International AIDS Society conference update. Compound targeting Rev protein promising. Orphaned approach still looking for home.

While clinicians and HIV/AIDS patients anxiously watch the trend of the virus developing resistance to multiple antiretroviral therapies, the question remains whether new drug research will continue to save the day. Some suggest there will need to be multiple new classes of antiretroviral drugs developed in order to stretch further the life span of longtime HIV patients. One potential new class would target the Rev protein, an approach that has received very little attention from the research and pharmaceutical communities.

Defective rev response element (RRE) and rev gene in HAART treated AIDS patients with discordance between viral load and CD4+ T-cell counts.

BACKGROUND: Highly Active Antiretroviral Therapy (HAART) has emerged as one the most effective method for treating AIDS patients. However, after variable period of treatment, many AIDS patients on HAART show elevated PCR viral load without a corresponding decline in CD4+ T-cells. Our earlier studies have revealed that the viruses present in the plasma of such patients are not infectious. OBJECTIVE: The aim of this study is to characterize the changes in the regulatory genes of HIV-1, namely tat, rev and rev response element (RRE) isolated from the plasma of such AIDS patients and to assess their role in role in affecting viral infectivity, hence its contribution, in the 'contradictory phenomenon' of high viral load and high CD4+ T-cell counts. STUDY DESIGN: The viral RNA was isolated from the plasma of HAART patients when they exhibited high plasma viral load and high CD4+ T-cell counts. The target regulatory genes were amplified by RT-PCR and sequenced. Sequences were also obtained from the proviral DNA from the peripheral blood mononuclear cells (PBMCs) of the study subjects. The sequences were compared with the wild type viral sequence to look for the changes induced in them due to HAART regime. RESULTS AND CONCLUSION: Our data revealed that RRE was missing in the viral particles isolated from the plasma of all study subjects. In two patients, the second exon of the rev gene was missing thereby leading to defective Rev protein. In another patients, Rev synthesis was prematurely stopped due to G135T substitution in the amino terminal domain. No such changes were observed in the corresponding proviral DNA. These changes are likely to result in the assembly of non-infectious virus due to lack of envelope proteins. Absence of RRE and Rev protein also leads to transport and packaging of multiply spliced transcripts into the virions instead of complete genomic RNA.

Inhibition of HIV-1 replication by cell-penetrating peptides binding Rev.

New therapeutic agents able to block HIV-1 replication are eagerly sought after to increase the possibilities of treatment of resistant viral strains. In this report, we describe a rational strategy to identify small peptide sequences owning the dual property of penetrating within lymphocytes and of binding to a protein target. Such sequences were identified for two important HIV-1 regulatory proteins, Tat and Rev. Their association to a stabilizing domain consisting of human small ubiquitin-related modifier-1 (SUMO-1) allowed the generation of small proteins named SUMO-1 heptapeptide protein transduction domain for binding Tat (SHPT) and SUMO-1 heptapeptide protein transduction domain for binding Rev (SHPR), which are stable and efficiently penetrate within primary lymphocytes. Analysis of the antiviral activity of these proteins showed that one SHPR is active in both primary lymphocytes and macrophages, whereas one SHPT is active only in the latter cells. These proteins may represent prototypes of new therapeutic agents targeting the crucial functions exerted by both viral regulatory factors.

Effects of 1,8-diaminooctane on the FIV Rev regulatory system.

Proper function of the Rev regulatory system is essential for the replication of lentiviruses, including feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV-1). Specifically, Rev affects the overall stability of viral mRNAs that encode necessary structural and enzymatic proteins. In turn, the eukaryotic initiation factor (eIF-5A) is indispensable for Rev function and is the only known protein whose biologically active form requires the unique amino acid, hypusine. Because 1,8-diaminooctane blocks the formation of hypusine by disrupting the cellular enzyme, deoxyhypusine synthase, thereby preventing activation of eIF-5A, we investigated the effects of 1,8-diaminooctane on posttranscriptional regulation. These are the first results to demonstrate that diaminooctane significantly reduced viral replication in a dose-dependent manner, even under conditions of contact inhibition, diminishing the compound's effect on cell proliferation. Similarly, the addition of increased concentrations of diaminooctane caused a reduction in the expression of a Rev-dependent CAT system without affecting a Rev-independent CAT system. At the RNA level, exposure of chronically infected CrFK cells to increasing concentrations of diaminooctane substantially decreased the levels of unspliced and singly spliced viral mRNAs and increased the relative amounts of multiply spliced transcripts in the cytoplasm. The findings of this study are the first demonstration that FIV, similar to HIV-1, requires eIF-5A for efficient Rev function and that small molecule intervention can indirectly target this lentivirus regulatory system.

Inhibition of the HIV-1 rev-RRE complex formation by unfused aromatic cations.

RNA viruses cause a wide range of human diseases. Development of new agents to target such viruses is an active area of research. Towards this goal, a series of diphenylfuran cations as potential inhibitors of the Rev-RRE complex have been designed and synthesized. Analysis of the interaction of the diphenylfurans with RRE and TAR RNA model systems by gel shift assays indicates that they exhibit both sequence and structure-dependent binding modes. Our results show a strong interaction between the diphenylfuran ring system and RRE bases, while the TAR interactions are much weaker with the compounds that are the best inhibitors of Rev-RRE.

Inhibition of acute-, latent-, and chronic-phase human immunodeficiency virus type 1 (HIV-1) replication by a bistriazoloacridone analog that selectively inhibits HIV-1 transcription.

Nanomolar concentrations of temacrazine (1,4-bis[3-(6-oxo-6H-v-triazolo[4,5,1-de]acridin-5-yl)amino-propyl ]piperazine) were discovered to inhibit acute human immunodeficiency virus type 1 (HIV-1) infections and suppress the production of virus from chronically and latently infected cells containing integrated proviral DNA. This bistriazoloacridone derivative exerted its mechanism of antiviral action through selective inhibition of HIV-1 transcription during the postintegrative phase of virus replication. Mechanistic studies revealed that temacrazine blocked HIV-1 RNA formation without interference with the transcription of cellular genes or with events associated with the HIV-1 Tat and Rev regulatory proteins. Although temacrazine inhibited the in vitro 3' processing and strand transfer activities of HIV-1 integrase, with a 50% inhibitory concentration of approximately 50 nM, no evidence of an inhibitory effect on the intracellular integration of proviral DNA into the cellular genome during the early phase of infection could be detected. Furthermore, temacrazine did not interfere with virus attachment or fusion to host cells or the enzymatic activities of HIV-1 reverse transcriptase or protease, and the compound was not directly virucidal. Demonstration of in vivo anti-HIV-1 activity by temacrazine identifies bistriazoloacridones as a new class of pharmaceuticals that selectively blocks HIV-1 transcription.

Inhibition of Rev·RRE complexation by triplex tethered oligonucleotide probes.

We have described a class of molecules, called tethered oligonucleotide probes (TOPs), that bind RNA on the basis of both sequence and structure. TOPs consist of two short oligonucleotides joined by a tether whose length and composition may be varied using chemical synthesis. In a triplex TOP, one oligonucleotide recognizes a short single-stranded region in a target RNA through the formation of Watson-Crick base pairs; the other oligonucleotide recognizes a short double-stranded region through the formation of Hoogsteen base pairs. Binding of triplex TOPs to an HIV-1 Rev Response Element RNA variant (RREAU) was measured by competition electrophoretic mobility shift analysis. Triplex TOP.RREAU stabilities ranged between -9.6 and -6.1 kcal mol-1 under physiological conditions of pH, salt, and temperature. Although the most stable triplex TOP.RREAU complex contained 12 contiguous U.AU triple helical base pairs, complexes containing only six or nine triple helical base pairs also formed. Triplex TOPs inhibited formation of the RRE.Rev complex with IC50 values that paralleled the dissociation constants of the analogous triplex TOP.RREAU complexes. In contrast to results obtained with TOPs that target two single-stranded RRE regions, inhibition of Rev.RREAU complexation by triplex TOPs did not require pre-incubation of RREAU and a TOP: triplex TOPs competed efficiently with Rev for RREAU and inhibited RREAU.Rev complexation at equilibrium.

Modulation of the Rev-RRE interaction by aromatic heterocyclic compounds.

The HIV-1 Rev protein regulates the nucleocytoplasmic distribution of viral precursor RNAs that encode HIV-1 structural proteins. Rev-mediated viral RNA expression requires a sequence-specific interaction between Rev and a viral RNA sequence, the Rev responsive element (RRE). Because the Rev-RRE interaction is essential for HIV-1 replication, anti-viral agents that selectively block this interaction may be effective anti-HIV-1 therapeutics. Here, we show that certain aromatic heterocyclic compounds, in particular, a tetracationic diphenylfuran, AK.A, can block binding of Rev to its high-affinity viral RNA binding site. AK.A abolishes Rev-RRE interactions at concentrations as low as 0.1 microM. Inhibition appears to be selective and results from competitive binding of the drug to a discrete region within the Rev binding site. Interestingly, the molecular basis for the AK.A-RNA interaction, as well as the mode of RNA binding differs from previously described aminoglycoside Rev inhibitors. Analysis of a variety of aromatic heterocyclic compounds and their derivatives reveals stereo-specific features required for the inhibition. Our results further demonstrate the feasibility of identifying and designing small molecules that selectively block viral RNA-protein interactions.

Human immunodeficiency virus type 1 Rev function requires continued synthesis of its target mRNA.

Synthesis of human immunodeficiency virus structural proteins is dependent on expression of the virus-encoded Rev protein due to the constitutive nuclear sequestration of mRNAs coding for the structural proteins. The pathway by which Rev, through interaction with the Rev-responsive element (RRE) within the mRNA, achieves export of the mRNA remains unclear. To probe the mechanism by which Rev induces nuclear export of its target mRNAs, the effect of inhibiting mRNA synthesis on the function of Rev was examined. Two approaches to address this issue were pursued: (i) the use of general transcription inhibitors such as 5,6-dichlorobenzimidazole riboside (DRB) and actinomycin D, and (ii) the more selective modulation of target gene transcription permitted by the use of a tetracycline-regulated promoter. Addition of either DRB or actinomycin D inhibited Rev action despite the presence of significant quantities of the target mRNA throughout the course of drug treatment. Furthermore, prolonged DRB treatment was found to improve rather than diminish the induction observed. Subsequent analysis using the tetracycline-modulated promoter demonstrated that Rev function was dependent on the transcription rate of the target mRNA and independent of target mRNA concentration. These data strongly indicate that Rev functions through interaction with newly synthesized target mRNA, facilitating its export by preventing its interaction with the host factors that effect nuclear sequestration.

Design and analysis of RNA structure-specific agents as potential antivirals.

A number of pathogenic RNA viruses, such as HIV-1, have extensive folded RNA conformations with imperfect A-form duplexes that are essential for virus function, and could serve as targets for structure-specific antiviral drugs. A method for the discovery of such drugs involves evaluation of the interactions with RNA of a wide variety of compounds that are known to bind to nucleic acids by different mechanisms. This approach has been initiated by using corresponding sequence RNA and DNA polymers as initial test systems for analysis of RNA binding strength and selectivity. Compounds that bind exclusively in the minor groove in AT sequences of DNA do not have significant interactions with RNA. Polycations, however, can show significant RNA affinity and binding selectivity, probably through complex formation in the RNA major groove. Some intercalators and a group of diphenylfuran cations have strong interactions with RNA that are very dependent on compound structure. RNA hairpin model systems for the RRE binding site of HIV-1 Rev protein were constructed for more detailed investigations. The diphenylfuran cations bind strongly to RRE and selectively inhibit Rev binding. CD, NMR, and fluorescence binding studies indicate that the active compounds bind in the internal loop region of RRE (with binding constants > 10(7)M-1), and cause a conformational change in the RNA. None of the standard nucleic acid binding modes appears to fit the results for complexes of the active compounds with RRE, and it is proposed that the diphenylfuran system threads through the internal loop region of RRE. Such a model allows contacts of the furan cationic substituents with both grooves of RRE in addition to the intercalation interactions with the bases.

Niruriside, a new HIV REV/RRE binding inhibitor from Phyllanthus niruri.

During the screening of natural products for their ability to inhibit the binding of HIV-REV protein to [33P]-labeled RRE RNA, one novel compound, niruriside (1), was isolated from the MeOH extract of the dried leaf of Phyllanthus niruri L. by bioassay-guided fractionation. The structure of niruriside was determined by spectroscopic methods. Niruriside showed specific inhibitory activity against the binding of REV protein to RRE RNA with an IC50 value of 3.3 microM; however, niruriside did not protect CEM-SS cells from acute HIV infection at concentrations up to 260 microM using an XTT dye reduction assay.

The roles of nucleolar structure and function in the subcellular location of the HIV-1 Rev protein.

The human immunodeficiency virus 1 (HIV-1) Rev transactivator protein plays a critical role in the regulation of expression of structural proteins by controlling the pathway of mRNA transport. The Rev protein is located predominantly in the nucleoli of HIV-1 infected or Rev-expressing cells. Previous studies demonstrated that the Rev protein forms a specific complex in vitro with protein B23 which is suggested to be a nucleolar receptor and/or carrier for the Rev protein. To study the role of the nucleolus and nucleolar proteins in Rev function, transfected COS-7 or transformed CMT3 cells expressing the Rev protein were examined for subcellular locations of Rev and other proteins using indirect immunofluorescence and immunoelectron microscopy. One day after transfection the Rev protein was found in most cells only in the nucleolar dense fibrillar and granular components where it colocalized with protein B23. These were designated class 1 cells. In a second class of cells Rev and B23 accumulated in the nucleoplasm as well as in nucleoli. Treatment of class 1 cells with actinomycin D (AMD) under conditions that blocked only RNA polymerase I transcription caused Rev to completely redistribute from nucleoli to the cytoplasm. Simultaneously, protein B23 was partially released from nucleoli, mostly into the nucleoplasm, with detectable amounts in the cytoplasm. In cells recovering from AMD treatment in the presence of cycloheximide Rev and B23 showed coincident relocation to nucleoli. Class 2 cells were resistant to AMD-induced Rev redistribution. Selective inhibition of RNA polymerase II transcription by alpha-amanitin or by DRB did not cause Rev to be released into the cytoplasm suggesting that active preribosomal RNA transcription is required for the nucleolar location of Rev. However, treatment with either of the latter two drugs at higher doses and for longer times caused partial disruption of nucleoli accompanied by translocation of the Rev protein to the cytoplasm. These results suggest that the nucleolar location of Rev depends on continuous preribosomal RNA transcription and a substantially intact nucleolar structure.

Nuclear export of the human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev is mediated by its activation domain and is blocked by transdominant negative mutants.

The human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev moves repeatedly between the cytoplasm, a perinuclear zone, the nucleoli, and nucleoplasmic speckles. In this study, we demonstrated by both indirect immunofluorescence and Western immunoblot analysis that nuclear exit of Rev transdominant negative mutants was defective compared with that of wild-type Rev. The basic and activation domains of Rev signal import and export, respectively, of Rev across the nuclear membrane. In cotransfection experiments, mutants containing mutations of Rev inhibited the nuclear egress of wild-type Rev, thus revealing a novel transdominant negative phenotype.

Inhibition of human immunodeficiency virus type 1 Rev-Rev-response element complex formation by complementary oligonucleotides.

Complementary 18-mer oligodeoxynucleotides (oligonucleotides) specifically inhibited the formation of human immunodeficiency virus Rev-Rev-response element (RRE) complexes. Inhibition of Rev-RRE binding required blockage of G-7819 to G-7820 in band shift assays. Structural studies revealed both local and distal effects. RRE structure was also disrupted by oligonucleotides targeted to other minor stems, by altering RNA renaturation conditions, or by reducing Rev concentrations--indicating a dynamic RRE structure and involvement of a minor RRE stem in the maturation of initial Rev-RRE complexes. Thus, complementary oligonucleotides alter RRE structure and may prove useful for the design of therapeutic anti-RRE oligonucleotides.