CBP80/20-dependent translation initiation factor (CTIF) inhibits HIV-1 Gag synthesis by targeting the function of the viral protein Rev.

Translation initiation of the human immunodeficiency virus type-1 (HIV-1) full-length RNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the full-length RNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF inhibits HIV-1 and HIV-2 Gag synthesis from the full-length RNA. Our results indicate that CTIF associates with HIV-1 Rev through its N-terminal domain and is recruited onto the full-length RNA ribonucleoprotein complex in order to interfere with Gag synthesis. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that Rev interferes with the association of CTIF with CBP80 indicating that CTIF and Rev compete for the CBC subunit.

A small-molecule inhibitor of HIV-1 Rev function detected by a diversity screen based on RRE-Rev interference.

The Rev protein of HIV-1 binds to the Rev Recognition Element (RRE) in the virus RNA to promote nuclear export of unspliced and partially spliced transcripts, an essential step in the virus transmission cycle. Here, we describe the screening of a library of chemically diverse compounds with an assay based on monitoring the interaction between the RNA-binding α-helix of Rev and its high-affinity binding site in the RRE. This screen allowed the identification of a benzofluorenone compound that inhibited the formation of the full-length RRE-Rev ribonucleoprotein by associating to the RRE, and blocked HIV-1 transcription and Rev action in cells. This molecule, previously studied as a cytostatic agent, had substantial antiretroviral activity. Together with other screening hits, it provides a new chemical scaffold for the development of antiretroviral agents based on blockage of HIV-1 RNA biogenesis.

Structure of an RNA Aptamer that Can Inhibit HIV-1 by Blocking Rev-Cognate RNA (RRE) Binding and Rev-Rev Association.

HIV-1 Rev protein mediates nuclear export of unspliced and partially spliced viral RNAs for production of viral genomes and structural proteins. Rev assembles on a 351-nt Rev response element (RRE) within viral transcripts and recruits host export machinery. Small (<40-nt) RNA aptamers that compete with the RRE for Rev binding inhibit HIV-1 viral replication. We determined the X-ray crystal structure of a potential anti-HIV-1 aptamer that binds Rev with high affinity (Kd = 5.9 nM). The aptamer is structurally similar to the RRE high-affinity site but forms additional contacts with Rev unique to its sequence. Exposed bases of the aptamer interleave with the guanidinium groups of two arginines of Rev, forming stacking interactions and hydrogen bonds. The aptamer also obstructs an oligomerization interface of Rev, blocking Rev self-assembly. We propose that this aptamer can inhibit HIV-1 replication by interfering with Rev-RRE, Rev-Rev, and possibly Rev-host protein interactions.

Identification of small molecule modulators of HIV-1 Tat and Rev protein accumulation.

BACKGROUND: HIV-1 replication is critically dependent upon controlled processing of its RNA and the activities provided by its encoded regulatory factors Tat and Rev. A screen of small molecule modulators of RNA processing identified several which inhibited virus gene expression, affecting both relative abundance of specific HIV-1 RNAs and the levels of Tat and Rev proteins. RESULTS: The screen for small molecules modulators of HIV-1 gene expression at the post-transcriptional level identified three (a pyrimidin-7-amine, biphenylcarboxamide, and benzohydrazide, designated 791, 833, and 892, respectively) that not only reduce expression of HIV-1 Gag and Env and alter the accumulation of viral RNAs, but also dramatically decrease Tat and Rev levels. Analyses of viral RNA levels by qRTPCR and RTPCR indicated that the loss of either protein could not be attributed to changes in abundance of the mRNAs encoding these factors. However, addition of the proteasome inhibitor MG132 did result in significant restoration of Tat expression, indicating that the compounds are affecting Tat synthesis and/or degradation. Tests in the context of replicating HIV-1 in PBMCs confirmed that 791 significantly reduced virus replication. Parallel analyses of the effect of the compounds on host gene expression revealed only minor changes in either mRNA abundance or alternative splicing. Subsequent tests suggest that 791 may function by reducing levels of the Tat/Rev chaperone Nap1. CONCLUSIONS: The three compounds examined (791, 833, 892), despite their lack of structural similarity, all suppressed HIV-1 gene expression by preventing accumulation of two key HIV-1 regulatory factors, Tat and Rev. These findings demonstrate that selective disruption of HIV-1 gene expression can be achieved.

Bioavailable inhibitors of HIV-1 RNA biogenesis identified through a Rev-based screen.

New antiretroviral agents with alternative mechanisms are needed to complement the combination therapies used to treat HIV-1 infections. Here we report the identification of bioavailable molecules that interfere with the gene expression processes of HIV-1. The compounds were detected by screening a small library of FDA-approved drugs with an assay based on measuring the displacement of Rev, and essential virus-encoded protein, from its high-affinity RNA binding site. The antiretroviral activity of two hits was based on interference with post-integration steps of the HIV-1 cycle. Both hits inhibited RRE-Rev complex formation in vitro, and blocked LTR-dependent gene expression and viral transcription in cellular assays. The best compound altered the splicing pattern of HIV-1 transcripts in a manner consistent with Rev inhibition. This mechanism of action is different from those used by current antiretroviral agents. The screening hits recognized the Rev binding site in the viral RNA, and the best compound did so with substantial selectivity, allowing the identification of a new RNA-binding scaffold. These results may be used for developing novel antiretroviral drugs.

A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.

BACKGROUND: Previously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex. RESULTS: To seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells. CONCLUSIONS: Nullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased levels of Rev-dependent viral transcripts. The outcomes support a role for DDX1 in maintenance of a Rev nuclear complex that transports viral RRE-containing mRNA to the cytoplasm. To our knowledge Nullbasic is the first anti-HIV protein that specifically targets the cellular protein DDX1 to block Rev's activity. Furthermore, our research raises the possibility that wild type Tat may play a previously unrecognized but very important role in Rev function.

A stably expressed llama single-domain intrabody targeting Rev displays broad-spectrum anti-HIV activity.

The HIV Rev protein mediates the transport of partially and unspliced HIV mRNA from the nucleus to the cytoplasm. Rev multimerizes on a secondary stem-loop structure present in the viral intron-containing mRNA species and recruits the cellular karyopherin CRM1 to export viral mRNAs from the nucleus to the cytoplasm. Previously we have identified a single-domain intrabody (Nb(190)), derived from a llama heavy-chain antibody, which efficiently inhibits Rev multimerization and suppresses the production of infectious virus. We recently mapped the epitope of this nanobody and demonstrated that Rev residues K20 and Y23 are crucial for interaction while residues V16, H53 and L60 are important to a lesser extent. Here, we generated cell lines stably expressing Nb(190) and assessed the capacity of these cell lines to suppress the replication of different HIV-1 subtypes. These cells stably expressing the single-domain antibody are protected from virus-induced cytopathogenic effect even in the context of high multiplicity of infection. In addition, the replication of different subtypes of group M and one strain of group O is significantly suppressed in these cell lines. Next, we analysed the natural variations of Rev amino acids in sequence samples from HIV-1 infected patients worldwide and assessed the effect of Nb(190) on the most prevalent polymorphisms occurring at the key epitope positions (K20 and Y23) in Rev. We found that Nb(190) was able to suppress the function of these Rev variants except for the K20N mutant, which was present in only 0.7% of HIV-1 sequence populations (n = 4632). Cells stably expressing the single-domain intrabody Nb(190) are protected against virus-induced cytopathogenic effect and display a selective survival advantage upon infection. In addition, Nb(190) suppresses the replication of a wide range of different HIV-1 subtypes. Large-scale sequence analysis reveals that the Nb(190) epitope positions in Rev are well conserved across major HIV-1 subtypes and groups. Altogether, our results indicate that Nb(190) may have broad potential as a gene therapeutic agent against HIV-1.

A cell-penetrating antibody fragment against HIV-1 Rev has high antiviral activity: characterization of the paratope.

The HIV-1 protein Rev oligomerizes on viral transcripts and directs their nuclear export. Previously, a Fab against Rev generated by phage display was used to crystallize and solve the structure of the Rev oligomerization domain. Here we have investigated the capability of this Fab to block Rev oligomerization and inhibit HIV-1 replication. The Fab itself did not have antiviral activity, but when a Tat-derived cell-penetrating peptide was appended, the resulting molecule (FabRev1-Tat) was strongly inhibitory of three different CCR5-tropic HIV-1 isolates (IC50 = 0.09-0.44 µg/ml), as assessed by suppression of reverse transcriptase activity in infected peripheral blood mononuclear cells, and had low cell toxicity (TC50 > 100 µg/ml). FabRev1-Tat was taken up by both peripheral blood mononuclear and HEK293T cells, appearing in both the cytoplasm and nucleus, as shown by immunofluorescence confocal laser scanning microscopy. Computational alanine scanning was used to identify key residues in the complementarity-determining regions to guide mutagenesis experiments. Residues in the light chain CDR3 (LCDR3) were assessed to be important. Residues in LCDR3 were mutated, and LCDR3-Tyr(92) was found to be critical for binding to Rev, as judged by surface plasmon resonance and electron microscopy. Peptides corresponding to all six CDR regions were synthesized and tested for Rev binding. None of the linear peptides had significant affinity for Rev, but four of the amide-cyclic forms did. Especially cyclic-LCDR3 (LGGYPAASYRTA) had high affinity for Rev and was able to effectively depolymerize Rev filaments, as shown by both surface plasmon resonance and electron microscopy.

The myxobacterial metabolite ratjadone A inhibits HIV infection by blocking the Rev/CRM1-mediated nuclear export pathway.

BACKGROUND: The nuclear export of unspliced and partially spliced HIV-1 mRNA is mediated by the recognition of a leucine-rich nuclear export signal (NES) in the HIV Rev protein by the host protein CRM1/Exportin1. This makes the CRM1-Rev complex an attractive target for the development of new antiviral drugs. Here we tested the anti-HIV efficacy of ratjadone A, a CRM1 inhibitor derived from myxobacteria. RESULTS: Ratjadone A inhibits HIV infection in vitro in a dose-dependent manner with EC50 values at the nanomolar range. The inhibitory effect of ratjadone A occurs around 12 hours post-infection and is specific for the Rev/CRM1-mediated nuclear export pathway. By using a drug affinity responsive target stability (DARTS) assay we could demonstrate that ratjadone A interferes with the formation of the CRM1-Rev-NES complex by binding to CRM1 but not to Rev. CONCLUSION: Ratjadone A exhibits strong anti-HIV activity but low selectivity due to toxic effects. Although this limits its potential use as a therapeutic drug, further studies with derivatives of ratjadones might help to overcome these difficulties in the future.

Characterization of novel inhibitors of HIV-1 replication that function via alteration of viral RNA processing and rev function.

Expression of the complete HIV-1 genome depends on the appropriate processing of viral RNA. Altering the balance of viral RNA processing impairs replication of the virus. In this report, we characterize two small molecule modulators of HIV-1 RNA processing, 8-azaguanine and 2-(2-(5-nitro-2-thienyl)vinyl)quinoline (5350150), which function by distinct mechanisms to suppress viral gene expression. Although only 8-Azaguanine dramatically decreased accumulation of HIV-1 unspliced and singly spliced RNAs and altered splice site usage, both compounds blocked Gag and Env expression without affecting production of Tat (p16) and Rev regulatory proteins. Subsequent analyses suggest that these compounds affect Rev-mediated RNA transport by different mechanisms. Both compounds induced cytoplasmic accumulation of Rev, suggesting that they function, in part, by impairing Rev function. This conclusion is supported by the determination that both drugs block the nuclear export of genomic HIV-1 RNA to the cytoplasm. Testing confirmed that these compounds suppress HIV-1 expression in T cells at doses below those previously used in humans for tumour chemotherapy. Together, our observations demonstrate that small molecules can be used to inhibit HIV-1 replication by altering another avenue of viral RNA processing, offering the potential for the development of novel therapeutics for controlling this disease.

A structural model of the HIV-1 Rev-integrase complex: the molecular basis of integrase regulation by Rev.

The HIV-1 Rev and integrase (IN) proteins control important functions in the viral life cycle. We have recently discovered that the interaction between these proteins results in inhibition of IN enzymatic activity. Peptides derived from the Rev and IN binding interfaces have a profound effect on IN catalytic activity: Peptides derived from Rev inhibit IN, while peptides derived from IN stimulate IN activity by inhibiting the Rev-IN interaction. This inhibition leads to multi integration, genomic instability and specific death of virus-infected cells. Here we used protein docking combined with refinement and energy function ranking to suggest a structural model for the Rev-IN complex. Our results indicate that a Rev monomer binds IN at two sites that match our experimental binding data: (1) IN residues 66-80 and 118-128; (2) IN residues 174-188. According to our model, IN binds Rev and its cellular cofactor, lens epithelium derived growth factor (LEDGF), through overlapping interfaces. This supports previous observations that IN is regulated by a tight interplay between Rev and LEDGF. Rev may bind either the IN dimer or tetramer. Accordingly, Rev is suggested to inhibit IN by two possible mechanisms: (i) shifting the oligomerization equilibrium of IN from an active dimer to an inactive tetramer; (ii) displacing LEDGF from IN, resulting in inhibition of IN binding to the viral DNA. Our model is expected to contribute to the development of lead compounds that inhibit the Rev-IN interaction and thus lead to multi-integration of viral cDNA and consequently to apoptosis of HIV-1 infected cells.

Systemic administration of combinatorial dsiRNAs via nanoparticles efficiently suppresses HIV-1 infection in humanized mice.

We evaluated the in vivo efficacy of structurally flexible, cationic PAMAM dendrimers as a small interfering RNA (siRNA) delivery system in a Rag2(-)/-γc-/- (RAG-hu) humanized mouse model for HIV-1 infection. HIV-infected humanized Rag2-/-γc-/- mice (RAG-hu) were injected intravenously (i.v.) with dendrimer-siRNA nanoparticles consisting of a cocktail of dicer substrate siRNAs (dsiRNAs) targeting both viral and cellular transcripts. We report in this study that the dendrimer-dsiRNA treatment suppressed HIV-1 infection by several orders of magnitude and protected against viral induced CD4(+) T-cell depletion. We also demonstrated that follow-up injections of the dendrimer-cocktailed dsiRNAs following viral rebound resulted in complete inhibition of HIV-1 titers. Biodistribution studies demonstrate that the dendrimer-dsiRNAs preferentially accumulate in peripheral blood mononuclear cells (PBMCs) and liver and do not exhibit any discernable toxicity. These data demonstrate for the first time efficacious combinatorial delivery of anti-host and -viral siRNAs for HIV-1 treatment in vivo. The dendrimer delivery approach therefore represents a promising method for systemic delivery of combinations of siRNAs for treatment of HIV-1 infection.

Effect of salt concentration on the conformation of TAR RNA and its association with aminoglycoside antibiotics.

RNA is an important biological target because it plays essential roles in many pathogenic and normal cellular processes. The design of inhibitors that target RNA involves optimization of noncovalent interactions, including van der Waals, hydrogen bond, and electrostatic interactions. Although sometimes regarded as nonspecific, electrostatic interactions are important in this optimization because the specific position of the phosphates may allow for specific charge-charge interactions with bound ligands. In this work, we have investigated the contribution of electrostatic interactions to the binding affinity of aminoglycoside antibiotics for TAR RNA. Because the charges in aminoglycoside antibiotics are provided by protonated amino groups, it is difficult to separate the contribution of hydrogen bonds and electrostatics to their binding specificity. Hence, we have investigated the dependence of the binding affinity on salt concentration, which should affect only the electrostatic contributions. Our results show that four aminoglycoside antibiotics (paromomycin, kanamycin-B, gentamycin, and tobramycin) bind TAR RNA with different affinities. Furthermore, the dependence of the binding affinity on salt concentration is different for kanamycin-B and paromomycin, with kanamycin-B showing a stronger dependence. Because all these antibiotics contain five positive charges, the results suggest that each antibiotic orients its charges in different ways when bound to TAR RNA. Our overall results support the idea that charge-charge interactions can contribute significantly to the specific binding of antibiotics to TAR RNA. Hence, the exact position of the charges should be considered in the design of any inhibitor of the interactions of TAR RNA.

Rev-derived peptides inhibit HIV-1 replication by antagonism of Rev and a co-receptor, CXCR4.

Rev, a viral regulatory protein of HIV-1, binds through its arginine-rich domain to the Rev-responsive element (RRE), a secondary structure in transcribed HIV-1 RNA. Binding of Rev to RRE mediates export of singly spliced or unspliced mRNAs from the nucleus to the cytoplasm. It has been previously shown that a certain arginine-rich peptide exhibits not only RRE-binding ability but also cell permeability and antagonism of CXCR4, one of the major coreceptors of HIV-1. Here we designed and synthesized arginine-rich peptides derived from the RNA-binding domain of Rev (Rev(34-50)) and evaluated their anti-HIV-1 activities. Rev(34-50)-A(4)C, comprising Rev(34-50) with AAAAC at the C-terminus to increase the alpha-helicity, inhibited HIV-1 entry by CXCR4 antagonism and virus production in persistently HIV-1-infected PM1-CCR5 cells. Interestingly, similar motif of human lymphotropic virus type I Rex (Rex(1-21)) also exerted moderate anti-HIV-1 activity. These results indicate that arginine-rich peptide, Rev(34-50)-A(4)C exerts dual antagonism against CXCR4 and Rev.

Prenylcoumarin with Rev-export inhibitory activity from Cnidii Monnieris Fructus.

By use of the fission yeast expressing the model fusion protein comprised of GST, SV40 T antigen NLS, GFP, and Rev-NES in the bioassay, the prenylcoumarin osthol (1) was disclosed as the new Rev-export inhibitor from the MeOH extract of Cnidii Monnieris Fructus. Furthermore, 1 was also found to inhibit export the genuine Rev in HeLa cells by indirect fluorescent antibody technique. By the competitive experiment using the biotinylated probe 3, osthol (1) was revealed to inhibit nuclear export of Rev through a NES non-antagonistic mode. Structure-activity relationship analysis of several analogs of 1 clarified that both prenyl side chain and double bond adjacent to the lactone carbonyl residue play an important role in the Rev-export inhibitory potency of 1.

Preintegration HIV-1 inhibition by a combination lentiviral vector containing a chimeric TRIM5 alpha protein, a CCR5 shRNA, and a TAR decoy.

Human immunodeficiency virus (HIV) gene therapy offers a promising alternative approach to current antiretroviral treatments to inhibit HIV-1 infection. Various stages of the HIV life cycle including pre-entry, preintegration, and postintegration can be targeted by gene therapy to block viral infection and replication. By combining multiple highly potent anti-HIV transgenes in a single gene therapy vector, HIV-1 resistance can be achieved in transduced cells while prohibiting the generation of escape mutants. Here, we describe a combination lentiviral vector that encodes three highly effective anti-HIV genes functioning at separate stages of the viral life cycle including a CCR5 short hairpin RNA (shRNA) (pre-entry), a human/rhesus macaque chimeric TRIM5 alpha (postentry/preintegration), and a transactivation response element (TAR) decoy (postintegration). The major focus on designing this anti-HIV vector was to block productive infection of HIV-1 and to inhibit any formation of provirus that would maintain the viral reservoir. Upon viral challenge, potent preintegration inhibition of HIV-1 infection was achieved in combination vector-transduced cells in both cultured and primary CD34(+) hematopoietic progenitor cell (HPC)-derived macrophages. The generation of escape mutants was also blocked as evaluated by long-term culture of challenged cells. The ability of this combination anti-HIV lentiviral vector to prevent HIV-1 infection, in vitro, warrants further evaluation of its in vivo efficacy.

New Rev-export inhibitor from Alpinia galanga and structure-activity relationship.

Bioassay-guided separation by use of the fission yeast expressing NES of Rev, an HIV-1 viral regulatory protein, disclosed 1'-acetoxychavicol acetate (ACA, 1) as a new inhibitor for nuclear export of Rev from the roots of Alpinia galanga. Both analysis for mechanism of action with biotinylated probe (2) and several synthesized analogs established crucial portions in 1 for Rev-export inhibitory activity.

Cessation of HIV-1 transcription by inhibiting regulatory protein Rev-mediated RNA transport.

The HIV-1 Rev protein, which traffics through nucleolus and shuttles between nucleus and cytoplasm, facilitates export of unspliced and singly spliced viral transcripts containing RRE RNA by the CRM1 export pathway. Inhibitions of the various stages of Rev-mediated RNA transport can arrest HIV-1 transcriptional process. The current understanding to the mechanism of Rev function, Rev-RRE interaction, as well as inhibitors hereof is reviewed.

Inhibition of the CRM1-mediated nucleocytoplasmic transport by N-azolylacrylates: structure-activity relationship and mechanism of action.

CRM1-mediated nucleocytoplasmic transport plays an important role in many cellular processes and diseases. To investigate the structural basis required for the inhibition of the CRM1-mediated nuclear export we have synthesized analogs of a previously identified small molecule lead compound and monitored their activity against the Rev function of the human immunodeficiency virus. Microscopy studies show that the active congeners of this series inhibit the nucleocytoplasmic transport of Rev and the co-localization between Rev and CRM1 in living cells. Mechanism of action studies show their interaction with the Cys528 residue of CRM1 involving a Michael-addition type of reaction. However, structure-activity relationship demonstrates strict constraints to the structure of the inhibitors, and shows that activity is not solely correlated to Michael-addition suggesting a more complex mechanism of action. Our results are suggestive for the existence of a well-defined interaction at the CRM1-NES binding site. In addition, the most selective congener inhibited the HIV-1 production in latently infected cells. These specific CRM1 inhibitors are of interest as tool for analyzing the mechanisms of post-transcriptional control of gene expression and provide insight in the design of new agents.