Rev Protein Research Articles

Over-expression of the HIV-1 Rev promotes death of nondividing eukaryotic cells

Expression of the human immunodeficiency virus type 1 (HIV-1) Rev protein is essential for completion of the viral life cycle. Rev mediates nuclear export of partially spliced and unspliced viral transcripts and therefore bears a nuclear localization signal (NLS) as well as a nuclear export signal (NES), which allow its nucleocytoplasmic shuttling. Attempts to express the wild-type Rev protein in eukaryotic human cultured cells have encountered difficulties and so far have failed. Here we show that accumulation of Rev, which occurs in nondividing Rev-expressing cells or when such cells reach confluency, results in death of these cells. Cell death was also promoted by addition of a cell permeable peptide bearing the Rev-NES sequence, but not by the Rev-NLS peptide. Our results probably indicate that binding of excess amounts of the Rev protein or the NES peptide to the exportin receptor CRM1 results in cells' death.

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.

Nucleocytoplasmic shuttling of HIV-1 integrase is controlled by the viral Rev protein.

In the current study we show that the Rev protein of Human Immunodeficiency Virus type 1 (HIV-1) inhibits nuclear import and mediates nuclear export of the HIV-1 integrase (IN) protein, which catalyzes integration of the viral cDNA. Interaction between IN and Rev in virus infected cells, resulting in the formation of a Rev-IN complex, has been previously described by us. Here we show that nuclear import of the IN, is inhibited by early expressed Rev. No nuclear import of IN was observed when Rev-overexpressing cells were infected by wild-type HIV-1. Similarly, no translocation of IN into nuclei was observed in the presence of Rev-derived peptides. On the other hand, massive nuclear import was observed following infection by a ΔRev virus or in the presence of peptides that promote dissociation of the Rev-IN complex. Our results show that IN is only transiently present within the nuclei of infected cells. Treatment of infected cells with leptomycin B caused nuclear retention of the Rev-IN complex. Removal of the leptomycin from these treated cells resulted in nuclear export of both Rev and IN. On the other hand, disruption of the nuclear located Rev-IN complex resulted in export of only the Rev protein indicating Rev-mediated nuclear export of IN. Our results suggest the involvement of Rev in regulating the integration process by limiting the number of integration events per cell despite the presence of numerous copies of viral cDNA.

Molecular and Biological Aspects of the Bovine Immunodeficiency Virus

The bovine immunodeficiency virus (BIV) was isolated in 1969 from a cow, R-29, with a wasting syndrome suggesting bovine leucosis. The virus, first designated bovine visna-like virus, remained unstudied until HIV was discovered in 1983. Then, it was demonstrated in 1987 that the bovine R-29 isolate was a lentivirus with striking similarity to the human immunodeficiency virus (HIV). Moreover, BIV has the most complex genomic structure among all identified lentiviruses shown by several regulatory/accessory genes encoding proteins, some of which are involved in the regulation of virus gene expression. This manuscript aims to review biological and molecular aspects of BIV, with emphasis on regulatory/accessory viral genes/proteins which are involved in virus expression.

Molecular and Biological Characterization of Equine Infectious Anemia Virus Rev

Equine infectious anemia virus (EIAV) is one of the most divergent members of the lentivirus subfamily of retroviruses and is considered a useful comparative model for molecular studies of lentivirus replication. The Rev protein of EIAV is functionally homologous with other lentiviral Revs and facilitates export of incompletely spliced viral mRNAs through a Crm1-dependent pathway. The trans- and cis-acting elements that mediate EIAV Rev function are similar to, but distinct from, the well-characterized elements in human immunodeficiency virus (HIV-1), the prototypical Rev protein. In addition, the EIAV rev sequence is highly variable in vivo, and changes in Rev phenotype correlate with changes in clinical stages of EIAV infection. This review summarizes the molecular biology of EIAV Rev-RRE interactions and the consequences of Rev variation in vivo. A comparative perspective of Rev activity may enhance understanding of an essential lentiviral protein and stimulate new strategies for treatment and prevention of lentivirus infections in vivo.

Identification of a Heterogeneous Nuclear Ribonucleoprotein-recognition Region in the HIV Rev Protein

The Rev protein is a key regulator of human immunodeficiency virus type 1 (HIV-1) gene expression. Rev is primarily known as an adaptor protein for nuclear export of HIV RNAs. However, Rev also contributes to numerous other processes by less well known mechanisms. Understanding the functional nature of Rev requires extensive knowledge of its cellular interaction partners. Here we demonstrate that Rev interacts with members of a large family of multifunctional host cell factors called hnRNPs. Rev employs amino acids 9-14 for specific binding to the heterogeneous nuclear ribonucleoproteins (hnRNP) A1, Q, K, R, and U. In addition, Rev interacts with hnRNP E1 and E2 by a different mechanism. The set of hnRNPs recognized by the N terminus of Rev feature RGG boxes. Exemplary testing of hnRNP A1 revealed a critical role of arginine residues within the RGG box for interaction with Rev. Finally, we demonstrate that expression levels of hnRNP A1, Q, K, R, and U influence HIV-1 production by persistently infected astrocytes, linking these hnRNPs to HIV replication. The novel interaction of HIV-1 Rev with functionally diverse hnRNPs lends further support to the idea that Rev is a multifunctional protein and may be involved in coupling HIV replication to diverse cellular processes and promoting virus-host cell interactions.

A Lentiviral Vector That Activates Latent Human Immunodeficiency Virus-1 Proviruses by the Overexpression of Tat and That Kills the Infected Cells

Despite the efficient HIV-1 replication blockage achieved with current highly active antiretroviral therapy (HAART) therapies, HIV-1 persists in the body and survives in a latent state that can last for the entire life of the patient. A long-lived reservoir of latently infected CD4(+) memory T cells represents the most important sanctuary for the virus and the greatest obstacle for viral eradication. In this work, we present an initial step toward a gene therapy approach aimed at the activation of latent provirus to induce the death of latently infected T cells. Latent HIV-1 infection is characterized by the failure of viral gene expression as a consequence of uninitiated or aborted transcription. We have constructed an HIV-1-based lentiviral vector (p5p53RTAT3) that expresses the viral trans-activating protein Tat in a drug-regulated manner and p53 in a Rev-dependent manner. We have demonstrated that the Tat-expressed protein from p5p53RTAT3 vector reactivates latent HIV-1 proviruses in J1.1 and ACH-2 cell lines and promotes p53-induced apoptosis in the presence of Rev. Our system was able to trigger the trans-activation of the provirus 5' long terminal repeat (LTR), stimulate the expression of the Rev protein from a tat-defective provirus, and provoke apoptosis selectively in the cells transfected with a tat-defective HIV-1 provirus in contrast to those with no HIV-1 provirus. However, the Rev-dependent p53 killing of latently infected cells was not effective enough for complete elimination of the awakened HIV-1 viruses. In summary, we have developed a vector system that is efficient in activating latent HIV-1 proviruses but that needs further improvement to kill infected cells.

Integration of HIV-1 DNA Is Regulated by Interplay between Viral Rev and Cellular LEDGF/p75 Proteins

The present work describes a novel interaction between the human immunodeficiency virus type 1 (HIV-1) Rev protein and the cellular lens epithelium-derived growth factor p75 (LEDGF/p75) protein in vitro and in virus-infected cells. Here we show, for the first time, that formation of an Rev-LEDGF/p75 complex is a crucial step in regulating viral cDNA integration. Coimmunoprecipitation experiments at various times after virus infection revealed that, first, an integrase enzyme (IN)-LEDGF/p75 complex is formed, which is then replaced by a Rev-LEDGF/p75 and Rev-IN complexes. This was supported by in vitro experiments showing that Rev promotes dissociation of the IN-LEDGF/p75 complex. Combination of the viral IN and the cellular LEDGF/p75 is required for proper integration of the viral cDNA into the host chromosomal DNA. Our findings demonstrate that integration of HIV-1 cDNA is regulated by an interplay between viral Rev and the host-cell LEDGF/p75 proteins.

Novel regulation of HIV-1 replication and pathogenicity: Rev inhibition of integration

Following fusion of the human immunodeficiency virus type-1 (HIV-1) with host cells' membrane and reverse transcription of the viral RNA, the resulted cDNA is integrated into the host genome by the viral integrase enzyme (IN). Quantitative estimations have revealed that only 1-2 copies are integrated per infected cell, although many copies of the viral RNA are reverse-transcribed. The molecular mechanism that restricts the integration degree has not, so far, been elucidated. Following integration, expressed partially spliced and unspliced transcripts are exported from the nuclei by the viral Rev protein. Here, we show that in virally infected cells, the Rev interacts with the IN forming a Rev-IN complex and consequently limits the number of integration events. Disruption of the Rev-IN complex by selected IN-derived peptides or infection by a Rev-deficient virus stimulate integration resulting in large numbers of integration event/cell. Conversely, infection of Rev-expression cells blocks integration and inhibits virus production. Increased integration appears to correlate with increased cell death of infected cultures. Our results thus demonstrate a new regulatory function of Rev and probably establish a link between Rev restriction of HIV-1 integration and protection of HIV-1-infected cells from premature cell death.

The Bovine Immunodeficiency Virus Rev Protein: Identification of a Novel Lentiviral Bipartite Nuclear Localization Signal Harboring an Atypical Spacer Sequence

The bovine immunodeficiency virus (BIV) Rev protein (186 amino acids [aa] in length) is involved in the nuclear exportation of partially spliced and unspliced viral RNAs. Previous studies have shown that BIV Rev localizes in the nucleus and nucleolus of infected cells. Here we report the characterization of the nuclear/nucleolar localization signals (NLS/NoLS) of this protein. Through transfection of a series of deletion mutants of BIV Rev fused to enhanced green fluorescent protein and fluorescence microscopy analyses, we were able to map the NLS region between aa 71 and 110 of the protein. Remarkably, by conducting alanine substitution of basic residues within the aa 71 to 110 sequence, we demonstrated that the BIV Rev NLS is bipartite, maps to aa 71 to 74 and 95 to 101, and is predominantly composed of arginine residues. This is the first report of a bipartite Rev (or Rev-like) NLS in a lentivirus/retrovirus. Moreover, this NLS is atypical, as the length of the sequence between the motifs composing the bipartite NLS, e.g., the spacer sequence, is 20 aa. Further mutagenesis experiments also identified the NoLS region of BIV Rev. It localizes mainly within the NLS spacer sequence. In addition, the BIV Rev NoLS sequence differs from the consensus sequence reported for other viral and cellular nucleolar proteins. In summary, we conclude that the nucleolar and nuclear localizations of BIV Rev are mediated via novel NLS and NoLS motifs.

Candidacidal effects of Rev (11-20) derived from HIV-1 Rev protein.

Rev is an essential regulatory protein for HIV-1 replication. Rev (11-20) is known as the significant region regarding the function of a nuclear entry inhibitory signal (NIS) of Rev. In this study, anticandidal effects and mechanism of action of Rev (11-20) were investigated. The result exhibited that Rev (11-20) contained candidacidal activities. To understand target site(s) of Rev (11-20), the intracellular localization of the peptide was investigated. The result showed that Rev (11-20) rapidly accumulated in the fungal cell surface. The cell wall regeneration test also indicated that Rev (11-20) exerted its anticandidal activity to fungal plasma membrane rather than cell wall. The fluorescent study using 1,6-diphenyl-1,3,5-hexatriene (DPH) further confirmed the membrane-disruption mechanism(s) of Rev (11-20). The present study suggests that Rev (11-20) possesses significant potential regarding therapeutic agents for treating fungal diseases caused by Candida species in humans.

Structural analysis of ribonucleopeptide aptamer against ATP

A ribonucleopeptide aptamer against ATP was obtained by the in vitro selection method. This ribonucleopeptide aptamer comprises a randomized and selected RNA linked to the Rev-responsive element (RRE) in complex with a peptide derived from an HIV Rev protein. The ribonucleopeptide aptamer selectively binds ATP in the presence of the Rev-derived peptide, exclusively. Here, we present the structural analysis of the ribonucleopeptide aptamer with NMR. The secondary structure of the RNA part of the aptamer, the selected RNA region linked to RRE, in the presence of the Rev-derived peptide was determined in an Ado-bound form. G:A and G:G base pairs, together with canonical base pairs, are formed in a duplex of RRE. The selected RNA region plays a crucial role in target binding. It has been found that the two U residues located in the selected RNA region trap Ado through the formation of the U:A:U base triple. This was directly confirmed by the HNN-COSY experiment through the detection of spin-spin couplings across the hydrogen bonds for Watson-Crick and Hoogsteen A:U base pairs in the U:A:U base triple.

Nuclear organization and the regulation of HIV-1 gene expression

Regulation of gene expression is profoundly involved in HIV-1 pathogenesis. This retrovirus integrates into host chromatin in order to transcribe and replicate it's genome. Lymphocytes harboring a quiescent but inducible provirus are a challenge to viral eradication in infected patients undergoing antiviral therapy. Therefore our understanding of the contribution of sub-nuclear positioning to viral gene expression may have far reaching implications also in the pathology of the infection. In order to gain insight in the conformation of chromatin at the site of HIV-1 integration we investigated lymphocytes carrying a single latent provirus. In the silenced state the provirus was consistently found at the nuclear periphery, associated in trans to pericentromeric heterochromatin in a significant number of quiescent cells. After induction of transcription, this association was lost, although the location of the transcribing provirus remained peripheral. These results unveil a novel mechanism of transcriptional silencing involved in HIV-1 post-transcriptional latency and reinforce the notion that gene transcription may occur also at the nuclear periphery. However, transcription is not the only limiting process during viral gene expression. Both unspliced and spliced viral RNAs need to be exported for efficient viral replication. In particular unspliced viral RNAs are retained in the nucleus until the viral Rev proteins mediate their export. Both RNA binding proteins and RNA hyperediting by adenosine deamination have been involved in the process of nuclear retention. In order to gain insights into this still poorly characterized pathway, we explored the proteome associated with the unspliced HIV-1 RNAs that are retained in the nucleus. We show that the viral RNA is associated with the host factors PSF/p54nrb and MATR3. PSF/p54nrb bind the viral RNA co-transcriptionally but MATR3 defines a subnuclear compartment where the viral RNA is delivered. Through this pathway, Rev is able to associate with unspliced HIV RNA directing its nuclear export. Interestingly PSF/p54nrb binding and localization to MATR3 foci occur independently of RNA hyperediting. Our findings reveal a novel cellular mechanism of nuclear retention that is hijacked by a virus.

An isostructural G-G to A-A substitution within the HIV RRE RNA switches the specificity towards arginine-rich peptides

The HIV Rev protein utilizes a short alpha-helical arginine-rich RNA-binding domain to bind deeply within the major groove of an internal loop region of the Rev-response element (RRE) RNA. A G48-G71 base-pair which covaries to an isostructural A48-A71 base pair has been shown to play an important structure role in Rev-RRE binding. On the other hand, a high affinity RRE-binding peptide aptamer, the K1 peptide, was shown to have low binding affinity towards the RRE A48-A71 mutant, suggesting that the K1 peptide was recognizing the G48-G71 base-pair. In this study, in an attempt to understand the basis for the recognition of the G48-G71 base-pair by the K1 peptide, the selection of peptides that bind to the RRE A48A71 (RREAA) mutant was carried out. As a result, a peptide specific for the mutant, the LDN1 peptide, was identified. The LDN1 peptide was found to bind to the internal loop region of the RREAA, as in the case of the K1-RRE interaction. However, amino acids important for LDN1-binding to RREAA, were found to be distinct from those important for K1-binding to the RRE. These results demonstrate how subtle changes in RNA structure can dramatically alter the peptide-binding specificity of an RNA.

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.

Thermodynamic Profiling of HIV RREIIB RNA–Zinc Finger Interactions

The interactions between the HIV Rev-responsive element (RRE) RNA and the HIV regulatory protein Rev, are crucial for the HIV life-cycle. Earlier, we showed that single C 2H 2 zinc fingers (znfs) have the same binding site as the Rev peptide and exhibit nanomolar affinity. In this study, the specific role of amino acid side chains and molecular processes involved with complex formation were investigated by perturbation of the binding energetics via changes in temperature, pH, buffers, and salt concentrations, as well as znf and RNA mutations, by isothermal titration calorimetry. Interestingly, despite the large cationic charge on the znfs, the number of interactions with the RNA phosphate backbone was lower than intuitively expected. The presence of binding induced protonation was established by ITC and localized by NMR to a histidine on the znf β-sheet. The Δ C p of znf–RNA binding was observed to be substantially negative and could not be accounted for by conventional solvent-accessible surface area models. An alternative model, based on the extent of hydrogen bond changes as a result of differences in ligand-induced water displacement at the binding site, provided reasonable explanation of the trends in Δ C p, as well as Δ H and Δ S. Our studies show that incorporation of favorable interactions at the solvent-excluded binding interface can be used to alleviate the unfavorable enthalpic penalties of displacing water molecules from the hydrated RNA surface.

Water, Shape Recognition, Salt Bridges, and Cation–Pi Interactions Differentiate Peptide Recognition of the HIV Rev-Responsive Element

Recognition of the human immunodeficiency virus Rev-responsive element (RRE) RNA by the Rev protein is an essential step in the viral life cycle. Formation of the Rev–RRE complex signals nucleocytoplasmic export of unspliced and partially spliced viral RNA. Essential components of the complex have been localized to a minimal arginine-rich Rev peptide and stem IIB of RRE. In vitro selection studies have identified a synthetic peptide known as RSG 1.2 that binds with better specificity and affinity to RRE than the Rev peptide. NMR structures of both peptide–RNA complexes of Rev and RSG 1.2 bound to RRE stem IIB have been solved and reveal gross structural differences between the two bound complexes. Molecular dynamics simulations of the Rev and RSG 1.2 peptides in complex with RRE stem IIB have been simulated to better understand on an atomic level how two arginine-rich peptides of similar length recognize the same sequence of RNA with such different structural motifs. While the Rev peptide employs some base-specific hydrogen bonding for recognition of RRE, shape recognition, through contact with the sugar-phosphate backbone, and cation–pi interactions are also important. Molecular dynamics simulations suggest that RSG 1.2 binds more tightly to the RRE sequence than Rev by forming more base-specific contacts, using water to mediate peptide–RNA contacts, and is held in place by a strong salt bridge network spanning the major groove of the RNA.

Novel Translation Products from Simian Immunodeficiency Virus SIVmac239 Env-Encoding mRNA Contain both Rev and Cryptic T-Cell Epitopes

Understanding the correlates of immune protection against human immunodeficiency virus and simian immunodeficiency virus (SIV) will require defining the entire cellular immune response against the viruses. Here, we define two novel translation products from the SIV env mRNA that are targeted by the T-cell response in SIV-infected rhesus macaques. The shorter product is a subset of the larger product, which contains both the first exon of the Rev protein and a translated portion of the rev intron. Our data suggest that the translation of viral alternate reading frames may be an important source of T-cell epitopes, including epitopes normally derived from functional proteins.

Structure of the HIV‐1 Rev response element alone and in complex with regulator of virion (Rev) studied by atomic force microscopy

The interaction of multiple HIV-1 regulator of virion (Rev) proteins with the viral RNA target, the Rev response element (RRE), is critical for nuclear export of incompletely spliced and unspliced viral RNA, and for the onset of the late phase in the viral replication cycle. The heterogeneity of the Rev-RRE complex has made it difficult to study using conventional structural methods. In the present study, atomic force microscopy is applied to directly visualize the tertiary structure of the RRE RNA alone and in complex with Rev proteins. The appearance of the RRE is compatible with the earlier proposed RRE secondary structure in dimensions and overall shape, including a stalk and a head interpreted as stem I, and stem-loops II-V in the secondary structure model, respectively. Atomic force microscopy imaging of the Rev-RRE complex revealed an increased height of the structure both in the stalk and head regions, which is in accordance with a binding model in which Rev binding to a high affinity site in stem IIB triggers oligomerization of Rev proteins through cooperative binding along stem I in RRE. The present study demonstrates that atomic force microscopy comprises a useful technique to study complex biological structures of nucleic acids at high resolution.

Transcriptional Errors in Human Immunodeficiency Virus Type 1 Generate Targets for T-Cell Responses

We measured T-cell responses to human immunodeficiency virus type 1 (HIV-1) cryptic epitopes encoded by regions of the viral genome not normally translated into viral proteins. T-cell responses to cryptic epitopes and to regions normally spliced out of the HIV-1 viral proteins Rev and Tat were detected in HIV-1-infected subjects.