Human Immunodeficiency Virus Type 1 Rev Protein Research Articles

HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment.

Nuclear RNA export pathways in eukaryotes are often linked to the fate of a given RNA. Therefore, the choice of export pathway should be well-controlled to avoid an unfavorable effect on gene expression. Although some RNAs could be exported by more than one pathway, little is known about how the choice is regulated. This issue is highlighted when the human immunodeficiency virus type 1 (HIV-1) Rev protein induces the export of singly spliced and unspliced HIV-1 transcripts. How these RNAs are exported is not well understood because such transcripts should have the possibility of utilizing CRM1-dependent export via Rev or cellular TAP/NXF1-dependent export via the transcription/export (TREX) complex, or both. Here we found that Rev suppressed TAP/NXF1-dependent export of model RNA substrates that recapitulated viral transcripts. In this effect, Rev interacted with the cap-binding complex and inhibited the recruitment of the TREX complex. Thus, Rev controls the identity of the factor occupying the cap-proximal region that determines the RNA export pathway. This ribonucleoprotein remodeling activity of Rev may favor viral gene expression.

HIV-1 Virus-Like Particles Produced by Stably Transfected Drosophila S2 Cells: a Desirable Vaccine Component

The development of a successful vaccine against human immunodeficiency virus type 1 (HIV-1) likely requires immunogens that elicit both broadly neutralizing antibodies against envelope spikes and T cell responses that recognize multiple viral proteins. HIV-1 virus-like particles (VLP), because they display authentic envelope spikes on the particle surface, may be developed into such immunogens. However, in one way or the other current systems for HIV-1 VLP production have many limitations. To overcome these, in the present study we developed a novel strategy to produce HIV-1 VLP using stably transfected Drosophila S2 cells. We cotransfected S2 cells with plasmids encoding HIV-1 envelope, Gag, and Rev proteins and a selection marker. After stably transfected S2 clones were established, HIV-1 VLP and their immunogenicity in mice were carefully evaluated. Here, we report that HIV-1 envelope proteins are properly cleaved, glycosylated, and incorporated into VLP with Gag. The amount of VLP released into culture supernatants is comparable to those produced by insect cells infected with recombinant baculoviruses. Moreover, cryo-electron microscopy tomography revealed average 17 spikes per purified VLP, and antigenic epitopes on the spikes were recognized by the broadly neutralizing antibodies 2G12, b12, VRC01, and 4E10 but not by PG16. Finally, mice primed with DNA and boosted with VLP in the presence of CpG exhibited anti-envelope antibody responses, including ELISA-binding, neutralizing, antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated viral inhibition, as well as envelope and Gag-specific CD8 T cell responses. Thus, we conclude that HIV-1 VLP produced by the S2 expression system has many desirable features to be developed into a vaccine component against HIV-1.

A novel role for the viral Rev protein in promoting resistance to superinfection by human immunodeficiency virus type 1

At the cellular level, cells infected with human immunodeficiency virus type 1 (HIV-1) exhibit immunity to a second infection by the virus that initiated the first infection or by related viruses [superinfection resistance (SIR)]. In the case of HIV infection, SIR was basically attributed to downregulation of the CD4 receptors. We have recently reported on an interaction between HIV-1 Rev and integrase (IN) proteins, which results in inhibition of IN activity in vitro and integration of cDNA in HIV-1-infected cells. A novel function for the viral Rev protein in controlling integration of HIV cDNAs was thus proposed. The results of the present work suggest involvement of the inhibitory Rev in sustaining SIR. A single exposure to wild-type HIV-1 resulted in one to two integrations per cell. The number of integrated proviral cDNA copies remained at this low level even after double infection or superinfection. SIR was dependent on Rev expression by the strain used for the first infection and was eliminated by peptides that disrupt intracellular complex formation between IN and Rev. The same lack of resistance was observed in the absence of Rev, namely following first infection with a DeltaRev HIV strain. The involvement of Rev, expressed from either unintegrated or integrated viral cDNA, in promoting SIR was clearly demonstrated. We conclude that SIR involves Rev-dependent control of HIV cDNA integration.

Insulin-like growth factor II mRNA binding protein 1 modulates Rev-dependent human immunodeficiency virus type 1 RNA expression

Human immunodeficiency virus type 1 (HIV-1) needs to overcome cellular counter mechanisms such as to successfully propagate itself. Results of our recent studies show that overexpression of insulin-like growth factor II mRNA binding protein 1 (IMP1) inhibits production of infectious HIV-1 particles through adversely affecting virus maturation. Here, we report that IMP1 interacts with HIV-1 Rev protein and its ectopic expression causes relocation of Rev from the nucleus to the cytoplasm. In accordance with this observation, ectopic expression of IMP1 severely diminishes Rev-dependent expression of CAT enzyme and disturbs HIV-1 RNA expression by causing accumulation of the multiple spliced viral RNA. Results of mutagenesis analysis further reveal that the KH4 domain represents the key element of IMP1 in modulating HIV-1 RNA expression. Taken together, these data suggest, in addition to hampering virus assembly, that IMP1 also has an effect on Rev-dependent viral RNA expression.

Design of beta-hairpin peptidomimetics that inhibit binding of alpha-helical HIV-1 Rev protein to the rev response element RNA.

The virally encoded Rev protein of human immunodeficiency virus type-1 (HIV-1) plays a critical role in viral replication by regulating the transport of unspliced and partially spliced viral RNA from the nucleus to the cytoplasm of infected cells.[1, 2] Rev binds first to a specific region of the HIV-1 mRNA, called the Rev responsive element (RRE) of stem loop IIb (Figure 1); subsequent to this initial binding event, approximately 10 additional Rev molecules oligomerize through protein–protein and protein–RNA interactions and coat the entire RRE.[3] Because of its essential role in viral replication, the interaction between Rev protein and the high-affinity RRE binding site represents an attractive yet unexploited target for antiviral therapy.

Quantitative analysis of shape-specific interactions of Rev response element with a positively charged Rev peptide by capillary electrophoresis

Human immunodeficiency virus type 1 (HIV-1) Rev protein is known to regulate the expression of proteins via binding to an RNA site termed the HIV Rev response element (RRE) presumably with a defined shape, mediated mainly by electrostatic interactions. We have developed a quantitative method based on CE-LIF detection for a systematic evaluation of interactions between a truncated RRE (tRRE) RNA and an HIV-1 Rev peptide. Employing a fluorescently labeled HIV-1 Rev protein fragment (RevF) as a probe, buffers were evaluated for the separation and detection as well as for the RNA shape-specific formation of the complex. Selection of an optimal buffer condition allowed us to perform quantitation of the tRRE-RevF complex formation and determine its dissociation constant. In addition, competitive inhibitions of the RNA-peptide interaction by some aminoglycosides were evaluated quantitatively by monitoring the complex peak, resulting in determination of IC(50) values. This sensitive and reliable CE-LIF-based method would be of interest in developing various screening systems for RNA interference in drug discovery.

Requirement of an additional Sam68 domain for inhibition of human immunodeficiency virus type 1 replication by Sam68 dominant negative mutants lacking the nuclear localization signal

Human immunodeficiency virus type 1 (HIV-1) replication requires active nuclear export of unspliced and incompletely spliced HIV-1 RNA transcripts. This process is evolutionally made possible by expression of HIV-1 Rev, one of the three HIV-1 proteins encoded by completely spliced HIV-1 RNAs. Evidence has accumulated to suggest that Sam68 plays an important role in HIV-1 replication through HIV-1 Rev protein. In the present study, we further examined the structure–function relationship of Sam68 protein in relation to HIV-1 replication. We identified a Sam68 domain located between aa269 and aa321 to be involved in the HIV-inhibitory effects of Sam68 dominant negative mutants lacking the nuclear localization signal (NLS). Deletion of this domain abrogated inhibition of HIV-1 replication by these mutants. HIV-1 Rev protein appeared to mediate the HIV-inhibitory effects of these mutants and by this domain, as assessed by Rev-dependent chloramphenicol acetyltransferase reporter gene assay, in trans rev-defective HIV-1 complementation assay, and RNase protection assay. The HIV-inhibitory mutants containing this domain were further found to have diminished binding affinity to the wild-type Sam68 and to be associated with cytoplasmic retention of exclusively nuclear localized wild type Sam68. Taken together, these results further ascertain the important role of Sam68 in HIV-1 Rev function and viral replication, and suggest that the HIV-inhibitory effects of Sam68 dominant negative mutants directly result from their binding to endogenous Sam68 and their interference with nuclear localization of endogenous Sam68.

Co-expression of a trans-dominant negative mutant of the human immunodeficiency virus type 1 (HIV-1) Rev protein affects the Rev-dependent splicing pattern and expression of HIV-1 RNAs.

Trans-dominant negative mutants of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev inhibit the function of wild-type Rev in a dose-dependent manner. This was previously shown to be caused by nuclear retention of the wild-type protein. In the present work, further analysis of the trans-dominant negative effect was performed using cotransfection experiments with different constructs encoding HIV-1 Rev and viral structural proteins together with a plasmid encoding a trans-dominant negative Rev mutant. Thus, one species of pre-mRNA was transcribed from the reporter plasmids. This pre-mRNA was then either spliced or exported by Rev as unspliced RNA for translation of the HIV structural proteins. An immunofluorescence assay and Western blot analysis were used for analysis of protein expression. In situ hybridization was applied for labelling of unspliced mRNA in transfected cells, and RNase protection analysis was used to determine the relative amount of unspliced versus spliced mRNAs. The experiments confirmed that the transdominant negative mutant inhibited nuclear export of unspliced mRNA. It was, in addition, demonstrated for the first time that the trans-dominant negative mutant also affected a Rev-dependent regulatory step connected with viral pre-mRNA splicing. As a consequence, proteins expressed from unspliced and singly spliced HIV mRNAs decreased while there was an increase in protein products encoded by spliced and alternatively spliced mRNAs.

Regulation of human immunodeficiency virus type 1 infectivity by the ERK mitogen-activated protein kinase signaling pathway.

ERK1 and ERK2 mitogen-activated protein kinases (MAPK) play a critical role in regulation of cell proliferation and differentiation in response to mitogens and other extracellular stimuli. Mitogens and cytokines that activate MAPK in T cells have been shown to activate human immunodeficiency virus type 1 (HIV-1) replication. Little is known about the signal transduction pathways that activate HIV-1 replication in T cells upon activation by extracellular stimulation. Here, we report that activation of MAPK through the Ras/Raf/MEK signaling pathway enhances the infectivity of HIV-1 virions. Virus infectivity was enhanced by treatment of cells with MAPK stimulators, such as serum and phorbol myristate acetate, as well as by coexpression of constitutively activated Ras, Raf, or MEK (MAPK kinase) in the absence of extracellular stimulation. Treatment of cells with PD 098059, a specific inhibitor of MAPK activation, or with a MAPK antisense oligonucleotide reduced the infectivity of HIV-1 virions without significantly affecting virus production or the levels of virion-associated Gag and Env proteins. MAPK has been shown to regulate HIV-1 infectivity by phosphorylating Vif (X. Yang and D. Gabuzda, J. Biol. Chem. 273:29879-29887, 1998). However, MAPK activation enhanced virus infectivity in some cells lines that do not require Vif function. The HIV-1 Rev, Tat, p17(Gag), and Nef proteins were directly phosphorylated by MAPK in vitro, suggesting that other HIV-1 proteins are potential substrates for MAPK phosphorylation. These results suggest that activation of the ERK MAPK pathway plays a role in HIV-1 replication by enhancing the infectivity of HIV-1 virions through Vif-dependent as well as Vif-independent mechanisms. MAPK activation in producer cells may contribute to the activation of HIV-1 replication when T cells are activated by mitogens and other extracellular stimuli.

Viruses, microorganisms and scientists meet the nuclear pore. Leysin, VD, Switzerland, February 26-March 1, 1998.

The modular architecture of the nuclear pore complex (NPC) consists of a membrane‐embedded basic framework (the ‘spoke complex’) made up of eight multidomain spokes with two rings on each face (Figure 1A). The ring facing the cytoplasm is decorated with eight ∼50 nm fibrils extending into the cytosol, and the nuclear ring is capped with a basket‐like assembly of eight thin, 50–100 nm filaments joined distally by a 30–40 nm diameter terminal ring. The center of the basic framework harbors the ‘gated transport channel’, which is involved in signal‐mediated bidirectional transport of macromolecules and is plugged to the central ‘transporter’, whose ultimate structure and functional role in mediated nuclear transport remains to be established. N.Pante and U.Aebi (Basel) reported their approaches to the functional roles of the different NPC components and to dissection of the steps of nuclear import and export at the NPC by direct visualization of gold‐labeled proteins and RNAs moving in or out of the cell nucleus through the NPC (Figure 1B). Figure 1. ( A ) Current consensus model of the membrane‐bound NPC. Its major structural components include the basic framework, the central plug or gated channel, the cytoplasmic and nuclear rings, and the cytoplasmic fibrils and nuclear basket. ( B ) Visualization of the different steps of nuclear import of an NLS‐bearing protein through the NPC. At the bottom are selected examples of cross‐sectioned NPCs with associated nucleoplasmin–gold particles (8 nm diameter), depicted at different stages of transport; at the top is a schematic diagram of the distinct steps visualized by electron microscopy. In the first step of transport, the NLS protein to be imported associates with the NLS receptor (importins α and β) (1). This step takes place in the cytoplasm and does not require physical interaction with any NPC component. In a second step, this ‘targeting complex’ docks …

Intracellular Trafficking and Interactions of the HIV-1 Tat Protein

Fusions of the human immunodeficiency virus type 1 (HIV-1) transactivator protein Tat to the green fluorescent protein (GFP) were used to study the intracellular localization, trafficking, and interactions of Tat in human cells. Tagging Tat with GFP did not change its nuclear localization or ability to act as a transactivator. Tat-GFP expressed at low levels was found in the nucleus, whereas overexpression resulted in nucleolar accumulation. A Tat-GFP hybrid protein containing in addition the HIV-1 Rev nuclear export signal (NES) localized predominantly to the cytoplasm. This shuttle protein, Tat-GFP-NES, transactivated the HIV-1 long terminal repeat. Thus a Tat molecule being only transiently present in the nucleus is active and nucleolar accumulation of Tat is not prerequisite for function. A coexpression assay previously used to define protein interaction domains in the HIV-1 Rev protein [R. H. Stauber, E. Afonina, S. Gulnik, J. Erickson, and G. N. Pavlakis (1998a).Virology251, 38–48.] indicated that Tat exists predominantly as a monomer and does not form stable multimers with B23 in living cells. Using a heterokaryon fusion assay, we found that Tat-GFP was able to shuttle between the nucleus and the cytoplasm. Tat therefore has the potential to perform functions in the nucleus as well as in the cytoplasm.

Posttranscriptional Regulation by the HIV-1 Rev Protein

Human Immunodeficiency Virus Type 1 (HIV-1) encodes a range of mRNA species derived by partial or complete splicing of a single initial RNA transcript. The nuclear export of the incompletely spliced class of HIV-1 RNAs, which encode critical viral structural proteins, is dependent on the Rev post-transcriptional regulatory protein, a viral RNA binding factor that is expressed early in the HIV-1 replication cycle. Rev contains a potent nuclear export signal that mediates the nucleocytoplasmic transport of bound RNAs subsequent to an interaction with components of the nuclear pore. It has now become evident that the nuclear export pathway utilized by HIV-1 Rev is also critical for the nuclear export of a range of cellular proteins and RNAs.

HIV Rev uses a conserved cellular protein export pathway for the nucleocytoplasmic transport of viral RNAs

Background: The structural proteins of human immunodeficiency virus type 1 (HIV-1) are encoded by intron-containing mRNAs that normally are retained in the nucleus. A viral regulatory protein, Rev, specifically induces the accumulation of these transcripts in the cytoplasm. Rev is an RNA-binding protein that also contains an ‘effector’ domain. The Rev effector domain has recently been shown to function as an autonomous nuclear export signal (NES) that, when fused to a foreign protein, will cause its rapid nuclear export. We and others have recently reported the cloning of a human protein (hRIP/Rab), that specifically interacts with the effector domain of Rev.Results Here we show that the NESs contained within two cellular proteins, PKI and IκB, which are not involved in RNA metabolism, also interact with hRIP. Fusion of these cellular sequences to the Rev RNA-binding domain reconstitutes a functional Rev protein. In addition to hRIP, these NESs also bind to several nuclear pore proteins located on the nucleoplasmic, membrane, and cytoplasmic side of the nuclear pore complex (NPC). We show that this protein export pathway is highly conserved by demonstrating that mammalian NESs also function in yeast.Conclusion Our results indicate that the HIV-1 Rev protein evolved to take advantage of a cellular protein export pathway in order to allow the nucleocytoplasmic transport of unspliced viral RNA. Our data suggest a model in which the export substrate is translocated through the NPC by sequential interactions with different nucleoporins. Finally, our experiments suggest a mechanism by which IκB can downregulate nuclear NFκB activity by causing its rapid export from the nucleus.

Recombinant human Fab antibody fragments to HIV-1 Rev and Tat regulatory proteins: Direct selection from a combinatorial phage display library

A human Fab phage display library has been produced from peripheral blood lymphocytes of an individual who was asymptomatic after 10 years of infection with human immunodeficiency virus type-1 (HIV-1). The library was panned against the HIV-1 Rev and Tat regulatory proteins and several clones, producing Fab binding to these proteins, were isolated (3 to Rev and 4 to Tat) with binding constants varying from 10 −6M to 10 −8M. DNA sequencing demonstrated two unique anti-Rev Fab clones, but the four anti-Tat Fab comprised only two unique IgG 1 heavy chain Fd fragments, illustrating redundancy of light chains. Peptide mapping of the epitopes recognized by these Fab indicated that three of the anti-Tat Fab were directed to the functional domain between amino acid residues 22–33 of the Tat molecule, and that binding was inhibited by reduction of this cysteine-rich region with dithiothreitol. The anti-Rev Fab were directed to sites adjacent to the Rev basic nucleolar localization sequence (residues 52–64) and to the Rev activation domain (residues 75–88). Binding constants were of a similar order to that of an anti-Rev single-chain Fv fragment (SFv) used successfully for intracellular immunization, and as such intracellular effects with the human anti-Tat and anti-Rev Fab are not precluded. These newly described human antibody fragments to HIV-1 regulatory proteins may be critical moieties for gene therapeutic protocols, to control HIV-1 replication in human cells.

Detection of intracellular HIV-1 Rev protein by flow cytometry

The Rev trans-activator protein plays a pivotal role in human immunodeficiency virus type 1 (HIV-1) replication by allowing expression of the viral structural proteins. We have developed a protocol to quantitatively assay intracellular steady state levels of Rev Ag (Rev wild type and RevM10 proteins) by flow cytometry. Three fixation and permeabilization techniques were compared. These protocols varied in the magnitude of the signal which could be detected, and in the ability to distinguish between Rev Ag positive and negative populations. This technology is applicable to a variety transduced or transfected cell types (species, lineage), and for cell lines and primary cells acutely infected with HIV-1. The assay is therefore a valuable tool both to analyze Rev protein expression levels in HIV-infected cells and to optimize delivery of the dominant-negative RevM10 gene for clinical gene therapy applications. In addition, a second, independent intracellular protein (HIV-Tat) has been detected using the same approach.

Inhibition of clinical human immunodeficiency virus (HIV) type 1 isolates in primary CD4+ T lymphocytes by retroviral vectors expressing anti-HIV genes.

Gene therapy may be of benefit in human immunodeficiency virus type 1 (HIV-1)-infected individuals by virtue of its ability to inhibit virus replication and prevent viral gene expression. It is not known whether anti-HIV-1 gene therapy strategies based on antisense or transdominant HIV-1 mutant proteins can inhibit the replication and expression of clinical HIV-1 isolates in primary CD4+ T lymphocytes. We therefore transduced CD4+ T lymphocytes from uninfected individuals with retroviral vectors expressing either HIV-1-specific antisense-TAR or antisense-Tat/Rev RNA, transdominant HIV-1 Rev protein, and a combination of antisense-TAR and transdominant Rev. The engineered CD4+ T lymphocytes were then infected with four different clinical HIV-1 isolates. We found that replication of all HIV-1 isolates was inhibited by all the anti-HIV vectors tested. Greater inhibition of HIV-1 was observed with transdominant Rev than with antisense RNA. We hereby demonstrated effective protection by antisense RNA or transdominant mutant proteins against HIV-1 infection in primary CD4+ T lymphocytes using clinical HIV-1 isolates, and this represents an essential step toward clinical anti-HIV-1 gene therapy.

Tat and Rev Differentially Affect Restricted Replication of Human Immunodeficiency Virus Type 1 in Various Cells

Human immunodeficiency virus type 1 (HIV-1) can infect various cell lines in culture and be maintained in a chronic state of restricted replication. These states of proviral latency are characterized by a predominance of spliced compared to unspliced viral RNA species. The proximate molecular mechanisms leading to restricted HIV-1 replication may differ in various cell lines. Importantly, recent studies have demonstrated that the site of integration is the critical parameter leading to provital latency in ACH-2 cells. Utilizing murine retrovital shuttle vectors, the HIV-1 Tat protein was demonstrated to dramatically increase HIV-1 expression in the restrictively infected U1 monocytic cell line but not in the ACH-2 T-lymphocytic line. The HIV-1 Rev protein only modestly increased vital expression in both of these cell types. Thus, these data support the hypothesis that the mechanisms which initiate and/or maintain restricted HIV-1 expression may differ in various cell types in cell culture, and possibly in vivo.

Synergistic Anti-Human Immunodeficiency Viral (HIV-1) Effect of the Immunomodulator Ampligen (Mismatched Double-Stranded RNA) with Inhibitors of Reverse Transcriptase and HIV-1 Regulatory Proteins

The potent antiviral effect of double stranded RNA, such as the mismatched poly(l)·poly(C12U) [Ampligen], 2′,3′-dideoxy-3′-fluorothymidine (FddThd) and antisense oligodeoxynucleotides (ODN) has been established in in vitro systems using cells infected with the human immunodeficiency virus type 1 (HIV-1). We report here that the immunomodulator poly(l)·poly(C12U) interacts synergistically with (1) the reverse transcriptase inhibitor FddThd (FIC value: 0.43), (2) the modified (5′- and 3′-end capped thioates) antisense ODN-4 directed against the splice acceptor site of the HIV-1/ tat gene (FIC value: 0.66) and (3) also with pyronin Y, a compound which prevents binding of HIV-1 Rev protein to the HIV-1 RRE element. These data suggest that combinations of poly(l)·poly(C12U), a stimulator of the natural antiviral protection system of the cells, with compounds targeting HIV1-specific processes should be considered as candidate treatments of AIDS patients.

Dominant negative mutants of human T-cell leukemia virus type I Rex and human immunodeficiency virus type 1 Rev fail to multimerize in vivo.

Human T-cell leukemia virus type I (HTLV-I) Rex and human immunodeficiency virus type 1 (HIV-1) Rev are essential gene products required for the replication of these two pathogenic human retroviruses. Both Rex and Rev act at a posttranscriptional level by binding to highly structured RNA-response elements, the Rex-response element in HTLV-I and the Rev-response element in HIV-1. Using a sensitive in vivo assay of protein-protein interaction, we now demonstrate that the HTLV-I Rex and HIV-1 Rev proteins readily form homomultimeric complexes in the absence of their cognate RNA-response elements yet fail to form heteromultimeric complexes with each other. Dominant negative mutations have been identified in both the rex and rev genes which presumably specify a critical activation or effector domain in each of these viral transactivators. Surprisingly, these dominant negative mutants of Rex and Rev fail to interact in vivo. These findings raise the possibility that the binding of nonfunctional monomers rather than functional multimers underlies the transdominant phenotype of these Rex and Rev mutants. Further, it seems likely that the assembly of functional and stable multimers of Rex and Rev in vivo may depend not only on the intrinsic multimerization domains of these proteins but also on the binding of a bridging cellular cofactor to the related activation domains present in each viral transactivator.

Mouse monoclonal antibodies recognizing the activation domains of HIV-1 rev transactivator

The rev protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 kDa apparent molecular weight, is essential to target the mRNA for virion polypeptides into the cytoplasm. So far, at least four necessary functional domains have been assigned to the HIV-1 rev protein: (1) one for RNA binding; (2) a second for nuclear/nucleolar localization that may be indistinguishable from the RNA binding motif; (3) two domains for multimerization; and (4) a putative activation domains (AD) that is suppressed in trans by dominant-negative mutant rev protein. We report three IgG 1κ mouse monoclonal antibodies (mabs) that were independently raised against rev protein expressed in Escherichia coli. Epitopes are mapped by immunoprecipitation and Western blot screening with 40 different rev mutant peptides. Surprisingly, monoclonal antibodies from all three hybridomas recognized the activation domains of HIV-1 rev.