The regulation of export of mRNA from nucleus to cytoplasm.

The past year has been marked by the discovery that the influenza virus NS1 protein belongs to the group of viral proteins that regulate the nuclear export of mRNA. This protein, like other viral proteins in this group, such as the Rev protein of human immunodeficiency virus 1 (HIV-1) and the complex of two adenovirus early proteins, has the potential to provide insights into the poorly understood process of the nuclear export of mRNA.

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.

The eps15 homology (EH) domain-based interaction between eps15 and hrb connects the molecular machinery of endocytosis to that of nucleocytosolic transport.

The Eps15 homology (EH) module is a protein-protein interaction domain that establishes a network of connections involved in various aspects of endocytosis and sorting. The finding that EH-containing proteins bind to Hrb (a cellular cofactor of the Rev protein) and to the related protein Hrbl raised the possibility that the EH network might also influence the so-called Rev export pathway, which mediates nucleocytoplasmic transfer of proteins and RNAs. In this study, we demonstrate that Eps15 and Eps15R, two EH-containing proteins, synergize with Hrb and Hrbl to enhance the function of Rev in the export pathway. In addition, the EH-mediated association between Eps15 and Hrb is required for the synergistic effect. The interaction between Eps15 and Hrb occurs in the cytoplasm, thus pointing to an unexpected site of action of Hrb, and to a possible role of the Eps15-Hrb complex in regulating the stability of Rev.

Localization of HIV-1 RNA in mammalian nuclei.

The Rev protein of human immunodeficiency virus type 1 (HIV-1) facilitates the nuclear export of unspliced and partially spliced viral RNAs. In the absence of Rev, these intron-containing HIV-1 RNAs are retained in the nucleus. The basis for nuclear retention is unclear and is an important aspect of Rev regulation. Here we use in situ hybridization and digital imaging microscopy to examine the intranuclear distributions of intron-containing HIV RNAs and to determine their spatial relationships to intranuclear structures. HeLa cells were transfected with an HIV-1 expression vector, and viral transcripts were localized using oligonucleotide probes specific for the unspliced or spliced forms of a particular viral RNA. In the absence of Rev, the unspliced viral RNAs were predominantly nuclear and had two distinct distributions. First, a population of viral transcripts was distributed as approximately 10-20 intranuclear punctate signals. Actinomycin D chase experiments indicate that these signals represent nascent transcripts. A second, stable population of viral transcripts was dispersed throughout the nucleoplasm excluding nucleoli. Rev promoted the export of this stable population of viral RNAs to the cytoplasm in a time-dependent fashion. Significantly, the distributions of neither the nascent nor the stable populations of viral RNAs coincided with intranuclear speckles in which splicing factors are enriched. Using splice-junction-specific probes, splicing of human beta-globin pre-mRNA occurred cotranscriptionally, whereas splicing of HIV-1 pre-mRNA did not. Taken together, our results indicate that the nucleolus and intranuclear speckles are not involved in Rev regulation, and provide further evidence that efficient splicing signals are critical for cotranscriptional splicing.

HIV-1 regulatory/accessory genes: keys to unraveling viral and host cell biology.

Human immunodeficiency virus type-1 (HIV-1) manipulates fundamental host cell processes in sophisticated ways to achieve optimum replicative efficiency. Recent studies have provided new details on the molecular interactions of HIV-1 with its host cell. For example, HIV-1 encodes a protein that regulates transcriptional elongation by interacting with a cellular cyclin-dependent kinase, another that activates the specific nuclear export of viral RNA, and several others that affect the intracellular trafficking of viral and host cell proteins. Detailed analysis of the interplay between these viral proteins and normal cellular activities has provided new insights into central questions of virology and host cell biology.

Nuclear retention of multiply spliced HIV-1 RNA in resting CD4+ T cells.

HIV-1 latency in resting CD4+ T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART). We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4+ T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS) HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4+ T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4+ T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB) was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4+ T cells. Overexpression of PTB in resting CD4+ T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4+ T cells. Virus culture experiments showed that overexpression of PTB in resting CD4+ T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.

Regulation of HIV gene expression by RNA-protein interactions.

Human immunodeficiency virus (HIV) gene expression is tightly controlled through the interaction of trans-acting regulatory proteins with the many cis-acting elements present in viral DNA and RNA. Two proteins encoded by HIV, referred to as Tat and Rev, are essential positive regulators of gene expression. Recent work shows that these proteins control HIV gene expression through interaction with RNA target elements present within the 5' untranslated leader sequence and envelope gene, respectively. There is evidence that these interactions in themselves are not sufficient to confer regulation without the presence of additional host cell factors.

A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA.

A common feature of gene expression in all retroviruses is that unspliced, intron-containing RNA is exported to the cytoplasm despite the fact that cellular RNAs which contain introns are usually restricted to the nucleus. In complex retroviruses, the export of intron-containing RNA is mediated by specific viral regulatory proteins (e.g., human immunodeficiency virus type 1 [HIV-1] Rev) that bind to elements in the viral RNA. However, simpler retroviruses do not encode such regulatory proteins. Here we show that the genome of the simpler retrovirus Mason-Pfizer monkey virus (MPMV) contains an element that serves as an autonomous nuclear export signal for intron-containing RNA. This element is essential for MPMV replication; however, its function can be complemented by HIV-1 Rev and the Rev-responsive element. The element can also facilitate the export of cellular intron-containing RNA. These results suggest that the MPMV element mimics cellular RNA transport signals and mediates RNA export through interaction with endogenous cellular factors.

Modulation of HIV-1 Rev protein abundance and activity by polyubiquitination with unconventional Lys-33 branching.

The HIV-1 Rev protein plays a key role in virus replication by allowing export to the cytoplasm of unspliced or singly-spliced RNAs. In this report, we investigated whether Rev is modified by ubiquitination or sumoylation. Whereas no evidence of sumoylation was obtained, transient expression experiments showed that ubiquitin conjugates to Rev as high molecular weight polyubiquitin chains. Mutation of the three lysine residues of Rev showed that the site of ubiquitin conjugation is Lys-115. Experiments with ubiquitin mutants including a single lysine at every seven possible position indicated that branching of the polyubiquitin chains mainly involves Lys-33. Mutation of Rev Lys-115 to arginine reduces markedly the steady state amount of the protein, but does not impair its ability to export RNA via the Rev response element. These observations support the notion that polyubiquitination of Rev stabilizes the viral protein but hinders its activity.

Antiviral properties of combination peptides of HIV-1 Rev NLS and NES.

Nuclear translocation signal has been identified as a mediator of protein shuttling between nuclear and cytoplasm. Here we report that the combination of peptides from nuclear localization signal (NLS) and nuclear export signal (NES) of HIV-1 Rev have an antiviral activity against the Herpes virus of turkey and Marek's disease virus serotype 1.

Analysis of the tumor suppressor activity of the K-rev-1 gene in human tumor cell lines.

Overexpression of the human K-rev-1 gene in v-Ki-ras-transformed NIH 3T3 cells has been reported to result in the reversal of transformation and tumor suppression. To address whether human K-rev-1 is a tumor suppressor gene of human tumor cells, we have systematically transfected epitope-tagged wild-type or activated mutant K-rev-1 complementary DNA expression vectors into a series human tumor cell lines that express an activated ras oncogene, namely HT1080, EJ, and SW480. Using the epitope-tag-specific monoclonal antibody, it is shown that the K-rev-1 protein localizes to the medial/trans-Golgi network. Ectopic expression of the wild-type or activated mutant K-rev-1 protein did not significantly affect the morphology or in vitro growth of any clones. Furthermore, all clones expressing the wild-type or activated mutant K-rev-1 protein were tumorigenic. Western blot analysis of tumor reconstitutes demonstrated that there was no decrease or loss of introduced K-rev-1 protein expression. The results in the present study demonstrate that expression of K-rev-1 does not reverse the transformed phenotype or significantly affect the tumorigenic phenotype of human tumor cell lines that express endogenous ras oncogenes.

A cis-acting peptide signal in human immunodeficiency virus type I Rev which inhibits nuclear entry of small proteins.

A peptide signal, which may control nucleo-cytoplasmic protein trafficking, was newly identified in human immunodeficiency virus type I (HIV-1) Rev, a lentiviral post-transcriptional transactivator. The sequence, in the amino-terminal portion of HIV-1 Rev, maintains a Rev mutant with a dysfunctional nuclear/nucleolar targeting signal outside of the nucleus, although this Rev molecule itself is small enough to pass through the nuclear pores. Transition of this sequence to the N-terminus of human T-lymphocytic leukemia/lymphoma virus type I (HTLV-I) p21x, which is usually located evenly distributed throughout the cell, resulted in capture of p21x in the cytoplasm. Mutational analysis clarified that a 14 residue peptide sequence was sufficient to display this inhibitory effect against nuclear entry. Furthermore, this HIV-1 Rev sequence was capable of inhibiting nuclear entry of a fragment of a human ribosomal protein, when it was fused to the carboxy terminus. The identified nuclear entry inhibitory signal (NIS) contains a conserved hydrophilicity motif, which forms an amphipathic helix. Significantly, this motif and its helical structure were shown to be important for NIS function and the HIV-1 Rev function itself. Possible roles for NIS as a molecular anchor are proposed herein.

General effect of Sam68 on Rev/Rex regulated expression of complex retroviruses.

We have previously demonstrated that overexpression of Sam68 functionally substitutes for, as well as synergizes with, HIV-1 Rev in RRE-mediated gene expression and virus replication. In addition, C-terminal deletion mutants of Sam68 exhibit a transdominant negative phenotype in HIV replication. We now report that Sam68 also enhances the activities of Rev-like proteins of other complex retroviruses (e.g. HTLV-1 and EIAV) on their respective RNA targets. Furthermore, we demonstrate that Sam68 can function alone as well as synergize with Rev-MS2 and/or Rex-MS2 chimeric proteins on expression mediated by the corresponding RRE-MS2 fusion RNA element. Additionally, dominant negative mutants of Sam68 also repressed the synergistic activation of Sam68 with Rex, E-Rev, and/or Rev-MS2/Rex-MS2 on their corresponding RNA targets. Thus, Sam68 may play an important role in the post-transcriptional regulation of all complex retroviruses. Oncogene (2000) 19, 4071 - 4074

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.

Construction and expression of a Rev-dependent TNF-R1 expressing HIV-infected-cell injurious vectors.

BACKGROUND: Rev is necessary for exporting unspliced and incompletely spliced intron containing HIV mRNAs and for HIV replication. The aim of this study is to develop a kind of selective suicide construct that can specifically and directly induce HIV infected cells into apoptosis based on the high affinity of Rev and Rev response element (RRE). METHODS: Molecular-cloning technique was used to synthesis Rev dependent TNF-R1 expression construct pDM128-TNF-R1 (pT128) that contains RRE and TNFR1 gene. Restriction digestion, Polymerase Chain Reaction (PCR) and DNA sequencing were processed and the exactness and correctness of the inserted TNF-R1 gene in pT128 were confirmed repeatedly. The expression of pT128 co-transfected with different combination of other plasmids by calcium phosphate-DNA co-precipitation in Helas and by gene gun transfection in keratinocytes was further tested by flow-cytometry and cell counted under microscope. RESULTS: The new plasmid specifically expressed TNF-R1 in Helas when co-transfected with pRev but did not when without pRev. Indirect expression of TNF-R1 from pT128 was slower than the direct expression of that from Hu p60 TNFR1 in pDC302 (pT60), but all those pT60 or pT128 transfected cells showed apoptosis at last while TNF-R1 was sufficiently expressed. Other kinds of Rev expression construct such as pAD8 and a chimeric HIV vaccine also can switched on the selective expression of pT128. Not only Rev-dependent expression in Helas, pT128 also normally expressed its TNF-R1 in keratinocytes. Co-transfected with pRev or pAD8 that expressed Rev, pT128 expressed TNF-R1 and induced apoptosis of green fluorescent keratinocytes in skin explant. The number of green fluorescent keratinocytes co-transfected by pT128 plus pRev or pAD8 was gradually outnumbered by that co-transfected by pT128 only. The difference was more significant after culturing for 72 hours. CONCLUSIONS: Rev dependent pT128 is able to selectively induce apoptosis of HIV-infected or Rev-expressed target cells by expression of TNF-R1. The new strategy based on manipulation of the regulatory protein of HIV may be valuable in design of new HIV vaccine.

Novel Function of Rev-erbα in Promoting Brown Adipogenesis.

Brown adipose tissue is a major thermogenic organ that plays a key role in maintenance of body temperature and whole-body energy homeostasis. Rev-erbα, a ligand-dependent nuclear receptor and transcription repressor of the molecular clock, has been implicated in the regulation of adipogenesis. However, whether Rev-erbα participates in brown fat formation is not known. Here we show that Rev-erbα is a key regulator of brown adipose tissue development by promoting brown adipogenesis. Genetic ablation of Rev-erbα in mice severely impairs embryonic and neonatal brown fat formation accompanied by loss of brown identity. This defect is due to a cell-autonomous function of Rev-erbα in brown adipocyte lineage commitment and terminal differentiation, as demonstrated by genetic loss- and gain-of-function studies in mesenchymal precursors and brown preadipocytes. Moreover, pharmacological activation of Rev-erbα activity promotes, whereas its inhibition suppresses brown adipocyte differentiation. Mechanistic investigations reveal that Rev-erbα represses key components of the TGF-ß cascade, an inhibitory pathway of brown fat development. Collectively, our findings delineate a novel role of Rev-erbα in driving brown adipocyte development, and provide experimental evidence that pharmacological interventions of Rev-erbα may offer new avenues for the treatment of obesity and related metabolic disorders.

Activation of a diphtheria toxin A gene by expression of human immunodeficiency virus-1 Tat and Rev proteins in transfected cells.

Expression of a gene encoding the diphtheria toxin A (DT-A) fragment, controlled by tissue specific regulatory elements, has previously been used to kill selected cell populations. Here, we have examined the feasibility of controlling DT-A expression using regulatory systems from the human immunodeficiency virus (HIV-1) genome. Plasmids were constructed which express either DT-A or, as a model system, the luciferase (luc) reporter gene, under control of HIV-1 long terminal repeat (LTR) sequences (-167 to +80). While trans-activation by expression of the viral protein Tat was demonstrated, significant basal expression was observed. To reduce basal expression, cis-acting negative regulatory elements from the env region of the HIV-1 genome were inserted in the 3' untranslated region of both the luc and DT-A constructs. This dramatically reduced basal expression from the HIV LTR, and now both viral regulatory proteins Tat and Rev were required for maximal trans-activation. Such regulation of DT-A expression might be therapeutically applied to selectively kill HIV-infected cells in acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC).

Constitutive expression of chimeric neo-Rev response element transcripts suppresses HIV-1 replication in human CD4+ T lymphocytes.

We have previously reported that chimeric neomycin phosphotransferase (neo)-Rev response element (RRE) transcripts suppress the function of the human immunodeficiency virus type 1 (HIV-1) Rev trans-activator protein in HeLa cells. In an extension of these experiments, human CD4+ CEM cells (G418-resistant cell populations and clonal isolates) stably expressing chimeric neo-RRE genes (2, 3, or 6 RRE copies) were generated using retroviral-mediated gene transfer. The transduced CEM clones were infected with the HIV-1 HTLVIIIB isolate and the following three phenotypes were observed: (i) the transduced CEM cells were readily infected with HIV-1 indistinguishable from the control CEM cells; (ii) the appearance of HIV-1 replication markers was significantly delayed; (iii) no signs of HIV-1 replication were detectable although proviral HIV-1 DNA sequences could be detected in these cells. Furthermore, HIV antigen expression was limited in neo-resistant CEM cell populations inoculated with the HIV-1 HTLVIIIB isolate. Only 10% of the CEM-pX17-3xRRE cells and 20% of the CEM-pX17-2xRRE cells displayed HIV-1 antigens 43 days after challenge and had retained CD4 surface expression on 47% and 64% of the cells, respectively. In sharp contrast, 80% of the CEM-pX17 or the CEM-pX17-6xRRE cells expressed HIV-1 antigens but no CD4 antigens were detectable in these cultures. These results clearly indicate that RRE decoys could be developed into an effective somatic gene therapy approach against HIV-1 induced acquired immunodeficiency syndrome (AIDS).

Development of an Rev-independent, minimal simian immunodeficiency virus-derived vector system.

Lentiviral vectors are attractive candidates for gene therapy because of their ability to integrate into nondividing cells. To date, conventional HIV-1-based vectors can be produced at higher titers, but concerns regarding their safety for human use exist because of the possibility of recombination leading to production of infectious virions with pathogenic potential. Development of lentivirus vectors based on nonhuman lentiviruses constitutes an active area of research. We described a novel HIV-SIV hybrid vector system in which an HIV-1-derived transfer vector is encapsidated by SIVmac1A11 core particles and pseudotyped with VSV glycoprotein G. In an effort to further develop this vector system, we modified the packaging plasmid by deletion of the SIV accessory genes. Specifically, versions of the packaging plasmid (SIVpack) lacking vif, vpr, vpx, and/or nef were constructed. Our results indicate that, as with HIV-1-based packaging plasmids, deletion of accessory genes has no significant effect on transduction in either dividing or nondividing cells. The SIV packaging plasmid was also modified with regard to the requirement for RRE and rev. Deletion of the RRE and rev from SIVpack led to dramatic loss of transduction ability. Introduction of the 5' LTR from the spleen necrosis virus to packaging plasmids lacking RRE/Rev was then sufficient to fully restore vector titer. A minimal SIV transfer vector was also developed, which does not require RRE/Rev and exhibits no reduction in transduction efficiency in two packaging systems. The SIV-based vector system described here recapitulates the biological properties of minimal HIV-1-derived systems and is expected to provide an added level of safety for human gene transfer. We suggest that the SIV-derived vector system will also be useful to deliver anti-HIV-1 gene therapy reagents that would inhibit an HIV-1-derived vector.

Molecular and virological effects of intracellular anti-Rev single-chain variable fragments on the expression of various human immunodeficiency virus-1 strains.

A variety of genetic therapies or intracellular immunization techniques hold promise as modalities to inhibit human immunodeficiency virus type 1 (HIV-1) replication in vivo. We have recently demonstrated that a single-chain variable fragment (SFv) construct, derived from a monoclonal antibody that binds to the HIV-1 regulatory protein Rev, can be expressed intracellularly and potently inhibits HIV-1 replication. This single-chain intracellular antibody, which avidly binds to the effector domain of Rev, is now demonstrated to dramatically inhibit various diverse laboratory and primary clinical strains of HIV-1. Potent suppression of HIV-1 replication by this modality is maintained over several months in long-term cultures. As well, the intracellular expression of anti-Rev SFv is shown to alter HIV-1 replication by specifically affecting Rev function. Importantly, no alterations in HIV-1 internalization, reverse transcription, or initial transcription of multiply spliced viral mRNAs are demonstrated in SFv-immunized cells, as compared to controls. Thus, these studies extend the understanding of the molecular mechanisms involved in the inhibition of lentivirus replication, by these intracellular antibody constructs.