic representation of the HIV-1 genome. Gag-pol and Envelope (gp 160) are the classical retroviral proteins. Note the overlapping reading frames. In addition to these structural proteins and enzymes, HIV has a number of accessory proteins that have crucial functions in the replication cycle and pathogenesis: Tat, Rev, Nef, Vif, Vpr and Vpu. Rev is expressed very early in the life cycle, and is the product of a fully spliced mRNA.
review current efforts in this field.
3. REV STRUCTURE AND FUNCTION 3.1 Rev domains
Rev is a 116 amino acid RNA binding phosphoprotein that binds a cis-acting RNA regulatory element contained within the env mRNA, termed the Rev response element (RRE) (8, 9, 10, 11). Mutational analyses of Rev have revealed several discrete domains: i) an amino terminal domain that determines RRE binding and nuclear localization, ii) an oligomerization domain, flanking the RRE binding domain, and iii) a carboxy terminal domain that acts as a nuclear export signal (NES) and binding site for cellular proteins, known as the activation domain (Figure 2).
The arginine-rich motif, located between amino acids 35 and 50 in the Rev protein, is responsible for nuclear localization as well as for the sequence-specific interaction with the RRE (10, 12, 13, 14, 15). A 17 amino acid peptide from this highly basic domain has been shown by circular dichroism to form an a-helix that binds the RRE with the same affinity as the full-length protein (15). The sequences immediately adjacent to this basic domain are critical for Rev oligomerization, which is required for full activity in vivo (13, 16, 17, 18). Subsequent to binding, Rev monomers multimerize on the RRE, in a process mediated by both protein-protein interactions, and protein-RNA interaction (13, 19, 20). Cellular cofactors binding to the activation domain facilitate multimerization (21, 18).
In addition to the nuclear localization and RNA binding domain, a protein activation domain which is required to mediate Rev effector functions in vivo is located at the carboxy terminus (22-26). A leucine-rich region has been identified as the critical part of this domain, which is required for interaction with cellular protein(s) involved in the transport of HIV mRNAs (24, 25). This domain also acts as a nuclear export signal (NES) (27, 28, 29, 30). NES have been identified in Rev proteins from non-primate lentiviruses (30), as well as in
several cellular proteins: the inhibitor of cAMP-dependent protein kinase (PKI) (28), the fragile X mental retardation protein (FMRP) (31), and the amphibian transcription factor IIIA (32). Unlike the better known nuclear localization sequences (NLS), this domain contains critical hydrophobic residues (28), and like the NLS, all these peptide domains are functionally interchangeable (28, 30, 31, 32) and capable of directing the export of unrelated proteins (27, 28).
3.2 Rev-RRE interactions
Rev represents a paradigm for the arginine-rich family of RNA binding proteins, and one of the best studied. The target for Rev binding, the RRE, is a highly structured 234 nucleotide RNA that forms an array of stem-loops (33, 34, 35). It has been demonstrated that the Rev binding site is located in a 13-nucleotide bulge structure in stem-loop IIB, shown in Figure 3 (11, 13, 14, 19, 20, 36, 37) (Figure 3). The secondary and tertiary structure of the RRE has been deduced from the in vitro selection of randomized RREs (38,39) and variation of these sequences (40). Two purine-purine pairs within the internal bulge of stem-loop IIB have been identified (37, 39- 42). These non-canonical base pairs open the major groove of the A-form RNA double helix, making the bases more accessible to the arginine-rich, positively- charged Rev peptide (39, 41).
In addition to these in vitro studies, a genetic strategy has been used to isolate Rev “suppressor” mutations that alleviated the deleterious effects of mutations in stem-loop IIB of the RRE (43). Taken together, these studies suggested that the arginine-rich a-helix of Rev docked into the major groove of the RNA double helix in the bulge of stem-loop IIB. The three dimensional structure of the high affinity RRE site (stem-loop IIB) complexed with the arginine-rich Rev peptide has recently been determined by nuclear magnetic resonance (NMR) techniques (44, 45). These studies confirm the purine-purine base pairs, separated by a non-conserved residue in the bulge, that cause the backbone to twist in an S-shaped fold. As predicted, the major groove
Figure 2. Rev domains.The RRE binding domain (hatched box) is located between amino acid 35-50 in HIV-1 Rev, and is rich in arginine residues. This domain is flanked by sequences important for oligomerization (shaded). The black box represents the activation domain or nuclear export signal (NES). The sequences of other NES in related lentiviruses are shown in the insert.
is doubled in width, allowing the arginine-rich a-helix to fit, making contacts with the phosphate backbone and with purine residues (46).
3.3 Cellular factors that interact with Rev
Rev is functional in a wide variety of eukaryotic cells, including yeast, Drosophila, Xenopus oocytes, and mammals (25, 47, 48, 49). Thus, the Rev cellular cofactor(s) may be evolutionary conserved proteins, essential for the function of normal cells. Several cellular factors have been described to interact with Rev. The murine protein YL2 and its human homologue p32 were shown to interact with the basic domain of Rev, the same domain that interacts with the RRE and contributes to the oligomerization process (50, 51, 52). The p32 protein associates with ASF/SF2, an essential splicing factor (53), and is thought to function as a link to the cellular splicing machinery. Rev has been shown to recruit ASF/SF2 itself to the Rev-RRE complex in vitro, thus causing inhibition of splicing (52). However, ASF/SF2 is not specific for Rev, since it also binds to the basic RNA binding domain of Tat (54), and it does not bind to the activation or effector domain of Rev, which has been shown to be essential for Rev function (22-26).
Figure 3. Structure of the Rev Responsive element (RRE). The stem-loop structure of the HIV-1 RRE is depicted here. Stem-loop IIB (SLIIB) (shaded) contains the Rev binding site (RBE), shown within the box. The two non-canonical purine-purine base pairs are indicated.
The Rev leucine-rich effector domain was considered a more likely candidate for interaction with cellular factors specific for Rev function, since mutations in this domain (Figure 2) abrogate Rev function whether fused to its own RRE-binding domain or to heterologous RNA binding sequences (55). In further support of this model, non-functional Rev mutants in the activation domain which contain an intact RNA binding domain, exhibit a potent dominant-negative effect (12, 23, 25). At least two cellular proteins have been shown to bind to the activation domain of Rev: the eukaryotic initiation factor 5A (eIF-5A) (56), and a novel class of nuclear pore-associated proteins (57, 58, 59, 60). Although the role of eIF5A in mediating Rev function is not completely understood, it has been shown that non-functional mutants of this protein that still retain their ability to bind Rev inhibit Rev-mediated nuclear export, in yeast and in human T cells (61). A novel yeast cellular protein that is part of the nuclear pore complex, called Rip1p (59), and its mammalian homologue , hRIP/Rab (57, 58) were found to bind to the activation domain of Rev, as well as to that of HTLV-I Rex (58), as required for a true cofactor of HIV-1 Rev function, since Rev and Rex, together with the Rev proteins from other lentiviruses have functionally equivalent activation domains (58). The RIP/Rab protein contains a series of repeats containing the amino acids phenylalanine and glycine, known as FG repeats. These repeats are characteristic of a class of nuclear pore proteins called FG nucleoporins (62). Rev has also recently been shown to interact with multiple FG nucleoporins in yeast and in mammalian cells (60), and the ability of Rev mutants to interact with these proteins correlates with their ability to promote nuclear export of RNA (60). These cellular proteins are important in the nuclear export process, and they have been shown to bind other NES in cellular proteins, such as PKI (29, see Section 3.1).
3.4 Mechanism of action of Rev
Two main hypotheses have been proposed for the mechanism by which Rev causes the relocalization of unspliced or partially spliced viral mRNAs in the cytoplasm: 1. inhibition of some aspect of pre-mRNA splicing by Rev, leading to increased mRNA transport to the cytoplasm, and 2. direct effect of Rev to increase the nuclear export of pre-mRNA species.
Most of the evidence in favor of the role of Rev in inhibition of splicing was originated in in vitro experimental systems. These studies showed that inefficient splicing is a pre-requisite for Rev function and that Rev inhibits the splicing of RRE-containing introns (63, 64). An arginine-rich peptide from the NLS/RNA binding domain of Rev has been shown to block the entry of the essential U4/U6.U5 small ribonuclear protein complex in the spliceosome assembly in vitro (65). However, this block does not require the presence of the Rev activation domain, that has been shown to be essential for Rev function in vivo. In addition, it has not been demostrated that this in vitro-observed inhibition of splicing is required in vivo for Rev function.
Although both models are plausible and not necessarily mutually exclusive, a recent large body of data points to RNA export rather than splicing as the mechanism of action of Rev, and a direct effect of Rev on the cellular nuclear transport machinery has now been demonstrated (49, 57-60). Earlier evidence in support of a role of Rev in nuclear export stemmed from the fact that no incompletely spliced viral mRNAs are exported to the cytoplasm in the absence of Rev, in human T cells containing stably integrated proviruses (66). Moreover, a sequence from an unrelated retrovirus, the Mason-Pfizer monkey virus, was shown to enable Rev-independent HIV replication, possibly by interacting with a cellular factor that plays a role in mRNA transport analogous to that of the Rev protein (67). More recently, the simultaneous discovery of the nucleoporin RIP/Rab by three independent laboratories (57-59) as a cellular cofactor for Rev function confirmed that Rev plays a direct role in the nuclear export of pre-mRNAs. As described in Section 3.1, several cellular proteins have been shown to contain nuclear export signals (NES) functionally homologous to that of Rev (28, 31, 32). Taken together, this evidence indicates that Rev acts as an adaptor to allow RRE-containing viral mRNAs to access a pre-existing cellular export pathway (29).
4. Rev as target for therapeutic intervention
As described above, Rev function is essential for viral replication. No cellular homologs of Rev have been described so far. Several steps are required for Rev function: binding to the RRE, oligomerization of Rev monomers, and interaction with cellular factors from the nuclear transport machinery. Each of these steps provide potentially specific targets for therapeutic intervention, and the fact that the structural contacts for RRE binding, as well as the role of Rev in nuclear export and its interaction with cellular proteins are now well understood makes this protein a very attractive therapeutic opportunity for the treatment of HIV infection and AIDS. Even though Rev represents an excellent viral target, no anti-Rev compounds have yet entered clinical trials, although some clinical studies have been initiated using gene therapy approaches involving Rev (see 68 for review of these efforts). In this Section, I will review the results of gene therapy, antisense and drug discovery efforts focused on the Rev protein.
4.1 Rev as a target for gene therapy
The resistance of HIV to anti-viral drugs, especially in the early days of single-drug regimes, has prompted a search for alternative methods of therapy. One approach has been gene therapy, meaning the transfer of antiviral genes to infected cells. This strategy is based on the notion that these “therapeutic” genes will render target cells resistant to HIV replication. Gene therapy can be based on the expression of suppressor proteins, or on expression of anti-viral RNA or DNA molecules. Some excellent review articles on gene therapy of AIDS have been published in the past few years (68-71). An extensive review of the anti-HIV gene therapy approaches is beyond the scope of this article, and I will focus on strategies involving Rev.
4.1.1 Protein-based suppressors of Rev function
One of the most advanced protein-based approaches involves the Rev mutant M10, a trans-dominant negative mutant with amino acid substitutions at positions 78 and 79 in the NES/activation domain, that retains the ability to bind to the RRE and multimerize, but is unable to effects its role in transport of pre-mRNAs (25, 72). Because of its trans-dominant negative phenotype, the M10 protein inhibits HIV replication when expressed in stable cell lines (73-76). In HIV infected patients Rev M10-transduced T cells showed increased survival compared to T cells transduced with a vector expressing a deletion mutant of Rev M10 (77). High levels of M10 are required to inhibit viral replication in primary cells, and the choice of vectors is therefore critical to the success of gene therapy (78). A Phase I clinical trial taking into account these parameters has been initiated by Systemics, Inc. (Palo Alto, CA).
Another protein-based strategy that has been explored is expression of an anti-Rev single-chain antibody (79). This single-chain antibody, or SFv, was expressed from a construct consisting of both light and heavy chain variable regions of an anti-Rev monoclonal antibody. Intracellular expression of this SFv resulted in a level of inhibition of HIV replication comparable to that shown with the Rev M10 transdominant mutant (79). This antibody appears to sequester Rev in the cytoplasm, thus preventing it from exerting its function in nuclear transport.
4.1.2 Intracellular expression of RNA-based Rev inhibitors
A large portion of the anti-HIV gene therapy efforts is based on RNA-based suppressors of viral replication, like ribozymes and RNA decoys. Ribozymes are RNA molecules that can be engineered to cleave RNA at specific sites (80). Retroviral vectors expressing hammerhead ribozymes targeted against different regions of the HIV genome have been shown to inhibit viral replication in transduced cells (81-83). A hammerhead ribozyme targeting the common exon of the Tat and Rev genes has been shown to inhibit HIV replication in a human T cell line (84). Because ribozymes are extremely sequence specific, mutations in the virus would rapidly result in resistance. To address this concern, combination strategies with ribozymes that target different sites, or with ribozymes together with other antiviral genes, such as RNA decoys, have been proposed. In fact, a fusion molecule consisting of a ribozyme targeting the U5 region of the HIV LTR and an RNA decoy representing stem-loop IIB of the RRE, has been shown to be more efficient than ribozymes or RNA decoys alone (85, 86). The expression of antisense RRE decoys in retroviral vectors is also being explored as of potential therapeutic value (87-89). The effects of stable expression of antisense RNA targeting the Rev, Tat, and Vpu genes on viral replication has also been investigated, and showed to be of limited efficacy (90, 91).
Although gene transfer for the treatment of HIV infections is an attractive alternative or complement to the use of antiviral drugs, it is still not a reality, and many problems related to gene delivery and level of expression remain to be solved (69). More classical antiviral approaches, such as drug discovery, are being pursued, extending the efforts towards other viral targets, and one of these is Rev. The next Sections will describe antisense and drug screening targeting the Rev protein, that do not involve gene transfer.
4.2 Inhibiting Rev function via antisense oligonucleotides and other nucleic-acid molecules
The antisense RNA strategy was inspired by a naturally occurring mechanism of gene regulation in prokaryotes (92). The specificity of Watson-Crick base pairing made antisense molecules very attractive as potential therapeutic agents. A vast amount of literature exists on the application of this strategy to human diseases, including viral infections (reviewed in 93). Both unmodified and modified antisense oligonucleotides directed against various HIV RNA sequences have been shown to inhibit viral replication, both in a sequence-specific and in a non-sequence specific manner. A synthetic phosphorothioate oligodeoxynucleotide targeting Rev mRNA has been shown to have antiviral activity in chronically infected cells, inhibiting HIV replication by 80% at 25 mM (94), possibly through inhibition of translation. Since very early on, oligonucleotides complementary to the RRE sequences were shown to have the capability of disrupting Rev-RRE binding in vitro (95), several modified oligonucleotides targeting different stem-loops of the RRE have been tested for inhibition of viral replication (96, 97), and found to inhibit viral replication in a specific manner.
A novel nucleic acid-based approach towards inhibition of HIV infection by blocking Rev function has been the use of decoy RNA-DNA chimeric oligonucleotides containing the high affinity 13 nucleotide “bubble” structure of stem-loop IIB (see Figure 3) (98). These chimeric decoy bound the RRE with high affinity in vitro and were shown to inhibit HIV replication 40-70% at ~10 mM, using various assays (98).
In spite of the enthusiasm generated by the use of phosphorothioate oligonucleotides in the area of viral diseases, to date these strategies have met with limited success and significant issues remain in their potential use as therapeutic agents, including efficacy, cell permeability, delivery and cost. Because of the present limitations of both gene therapy and nucleic-acid-based antivirals, it is important that traditional approaches, such as screening for compounds with anti-Rev activity, are explored. The next Section will review the low molecular weight compounds and natural products that inhibit Rev.
4.3 Low-molecular weight compounds and natural products that inhibit Rev
Rev has been considered a promising target for therapeutic intervention of HIV infections since it was proven to be essential for HIV replication. The earliest attempts at intefering with its function were based on antisense technology, in the late 1980s (see previous Section). In the past few years, knowledge of the mechanism of action of Rev has increased rapidly, and it became clear that Rev offers several molecular targets for drug discovery. The very specific Rev-RRE interactions have been a preferred target for drug discovery, since it has no cellular counterpart. At the same time, other groups have focused on cell-based assays that would allow discovery of a drug that acts at the level of the interaction of Rev with the cellular transport machinery, as well as at the RNA-binding level. The next sections will describe the compounds and natural products that have been found to interfere with Rev function, as well as their potential usefulness as therapeutic agents. A list of these agents is presented in Table 1.
Table 1. Low molecular weight inhibitors of Rev function. The structure of the compounds described so far as Rev inhibitors is shown, as well as the molecular target and their effect on HIV replication assays.
Inhibition of HIV replication
Inhibition of nuclear export of Rev
4.3.1. Intercalating agents and other RNA-binding compounds
The first approaches towards anti-Rev drug discovery focused on the Rev-RRE interaction. Because Rev binds to an RNA target, intercalating agents with specificity or preference towards RNA were investigated
as potential Rev-RRE inhibitors. The intercalating dye pyronin Y was reported to completely block the formation of the Rev-RRE complex in vitro, at low mM concentrations (99). In agreement with previous reports that Rev-RRE binding is a pre-requisite for oligomerization (13, 19, 20) this intercalating agent also block the formation of multimeric complexes. Despite these strong in vitro effects, the dye failed to inhibit HIV replication in cytoprotection assays, in part because of its high levels of cellular toxicity (99). This result was not altogether surprising since pyronin Y is known to intercalate DNA in addition to RNA. Other intercalating agents, derivatives of diphenylfuran, were also reported to inhibit Rev-RRE interaction, by causing a conformational change in the RRE (100). Although these agents can be useful as probes to investigate the precise mechanism of Rev-RRE binding, intercalating agents are clearly not attractive molecules from a therapeutic point of view, because of their many toxic and mutagenic effects.
Non-intercalating compounds with previously known RNA binding properties were also candidates for inhibition of Rev-RRE binding. In this group of molecules, the aminoglycoside antibiotic neomycinB and some of its analogs were reported to disrupt Rev binding to the RRE in a specific manner (40, 101). Aminoglycoside antibiotics are known to act at the level of prokaryotic ribosomes, disrupting mRNA translation by binding to 16S RNA (102,103). In addition to binding to bacterial 16S RNA and to the RRE, these antibiotics have been also reported to interfere with splicing (104) and to bind to hammerhead ribozymes (104). The binding affinities of the aminoglycosides for their RNA targets are not very high, they are in the low mM range (40, 101, 104, 105), and therefore not surprisingly this binding has been shown to have a low degree of specificity or selectivity (106). Because of this, it is expected that a large number of cellular RNA molecules will bind to these compounds in the mM range. In fact, aminoglycoside antibiotics are known to be quite toxic to human (107). As with intercalating agents, these molecules are also not very interesting from a therapeutic point of view, due to toxicity and lack of specificity.
4.3.2 Screening approaches
A classical approach towards drug discovery has been the random screening of a vast number of synthetic organic compounds or fungal/plants extracts. This method of discovery, combined with the use of medicinal chemistry, has been very successful in discovering new activities resulting in the development of therapeutic agents. Not surprisingly, this approach has been utilized to discover compounds capable of inhibiting Rev function.
A small-molecule inhibitor of Rev was discovered at Sterling-Winthrop (now Sanofi-Winthrop), using a 96-well plate assay to measure Rev function in transfected cells (108). The assay measured production of the p24 protein from the HIV gag-pol gene as a result of Rev expression, in COS-1 cells. A series of structurally related compounds, 8-alkyl-2-(4-pyridyl)pyrido[2,3-d]pyrimidin-5(8H)-ones, were found to inhibit Rev-dependent p24 production with an IC50 in the low mM range. These compounds were also found to inhibit HIV-1 replication in a human T lymphoma line in the same concentration range. Because cytotoxicity was observed at concentrations of ~25 mM, these compounds are not likely to be of therapeutic use in their present form, although they could be considered leads for the design of less toxic, more potent derivatives. At the same time, this effort has provided proof that inhibitors of Rev can be found using classical screening approaches.
A screening of natural products using an in vitro Rev-RRE binding assay was carried out at Bristol-Myers Squibb. Three novel natural products, one from a plant and two from fungi, were discovered and isolated by bio-assay guided fractionation (109, 110). The plant metabolite, niruriside, was isolated from Phyllanthus niruri, a plant widely used in Indian traditional medicine. This compound was shown to inhibit binding of Rev to the RRE at an IC50 of 3 mM, while the IC50 on an unrelated protein-RNA binding system (the R17 coat protein/operator RNA) was greater than 130 mM (109). However , this compound did not protect CEM-SS cells from acute HIV infection (109). Likewise, the two fungal metabolites, harziphilone and fleephilone, from the fungus Trichoderma harzianum, were found to inhibit Rev-RRE binding by 50 % at 2-8 mM, but had no anti-HIV activity as tested in the cytoprotection assay (110). Because of this lack of antiviral activity, these natural products are not considered useful. It is not clear why these compounds failed to inhibit HIV replication: since they were discovered in an in vitro assay, it is therefore possible that these metabolites fail to enter the cell, are metabolized by it, or have masking cytotoxic effects. These concerns were not addressed by these publications.
In contrast with Rev-RRE binding approaches, or cell-based assays measuring Rev function, a recent effort to discover inhibitors of Rev has focused on nuclear export (111). Rev acts in conjunction with the cellular nuclear export machinery, and to function it needs to translocate from the nucleus to the cytoplasm (see Sections 3.3 and 3.4). Four antibiotics of the leptomycin-kazusamycin family were found to inhibit the export of Rev to the cytoplasm at nanomolar concentrations, in Rev-expressing HeLa cells treated with actinomycin D. Leptomycin B was found to be specific in its inhibition of the nuclear export pathway, while it had no effect on nuclear import processes (111). This antibiotic was also found to inhibit HIV-1 replication in primary human monocytes, with an IC50 of 0.6 nM (111). However, because of its long-term toxicity in tissue culture, leptomycin cannot be used therapeutically. Although this drug was shown to affect only Rev-dependent gene expression, it is possible that the transport of other cellular molecules (proteins or ribosomal or small nuclear RNAs) is also inhibited. The inhibition of a cellular pathway used by Rev could explain the toxic effects of this drug, and it raises the possibility that all Rev inhibitors that affect this Rev function will prove unsuitable as therapeutic agents. A greater knowledge of the nuclear export pathway used by different cellular protein and mRNA species will be necessary to evaluate this hypothesis.
Although none of the compounds discovered to inhibit Rev function is currently being pursued as potential drugs, it is important to point out that drug discovery is a laborious and sometimes slow process, and that Rev has only recently become a target for discovery and development. At Oncogene Science, Inc., we are carrying out high throughput screening seeking Rev inhibitors, using a cell-based assay similar to the one used at Sterling-Winthrop (108). This program is in the early phases, and we hope to contribute new entities with new activities.
5. CONCLUSIONS AND PERSPECTIVE
The recent excitement generated by the combination therapies using reverse trancriptase inhibitors and protease inhibitors has produced renewed interest in the biotechnology/pharmaceutical industry to search for new therapies targeting other viral proteins, such as integrase. Because of its crucial function in HIV replication, Rev represents an attractive target. Gene therapy and nucleic acid-based approaches have been the primary focus of both academic and industry researchers in this area. Although these novel therapies are very promising, they are still in early stages. The encouraging results of these approaches in tissue culture systems validate Rev as a target for anti-HIV intervention. Using more classical approaches, some Rev inhibitors have been discovered, although none of them appears to be likely to be developed into a therapeutic agents. All of these approaches are in early phases, and these first attempts represent proof-of-principle experiments indicating that Rev-RRE interactions can be disrupted by small molecules, and that Rev interactions with the cellular machinery of nuclear export are also a valid molecular target for drug discovery.
6. ACKNOWLEDGMENTS The author’s drug discovery research is partly supported by an Small Business Innovative Research (SBIR) grant from NIAID, “Inhibition of the HIV-1 Rev transactivator”, grant number 5 R44 AI34685-03
7. REFERENCES 1. Cullen, B. R. Regulation of human immunodeficiency virus replication.Annu. Rev. Microbiol. 45, 219-250 (1991)
2. Haseltine, W. A. Molecular biology of the human immunodeficiency virus type 1. FASEB 5, 2349-2360 (1991)
3. Vaishnav, Y. N. and Wong-Staal, F. The biochemistry of AIDS. Annu. Rev. Biochem. 60, 577-630 (1991)
4. Cullen, B. R. and Malim, M. H. The HIV 1 Rev protein: prototype of a novel class of eukaryotic post transcriptional regulators.TIBS 16, 346-350 (1991).
5. Malim, M. H., Hauber, J., Fenrick, R. and Cullen, B. R. Immunodeficiency virus rev trans activator modulates the expression of the viral regulatory genes. Nature 335, 181-183 (1988).
6. Emerman, M., Vazeux, R. and Peden, K. The rev gene product of the human immunodeficiency virus affects envelope specific RNA localization. Cell 57, 1155-1165 (1989).
7. Hadzopoulou-Cladaras, M., Felber, B. K., Cladaras, C., Athanassopoulus, A., Tse, A. and Pavlakis, G. 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 (1989).
8. Rosen, C., Terwilliger, E., Dayton, A., Sodroski, J. and Haseltine, W. Intragenic cis acting art gene responsive sequences of the human immunodeficiency virus. Proc.Natl. Acad. Sci. USA 85, 2071-2075 .
9. Zapp, M. L. and Green, M. R. Sequence-specific RNA binding by the HIV-1 Rev protein. Nature 342, 714-716 (1989).
10. Olsen, H. S., Nelbock, P., Cochrane, A. W. and Rosen, C. A. Seconary structure is the major determinant for interaction of HIV rev protein with RNA. Science 247, 845-848 (1990).
11. Cochrane, A. W., Chen, C. H. and Rosen, C. A. Specific interaction of the human immunodeficiency virus Rev protein with a structured region in the env mRNA. Proc. Natl. Acad. Sci. USA 87, 1198-1202 (1990).
12. Malim, M. H., Bohnlein, S., Hauber, J. and Cullen, B. R. Functional dissection of the HIV 1 Rev trans activator derivation of a trans dominant repressor of Rev function. Cell 58, 205-214 (1989).
13. Malim, M. H. and Cullen, B. R. 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 (1991).
14. Kjems, J., Calnan, B. J., Frankel, A. D. and Sharp, P. A. Specific binding of a basic peptide from HIV 1 Rev. EMBO J. 11, 1119-1129 (1992).
15. Tan, R., Chen, L., Buettner, J.A., Hudson, D., and Frankel, A.D. RNA recognition by an isolated alpha helix. Cell 73, 1031-1040 (1993).
16. Olsen, H. S., Cochrane, A. W., Dillon, P. J., Nalin, C. M. and Rosen, C. A. 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 (1990b).
17. Zapp, M. L., Hope, T. J., Parslow, T. G. and Green, M. R. 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 (1991).
18. Madore, S. J., Tiley, L. S., Malim, M.H. and Cullen B. R. Sequence requirements for Rev multimerization in vivo. Virology 202,186-194 (1994).
19. Cook, K.S., Fisk, G. J., Hauber, J., Usman, N., Daly, T.J. and Rusche, J. R. Characterization of HIV-1 REV protein: binding stoichimetry and minimal RNA substrate. Nucleic Acids Res 19, 1577-1583 (1991).
20. Heaphy, S., Finch, J. T., Gait, M.J., Karn, J. and Singh, M. Human immunodeficiency virus type 1 regulator of virion expression, rev, forms nucleoprotein filaments after binding to a purine-rich “bubble” located within the rev-responsive region of viral mRNAs. Proc. Natl. Acad. Sci. USA 88, 7366-7370 (1991).
21. Bogerd, H. P. and Greene, W. C. Dominant negative mutants of human T-cell leukemia virus type I Rex and human immunodeficiency virus type 1 Rev fail to multimerize in vivo. J. Virol. 67, 2496-2502 (1993).
22. Mermer, B., Felber, B. K., Campbell, M. and Pavlakis, G. N. Identification of trans-dominant HIV-1 rev protein mutatns by direct transfer of bacterially produced protein into human cells. Nucleic Acids Res. 18, 2037-2044 (1990).
23. Venkatesh, L.K., and Chinnadurai, G. Mutants in a converved region near the carboxy-terminus of HIV-1 Rev identify functionally important residues and exhibit a dominant negative phenotype. Virology 178, 327-330 (1990).
24. Hope, T. J. , Bond, B. L., McDonald, D., Klein, N. P. and Parslow, T. Effector domains of human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type 1 Rex are functionally interchangeable and share an essential peptide motif. J. Virol. 65, 6001-6007 (1991).
25. Malim, M. H., McCarn, D. F., Tiley, L. and Cullen, B. R. Mutational definition of the human immunodeficiency virus type 1 Rev activation domain. J. Virol. 65, 4248-4254 (1991).
26. Weischelbraun, I., Farrington, G. K., Rusche, J. R., Boehnlein, E. and Hauber, J. Definition of the human immunodeficiency virus type Rev and human T-cell leukemia virus type I Rex protein activation domain by functional exchange. J. Virol. 66,2583-2587 (1992).
27. Fischer, U., Huber, J., Boelens, W.C., Mattaj, I.W. and Lührmann, R. The HIV 1 Rev activation domains is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82,475-483 (1995).
28. Wen, W., Meinkoth, J.L., Tsien, R.Y. and Taylor, S.S. Identification of a signal for rapid export of proteins from the nucleus. Cell 82, 463-473 (1995).
29. Fridell, R.A., Bogerd, H. P. and Cullen, B. R. Nuclear export of late HIV 1 mRNAs occurs via a cellular protein export pathway. Proc. Natl. Acad. Sci. USA 93,4421-4424 (1996).
30. Meyer, B.E., Meinkoth, J.L. and Malim, M.H. Nuclear transport of human immunodeficiency virus type 1, visna virus, and equine infectious anemia virus Rev protein: identification of a family of transferable nuclear export signals. J. Virol. 70,2350-2359 (1996).
31. Fridell, R.A., Benson, R.E., Hua, J., Bogerd, H.P. and Cullen, B.R. A nuclear role for the Fragile X mental retardation protein. EMBO J. 15,5408-5414 (1996).
32. Fridell, R.A., Fischer, U., Lührmann, R., Meyer, B.E., Meinkoth, J.L., Malim, M.H., and Cullen, B. R. Amphibian transcription factor IIIA proteins contain a sequence element functionally equivalent to the nuclear export signal of human immunodeficiency virus type 1 Rev. Proc. Natl. Acad. Sci. USA 93, 2936-2940 (1996).
33. Dayton, E. T., Powell, D. M. and Dayton, A. I. Functional analysis of CAR, the target sequence for the Rev protein of HIV-1. Science 246, 1625-1629 (1989).
34. Heaphy, S., Dingwall, C., Ernberg, I., Gait, M. J., Green, S. M., Karn, J., Lowe, A. D., Singh, M. and Skinner, M. A. HIV-1 regulator of virion expression (Rev) protein binds to an RNA stem-loop structure located within the Rev response element region. Cell 60, 685-693 (1990).
35. Malim, M. H., Tiley, L. S., McCarn, D. F., Rusche, J. R., Hauber, J. and Cullen, B.R. HIV 1 structural gene expression requires binding of the Rev trans activator to its RNA target sequence. Cell 60, 675-683 (1990).
36. Tiley, L.S., Malim, M.H., Tewary, H.K., Stockley, P.G and Cullen, B.R. Identification of a high affinity RNA binding site for the human immunodeficiency virus type 1 Rev protein. Proc. Natl. Acad. Sci. USA 89, 758-762 (1992).
37. Bartel, D.P., Zapp, M.L., , Green, M.R. and Szostak, J.W. HIV-1 Rev regulation involves recognition of non-Watson-Crick base pairs in viral RNA. Cell 67,529-536 (1991).
38. Giver, L., Bartel, D.P., Zapp, M.L. and Ellington, D.A. Selection and design of high-affinity RNA ligands for HIV-1 Rev. Gene 137, 19-24 (1993).
39. Leclerc, F., Cedegren, R. and Ellington, A.D. A three-dimensional model of the Rev-binding element of HIV-1 derived from analyses of aptamers. Nature Struct. Biol. 1, 293-300 (1994).
40. Werstruck, G., Zapp, M.L. and Green, M.R. A non-canonical base pair withint the human immunodeficiency virus rev-responsive element is involved in both rev and small molecule recognition. Chem. Biol. 3, 129-137 (1996).
41. Battiste, J.L., Tan, R., Frankel, A.D. and Williamson, J.R. Assignment and modeling of the Rev response element RNA bound to a Rev peptide using 13C-heteronuclear NMR. J. Biomol. Nucl. Magn. Reson. 6,375-389 (1995).
42. Iwai, S., Pritchard, C., Mann, D.A., Karn, J. and Gait, M.J. Recognition of the high affinity binding site in rev-response element RNA by the human immunodeficiency virus type-1 rev protein. Nucl. Acids. Res. 20, 6465-6472 (1992).
43. Jain, C. and Belasco, J.G. A structural model for the HIV-1 Rev-RRE complex deduced from altered-specificity rev variants isolated by a rapid genetic strategy. Cell 87, 115-125 (1996).
44. Ye, X., Gorin, A., Ellington, A.D. and Patel, D.J. Deep penetration of an alpha-helix into a widened RNA major groove in the HIV-1 rev peptide-RNA aptamer complex. Nature Struct. Biol. 3, 1026-1033 (1996).
45. Battiste, J.L., Mao, H., Rao, N.S., Tan, R., Muhandiram, D.R., Kay, L.E., Frankel, A.D. and Williamson, J.R. Alpha helix-RNA major groove recognition in an HIV-1 rev peptide-RRE RNA complex. Science 273, 1547-1551 (1996).
46. Grate, D. and Wilson, C. Role REVersal: understanding how RRE RNA binds its peptide ligand. Structure 5 7-11 (1997).
47. Ivey-Hoyle, M. and Rosenberg, M Rev-dependent expression of human immunodeficiency virus type 1 gpl60 in Drosophila melanogaster cells. Mol. Cell. Biol. 10,6152-6159 (1990).
48. Fischer, U., Meyer, S., Teufel, M., Heckel, C., Lührmann, R. and Rautmann, G. Evidence that HIV-1 Rev directly promotes the nuclear export of unspliced RNA. EMBO J. 13,4105-4112 (1994).
49. Stutz, F. and Rosbash, M. A functional interaction between Rev and yeast pre-mRNA is related to splicing complex formation. EMBO J. 13,4096-4104 (1994).
50. Luo, Y., Yu, H. and Peterlin, B.M. Cellular protein modulates effects of human immunodeficiency virus type 1 Rev. J.Virol. 68, 3850-3856 (1994).
51. Tange, T.O., Jensen, T.H. and Kjems, J. In vitro interaction between human immunodeficiency virus type 1 Rev protein and splicing factor ASF/SF2-associated protein, p32. J. Biol. Chem. 271, 10066-10072 (1996).
52. Powell, D.M., Amaral, M.C., Yu, J.Y., Maniatis, T. and Greene, W.C. HIV Rev-dependent binding of SF2/ASF to the Rev response element: possible role in Rev-mediated inhibition of HIV RNA splicing. Proc. Natl. Acad. Sci. USA 94, 973-978 (1997).
53. Krainer, A.R., Mayeda, A., Kozak, D. and Binns, G. Functional expression of cloned human splicing factor SF2, homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators. Cell 66, 383-394 (1991).
54. Fridell, R.A., Harding, L.S., Bogerd, H.P. and Cullen, B.R. Identification of a novel human zinc finger protein that specifically interacts with the activation domain of lentiviral Tat proteins. Virology 209, 347-357 (1995).
55. McDonald, D., Hope, T.J. and Parslow, T.G. Posttranscriptional regulation by the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins through a heterologous RNA binding site. J. Virol. 66, 7232-7238 (1992).
56. Ruhl, M., Himmelspach, M., Bahr, G. M., Hammerschmid, F., Jaksche, H., Wolff, B., Aschauer, H., Farrington, G. K., Probst, H., Bevec, D. and Hauber, J. Eukaryotic initiation factor 5A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating trans-activaton. J. Cell Biol. 123, 1309-1320 (1993).
57. Fritz, C.C., Zapp, M.L. and Green, M.R. A human nucleoporin-like protein that specifically interacts with HIV Rev. Nature 376,530-533 (1995).
58. Bogerd, H. P., Fridell, R. A., Madore, S. and Cullen, B. R. Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins. Cell 82,485-494 (1995).
59. Stutz, F., Neville, M. and Rosbash, M. Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast. Cell 82,495-506 (1995).
60. Stutz, F., Izaurralde, E., Mattaj, I.W. and Rosbash, M. 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 (1996).
61. Bevec, D., Jaksche, H., Oft, M., Wöhl, T., Himmelspach, M., Pacher, A., Schebesta, M., Koettnitz, K., Dobrovnik, M., Csonga, R., Lottspeich, F. Hauber, J. Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A. Science 271, 1858-1860 (1996).
62. Rout, M.P. and Wente, S. R. Trends Cell Biol. 4, 357-365 (1994).
63. Chang, D.D. and Sharp, P.A. Regulation by HIV Rev depends upon recognition of splice sites. Cell 59, 789-795 (1989).
64. Kjems, J., Frankel, A. D. and Sharp, P. A. Specific regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev. Cell 67, 169-178 (1991).
65. Kjems, J. and Sharp. P.A. The basic domain of Rev from human immunodeficiency virus type 1 specifically blocks the netry of U4/U6.U5 small nuclear ribonucleoprotein in spliceosome assembly. J. Virol. 67, 4769-4776 (1993).
66. Malim, M. H. and Cullen, B. R. Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins. Mol. Cell. Biol. 13, 6180-6189 (1993).
67. Bray, M., Prasad, S., Dubay, J. W., Hunter, E., Jeang, K.-T., Rekosh, D. and Hammarskjold, M.-L. A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent. Proc. Nat. Acad. Sci. USA 91, 1256-1260 (1994).
68. Bridges, S.H. and Sarver, N. Gene therapy and immuno restoration for HIV disease. The Lancet 345, 427-432 (1995).
69. Yu, M., Poeschla, E., and Wong-Staal, F. Progress towards gene therapy for HIV infection. Gene Ther. 1, 13-26 (1994).
70. Dropulic, B. and Jeang, K.-T. Gene therapy for human immunodeficiency virus infection: genetic antiviral strategies and targets for intervention. Hum.Gene Ther. 5, 927-939 (1994).
71. Lever, A.M.L. Gene therapy for HIV infection. Brit. Med. Bull. 51, 149-166 (1995).
72. Stauber, R., Gaitinaris, G. A., and Pavlakis, G. N. Analysis of trafficking of Rev and transdominant Rev proteins in living cells using green fluorescent protein fusions: transdominant Rev blocks the export of Rev from the nucleus to the cytoplasm. Virology 213, 439-449 (1995).
73. Malim, M.H., Freimuth, W.W., Liu, J., Boyle, T.J., Lyerle, H.K., Cullen, B.R. and Nabel, G.J. Identification of a novel cellular cofactor for the Rev/Rex class of
retroviral regulatory proteins. J. Exp. Med. 176, 1197-1201 (1992).
74. Bevec, D., Dobrovnik, M., Hauber, J. and Böhnlein, E. Inhibition of human immunodeficiency virus type 1 replication in human T cells by retroviral-mediated gene transfer of a dominant-negative Rev trans-activator. Proc. Natl. Acad. Sci. USA 89, 9870-9874 (1992).
75. Bahner, I, Zhou, C., Yu, X.-J., Hao, Q.-L., Guatelli, J. C. and Kohn, D.B. Comparison of trans-dominant inhibitory mutant human immuodeficiency virus type 1 genes expressed in retroviral vectors in human T lymphocytes. J. Virol. 67, 3199-3207 (1993).
76. Escaich, S., Kalfoglou, C., Plavec, I, Kaushal, S., Mosca, J.D. and Böhnlein, E. Hum. Gene Ther. 6,625-634 (1995).
77. Woffendin, C., Ranga, U., Yang, Z.-Y., Xu, L. and Nabel, G.J. Expression of a protective gene-prolongs survival of T cells in human immunodeficiency virus-infected patients. Proc. Natl. Acad. Sci. USA 93, 2889-2894 (1996).
78. Plavec, I., Agarwal, M., Ho, K.E., Pineda, M., Auten, J., Baker, J., Matsuzaki, H., Escaich, S., Bonyhadi, M. and Böhnlein, E. High transdominant RevM10 protein levels are required to inhibit HIV-1 replication in cell lines and primary T cells: implication for gene therapy of AIDS. Gene Ther. 4, 128-139 (1997).
79. Duan, L., Bagasra, O., Laughlin, M.A., Oakes, J.W. and Pomerantz, R.J. Potent inhibitor of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody. Proc. Natl. Acad. Sci. USA 91, 5075-5079 (1994).
80. Cech, T.R., Bass, B.L. Biological catalysis by RNA. Annu. Rev. Biochem. 55, 599-629 (1986).
81. Sarver, N., Cantin, E.M., Chang, P.S., Zaia, J. A., Ladne, P.A., Stephens, D.A. and Rossi, J.J. Ribozymes as potential anti-HIV-1 therapeutic agents. Science 247, 1222-1225 (1990).
82. Weerasinghe, M., Liem, S.E., Asad, S., Read, S.E. and Joshi, S. Resistance to human immunodeficiency virus type (HIV-1) infection in human CD4+ lymphocyte-derived cell lines conferred by using retroviral vectors expressing an HIV-1 RNA-specific ribozyme. J. Virol. 65, 5531-5534 (1991).
83. Dropulic, B., Lin, N.H., Martin, M.A. and Jeang, K.-T. Functional characterization of a U5 ribozyme: intracellular suppression of human immunodeficiency virus type 1 expression. J. Virol. 66, 1432-1441 (1992).
84. Zhou, C., Bahner, I, Larson, G., Zaia, J.A., Rossi, J. J. and Kohn, D.B. Inhibition of HIV-1 in human T-lymphocytes by retrovirally transduced anti-tat and rev hammerhead ribozymes. Gene 149, 33-39 (1994).
85. Yamada, O., Kraus, G., Luznik, L., Yu, M. and Wong-Staal, F. A chineric human immunodeficiency virus type 1 (HIV-1) minimal Rev response element-ribozyme molecular exhibits dual antiviral function and inhibits cell-cell transmission of HIV-1. J. Virol. 70, 1596-1601 (1996).
86. Gervaix, A., Li, X., Kraus, G. and Wong-Staal, F. Multigene antiviral vectors inhibit diverse human immunodeficiency virus type 1 clades. J. Virol. 71, 3048-3053 (1997).
87. Graham, G.J. and Maio, J.J. RNA transcripts of the human immunodeficiency virus transactivation response element can inhibit action of the viral transactivator. Proc. Natl. Acad. Sci. USA 87, 5817-5821 (1990).
88. Lee, T.C., Sullenger, B.A., Gallardo, H.F., Ungers, G.E. and Gilboa, E. Overexpression of RRE-derived sequences inhibits HIV-1 replication in CEM cells. New Biol. 4, 66-74 (1992).
89. Kim, J.H., McLinden, R.J., Mosca, J.D., Vahey, M.T., Greene, W.C. and Redfield, R.R. Inhibition of HIV replication by sense and antisense rev response elements in HIV-based retroviral vectors. J. Acquir. Immun. Defic. Syndr. Hum. Retrovir. 12, 343-351 (1996).
90. Rhodes, A. and James, W. Inhibition of human immunodeficiency virus replication in cell culture by endogenously synthesized antisense RNA. J. Gen. Virol. 71, 1965-1974 (1990).
91. Rhodes, A. and James, W. Inhibition of heterologous strains of HIV by antisense RNA. AIDS 5,145-151 (1991).
92. Green, P.J., Pines, O. and Inouye, M. The role of antisense RNA in gene regulation. Annu. Rev. Biochem. 55,569-597 (1986).
93. Stein, C.A. and Cheng, Y.C. Antisense oligonucleotides as therapeutic agents – is the bullet really magical? Science 261, 1004-1012 (1993).
94. Matsukara, M., Zon, G., Shinozuka, M., Robert-Guroff, T., Shimable, C., Stein, H., Mitsuya, H., Wong-Staal, F., Cohen, J.S. and Broder, S. Regulation of viral expression of human immunodeficiency virus in vitro by an antisense phosphorothioate oligodeoxynucleotide against rev (art/trs) in chronically infected cells. Proc. Natl. Acad. Sci. USA 86,4244-4248 (1989).
95. Chin, D.J. Inhibition of human immunodeficiency virus type 1 rev-rev-response element complex formation by complementary oligonucleotides. J. Virol. 66 ,600-607 (1992).
96. Li, G., Lisziewicz, Sun, D., Zon, G., Daefler, S., Wong-Staal, F., Gallo, R.C. and Klotman, M.E. Inhibition of Rev activity and human immunodeficiency virus type 1 replication by antisense oligdeoxynycleotide phosphorothioate analogs directed against the Rev-responsive element. J. Virol. 6882-6888 (1993).
97. Fenster, S.D., Wagner, R.W., Froehler, B.C. and Chin, D.J. Inhibition of human immunodeficiency virus type-1 env expression by C-5 propyne oligonucleotides specific for Rev-response element stem-loop V. Biochemistry 33, 8391-8398 (1994).
98. Nakaya, T., Iwai, S., Fujinaga, K., Sato, Y., Otsuka, E. and Ikuta, K. Decoy approach using RNA-DNA chimera oligonucleotides to inhibit the regulatory function of human immunodeficiency virus type 1 Rev protein. Antimicrob. Agents Chemother. 41, 319-325 (1997)
99. Schroder, H.C., Ushijima, H., Bek, A., Merz, H., Pfeifer, P., Muller, W. E. G. Inhibition of formation of Rev-RRE complex by pyronin Y. Antiviral Chem. Chemother 4, 103-109 (1993).
100. Ratmeyer, L., Zapp, M.L., green, M.R., Vinayak, R., Kumar, A., Boykin, D.W. and Wilson, W.D. Inhibition of HIV-1 Rev-RRE interaction by diphenylfuran derivatives. Biochemistry 35, 13689-13696 (1996).
101. Zapp, M. L., Stern, S. and Green, M. R. Small molecules that selectively block RNA binding of HIV-1 Rev protein inhibit Rev function and viral production. Cell 74, 969-978 (1993).
102. Woodcock, J., Moazed, D., Cannon, M., Davis, J. and Noller, H. F. Interaction of antibiotics with A- and P-site-specific bases in 16S ribosomal RNA. EMBO J. 10, 3099-3103 (1991).
103. Purohit, P., and Stern, S. Interactions of a small RNA with antibiotic and RNA ligands of the 30S subunit. Nature 370, 659-662 (1994).
104. von Ahsen, U., Davis, J., and Schroeder, R. Antibiotic inhibition of group I ribozyme function. Nature 353, 368-370 (1991).
105. Stage, T.K., Hertel, K.J. and Uhlenbeck, O.C. Inhibition of the hammerhead ribozyme by neomycin. RNA 1,95-101 (1995).
106. Wang, Y., Hamasaki, K. and Rando, R.R. Specificity of aminoglycoside binding to RNA constructs from the 16S rRNA decoding region and the HIV-RRE activator region. Biochemistry 36, 768-779 (1997).
107. Chambers, H.F. and Sanders, M.A. Antimicrobial agents: the aminoglycosides . In Goodman & Gilman’s The Pharmacological Basis of Therapeutics. Eds.: Hardman, J. G., Limbird, L.E., Molinoff, P.B., Ruddon, R.W. and Gilman, A.G. McGraw-Hill, NY (1996).
108. Ciccarelli, R. B.; Winter, L. A., Lorenz, R., Harris, A., Crawford, a. C., Bailey, T. R., Singh, B., Hammarskjold, M.-L., Rekosh, D. and Hughes, J. V. Inhibition of the cellular Rex response and HIV-1 replication by 8-Alkyl-2-(4-pyridyl)pyrido[2,3-d] pyrimidin-5(8H)-ones. Antiviral Chem. Chemother. 5, 169-175 (1994).
109. Qian-Cutrone, J., Huang, S., Trimble, J., Li, H., Lin, P.-F., Alam, M., Klohr, S. E. and Kadow, K.F. Niruriside, a new HIV REV/RRE binding inhibitor from Phyllanthus niruri. J. Nat. Prod. 59, 196-199 (1996).
110. Qian-Cutrone, J., Huang, S., Chang, L.-P., Pirnik, D.M., Klohr, S.E., Dalterio, R.A., Hugill, R., Lowe, S., Alam, M. and Kadow, K.F. Harziphilone and fleephilone, two new HIV REV/RRE binding inhibitors produced by Trichoderma harzianum. J. Antibiotics 49, 990-997 (1996).
111. Wolff, B., Sanglier, J.-J. and Wang, J. Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. Chem. Biol. 4, 139-147 (1997).