Regulation of Rev expression by the equine infectious anaemia virus tat-rev mRNA Kozak sequence and its potential influence on viral replication.

Rev, an important accessory protein of equine infectious anaemia virus (EIAV), induces the nuclear export of incompletely spliced viral mRNAs. Rev is translated from the tat-rev mRNA through leaky scanning of the tat CUG. In this study, the function of the Kozak sequence at the beginning of the rev ORF was investigated. Deletion or attenuation of the Kozak sequence resulted in expression of an N-terminal 11 aa-truncated Rev in addition to WT Rev. Truncated Rev displayed weaker promotion of Gag expression and processing than WT Rev. Furthermore, EIAV rescued from an infectious molecular clone (pEIAVUK3) with Kozak attenuation exhibited decreased viral replication in host cells in vitro. These results provide a new understanding of the relationship between EIAV Rev expression and viral replication.

Foamy virus vectors expressing anti-HIV transgenes efficiently block HIV-1 replication.

Gene therapy has the potential to control human immunodeficiency virus (HIV) in patients who do not respond to traditional antiviral therapy. In this study, we tested foamy virus (FV) vectors expressing three anti-HIV transgenes, both individually and in a combination vector. The transgenes tested in this study are RevM10, a dominant negative version of the viral rev protein, Sh1, a short hairpin RNA directed against a conserved overlapping sequence of tat and rev, and membrane-associated C46 (maC46), a membrane-attached peptide that blocks HIV cell entry. FV vectors efficiently transduce hematopoietic stem cells and, unlike lentivirus (LV) vectors, do not share viral proteins with HIV. The titers of the FV vectors described in this study were not affected by anti-HIV transgenes. On a direct comparison of FV vectors expressing the individual transgenes, entry inhibition using the maC46 transgene was found to be the most effective at blocking HIV replication. A clinically relevant FV vector expressing three anti-HIV transgenes effectively blocked HIV infection in primary macrophages derived from transduced, peripheral blood CD34-selected cells and in a cell line used for propagating HIV, CEMx174. These results suggest that there are potential benefits of using FV vectors in HIV gene therapy.

hnRNP E1 and E2 have distinct roles in modulating HIV-1 gene expression.

Pre-mRNA processing, including 5' end capping, splicing, and 3' end cleavage/polyadenylation, are events coordinated by transcription that can influence the subsequent export and translation of mRNAs. Coordination of RNA processing is crucial in retroviruses such as HIV-1, where inefficient splicing and the export of intron-containing RNAs are required for expression of the full complement of viral proteins. RNA processing can be affected by both viral and cellular proteins, and in this study we demonstrate that a member of the hnRNP E family of proteins can modulate HIV-1 RNA metabolism and expression. We show that hnRNP E1/E2 are able to interact with the ESS3a element of the bipartite ESS in tat/rev exon 3 of HIV-1 and that modulation of hnRNP E1 expression alters HIV-1 structural protein synthesis. Overexpression of hnRNP E1 leads to a reduction in Rev, achieved in part through a decrease in rev mRNA levels. However, the reduction in Rev levels cannot fully account for the effect of hnRNP E1, suggesting that hmRNP E1 might also act to suppress viral RNA translation. Deletion mutagenesis determined that the C-terminal end of hnRNP E1 was required for the reduction in Rev expression and that replacing this portion of hnRNP E1 with that of hnRNP E2, despite the high degree of conservation, could not rescue the loss of function.

Discriminating in vitro cell fusion from cell aggregation by flow cytometry combined with fluorescence resonance energy transfer.

Expression of fusion proteins in the plasma membrane enables cells to bind and fuse with surrounding cells to form syncytia. Cell fusion can have important functional outcomes for the interacting cells, as syncytia formation does in AIDS pathogenesis. Studies on cell fusion would be facilitated by a quantitative method able to discriminate between cellular aggregates and bona fide fused cells in a cell population. Flow cytometry with fluorescence resonance energy transfer is applied here for analyzing fusion of HIV-1 envelope-expressing cells with CD4+ Jurkat cells. Fusion partners were labeled with the vital lipophilic fluorescent probes DiO (green) and DiI (red) and FRET is manifested by an enhancement of the DiI red fluorescence intensity in double fluorescent cells, thus allowing discrimination between fused and aggregated cells. The inhibitory effect of anti-CD4 monoclonal antibodies and the inhibitory peptide T-20 upon cell fusion were readily quantified by this technique. This method allows the distinction of fused and aggregated cells even when they are at low frequencies.

Novel Pol II fusion promoter directs human immunodeficiency virus type 1-inducible coexpression of a short hairpin RNA and protein.

We demonstrate a novel approach for coexpression of a short hairpin RNA (shRNA) with an open reading frame which exploits transcriptional read-through of a minimal polyadenylation signal from a Pol II promoter. We first observed efficient inducible expression of enhanced green fluorescent protein along with an anti-rev shRNA. We took advantage of this observation to test coexpression of the transdominant negative mutant (humanized) of human immunodeficiency type 1 (HIV-1) Rev (huRevM10) along with an anti-rev shRNA via an HIV-1-inducible fusion promoter. The coexpression of the shRNA and transdominant protein resulted in potent, long-term inhibition of HIV-1 gene expression and suppression of shRNA-resistant mutants. This dual expression system has broad-based potential for other shRNA applications, such as cases where simultaneous knockdown of mutant and wild-type transcripts must be accompanied by replacement of the wild-type protein.

Application of the synthetic aminosugars for glycodiversification: synthesis and antimicrobial studies of pyranmycin.

A divergent approach was employed for the synthesis of aminosugars, from which a novel library of aminoglycoside antibiotics (pyranmycins) was synthesized. Pyranmycins have comparable antibacterial activity as neomycin, a clinically used aminoglycoside antibiotic, against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Mycobacterium smegmatis. In addition, pyranmycins, like streptomycin, are bacteriocidal while isoniazid (INH) is bacteriostatic. Therefore, pyranmycins may provide new therapeutic options in the treatment against tuberculosis. Several members of pyranmycins also manifest modest anti-Tat and anti-Rev activities, which may aid in the development of new anti-HIV agents. Although the antibacterial activity of pyranmycins against aminoglycoside resistant bacteria is less than expected, the synthetic methodologies of utilizing a library of aminosugars can be a model for future studies of glycodiversification or glycorandomization.

Impact of antigen expression kinetics on the effectiveness of HIV-specific cytotoxic T lymphocytes.

Recent studies indicate that the time required for virus-infected cells to become vulnerable for the activity of CTL is of significance for the capacity of CTL to control ongoing viral reproduction. To investigate whether this applies to the effectiveness of HIV-1-specific CTL, we measured virus production in cultures containing CD4(+) T cells inoculated with HIV at low multiplicity of infection, and CTL directed against an early protein, Rev, or a late protein, RT. The Rev-specific CTL prevented at least 2 log(10) more HIV-1 production, in 10 days, than similar numbers of RT-specific CTL. To study how CTL effectiveness depends on variations in the potency of effector functions and kinetics of HIV protein expression, we developed a mathematical model describing CTL-target cell interactions during successive infection cycles. The results show that substantially higher CTL-mediated target cell elimination rates are required to achieve control as there is less time for CTL to act before infected cells release progeny virions. Furthermore, in vitro experiments with HIV recombinant viruses showed that the RT-specific CTL were at least as effective as the Rev-specific CTL, but only if the RT epitope was expressed as part of the early protein Nef. Together these results indicate that CTL control ongoing HIV reproduction more effectively if they are able to recognize infected cells earlier during individual viral replication cycles. This provides rationale for immunization strategies that aim at inducing, boosting or skewing CTL responses to early regulatory proteins in AIDS vaccine development.

Visna virus-induced activation of MAPK is required for virus replication and correlates with virus-induced neuropathology.

It is well accepted that viruses require access to specific intracellular environments in order to proliferate or, minimally, to secure future proliferative potential as latent reservoirs. Hence, identification of essential virus-cell interactions should both refine current models of virus replication and proffer alternative targets for therapeutic intervention. In the present study, we examined the activation states of mitogen-activated protein kinases (MAPKs), ERK-1/2, in primary cells susceptible to visna virus and report that virus infection induces and sustains activation of the ERK/MAPK pathway. Treatment of infected cells with PD98059, a specific inhibitor of the ERK/MAPK pathway, abolishes visna virus replication, as evidenced by extremely low levels of Gag protein expression and reverse transcriptase activity in culture supernatants. In addition, although visna virus-induced activation of MAPK is detectable within 15 min, early events of viral replication (i.e., reverse transcription, integration, and transcription) are largely unaffected by PD98059. Interestingly, further examination demonstrated that treatment with PD98059 results in decreased cytoplasmic expression of gag and env, but not rev, mRNA, highly suggestive of an ERK/MAPK-dependent defect in Rev function. In vivo analysis of ERK-1/2 activation in brains derived from visna virus-infected sheep demonstrates a strong correlation between ERK/MAPK activation and virus-associated encephalitis. Moreover, double-labeling experiments revealed that activation of MAPK occurs not only in cells classically infected by visna virus (i.e., macrophages and microglia), but also in astrocytes, cells not considered to be major targets of visna virus replication, suggesting that activation of the ERK/MAPK pathway may contribute to the virus-induced processes leading to neurodegenerative pathology.

Gene combination raises broad human immunodeficiency virus-specific cytotoxicity.

DNA plasmid immunization has the important advantage over traditional vaccines of making it possible to combine selected genes into one vaccine. The efficacy of a combination of DNA plasmids encoding the nef, rev, and tat HIV-1 regulatory genes in inducing cellular immune responses was analyzed in asymptomatic HIV-1-infected patients. Patients initially selected for having low or no detectable immune responses to Nef, Rev, or Tat antigens developed MHC class I-restricted cytolytic activities as well as enhanced bystander effects. The induction of memory cells against target cells infected with the whole HIV-1 genome was analyzed by using a pseudovirus HIV-1/murine leukemia virus (MuLV), and target cells infected with vaccinia virus carrying the respective gene. The most remarkable change observed after immunization with the gene combination was an increase in cytotoxic T lymphocyte (CTL) precursors to target cells infected with the whole HIV-1 genome. Infection by the pseudotype HIV-1/MuLV virus should result in a multitude of HIV-1 peptides presented on the target cell surface, representative of the in vivo situation. An in vitro assessment of the expression of the single and combined gene products showed that this was consistent with the induction of CTL responses in vivo. No clinical advantage or adverse effects were noted. Therapeutic effects of such immunization may become measurable by structured therapy interruption.

Diminished production of human immunodeficiency virus type 1 in astrocytes results from inefficient translation of gag, env, and nef mRNAs despite efficient expression of Tat and Rev.

Astrocytes infected with human immunodeficiency virus type 1 (HIV-1) produce only minimal quantities of virus. The molecular events that limit acute-phase HIV-1 infection of astrocytes were examined after inducing acute-phase replication by transfection with the pNL4-3 proviral plasmid. The levels of HIV-1 mRNA were similarly high in both astrocytes and HeLa cells, but astrocytes produced approximately 50-fold less supernatant p24 than HeLa cells. We found that diminished HIV-1 production in astrocytes resulted from inefficient translation of gag, env, and nef mRNAs that were efficiently transported to the cytoplasm. Tat- or Rev-dependent reporter constructs showed no defect in Tat or Rev function in astrocytes compared with HeLa cells. HIV-1 mRNAs were correctly spliced, but only Rev and Tat proteins were efficiently translated from their native mRNAs. Pulse-chase labelling and immunoblot experiments revealed no defect in protein processing, but levels of Gag, Env, or Nef protein expressed were dramatically reduced in astrocytes compared to HeLa cells. These results demonstrate that inefficient translation of HIV-1 structural proteins underlies the restricted infection of astrocytes. The efficient expression of functional Tat and Rev by astrocytes may contribute to HIV-1 neuropathogenesis.

The yeast nucleoporin rip1p contributes to multiple export pathways with no essential role for its FG-repeat region.

The FG-repeat domain of the yeast Rip1 protein (Rip1p) was identified initially as a possible target for the nuclear export signal (NES) of the HIV-1 Rev protein in a yeast two-hybrid assay. Rip1p is inessential, associated with nuclear pore complexes, and structurally related to the FG-nucleoporin family of pore proteins. It contributes to HIV-1 Rev-mediated RNA export and is also important for the export of heat shock RNAs at 42 degrees C. We show here that Rip1p is essential for the export of heat shock RNAs, and this function is fulfilled by the unique carboxyl terminus of Rip1p with no substantial contribution from the FG-repeat region. Genetic interactions between Rip1p and the RNA export mediator Gle1p are described, which support a role of the carboxyl terminus of Rip1p in poly(A)+ RNA export. Finally, this domain of Rip1p also contributes to Rev-mediated RNA export. The data suggest that Rip1p promotes the nuclear export of different classes of substrates by contributing to optimal pore function.

Yeast heat shock mRNAs are exported through a distinct pathway defined by Rip1p.

We reported previously that heat or ethanol shock in Saccharomyces cerevisiae leads to nuclear retention of most poly(A)+ RNA but heat shock mRNAs (encoding Hsp70 proteins Ssa1p and Ssa4p) are efficiently exported in a process that is independent of the small GTPase Ran/Gsp1p, which is essential for most nucleocytoplasmic transport. To gain further insights into proteins essential or nonessential for export of heat shock mRNAs, in situ hybridization analyses to detect mRNA and pulse-labeling of proteins were used to examine several yeast mutant strains for their ability to export heat shock mRNAs following stress. Rip1p is a 42-kD protein associated with nuclear pore complexes and contains nucleoporin-like repeat sequences. It is dispensable for growth of yeast cells under normal conditions, but we report that it is essential for the export of heat shock mRNAs following stress. When SSA4 mRNA was induced from a GAL promoter in the absence of stress, it was efficiently exported in a strain lacking RIP1, indicating that Rip1p is required for export of heat shock mRNAs only following stress. Npl3p, a key mediator of export of poly(A)+ RNA, was not required for heat shock mRNA export, whereas Rss1p/Gle1p, a NES-containing factor essential for poly(A)+ RNA export, was also required for export of heat shock mRNAs after stress. High-level expression of the HIV-1 Rev protein, but not of Rev mutants, led to a partial block in export of heat shock mRNAs following stress. The data suggest a model wherein the requirement for Npl3p defines the mRNA export pathway, the requirement for Rip1p defines a pathway used for export of heat shock mRNAs after stress, and additional factors, including Rss1p/Gle1p and several nucleoporins (Rat7p/Nup159p, Rat2p/Nup120p, and Nup145p/Rat10p), are required in both pathways.

Oligomerization of HIV-1 Rev mutants in the cytoplasm and during nuclear import.

Oligomerization of Rev molecules has been shown to be required for Rev function. In addition to a Western blot assay monitoring dimer formation, a new in vivo assay analyzing formation of Rev heteromers in the cytoplasm and during nuclear import is presented here. The oligomerization assay is based upon the ability of Rev mutants with an intact nuclear localization signal (NLS) to interact specifically with mutants with a defective NLS and translocate such mutants to the nuclear compartments. Several of the mutants previously reported to be oligomerization defective were found to mediate nuclear and nucleolar localization of the NLS mutant. The Rev mutant previously named M4 was the only mutant tested that did not translocate the mutant with a defective NLS to the nucleus. Furthermore, the predominantly cytoplasmic localization of the M4 mutant suggests that oligomerization is important for effective nuclear import of Rev.

B-cell epitopes in HIV-1 Tat and Rev proteins colocalize with T-cell epitopes and with functional domains.

We describe the characterization of the B-cell epitopes of HIV-1 regulatory proteins Tat and Rev. The prevalence of antibodies to these proteins among human immunodeficiency virus (HIV)-1-infected individuals was examined by enzyme-linked immunosorbent assay (ELISA) and by Western blotting. The Tat and Rev antibody-positive sera were selected for epitope mapping performed with partially overlapping synthetic peptides bound to polyethylene pins. Eighteen and twelve percent of HIV-infected individuals had antibodies against Tat or Rev, respectively. In Tat, four epitopic regions were identified, situated within amino acids 6-10 (PRLEP), 21-37 (ACTNCYCKKCCFHCQVC), 39-58 (ITKALGISYGRKKRRQRRRA) and 74-82 (TSQSRGDPT). The most frequently recognized epitopic regions were located in the middle of the protein. In Rev, the two most frequently recognized epitopic regions were near the amino terminus of the protein within amino acids 12-20 (LIRTVRLIK) and 38-49 (RRNRRRRWRERQ). A third epitope was mapped around amino acids 55-62 (ISERILGT) and a fourth around amino acids 78-83 (LERLTU). To analyze the specificity of Tat and Rev epitopes, soluble synthetic peptides representing the identified epitopes were used in an ELISA assay, and the recognition of most epitopes was shown to be specific for HIV-1-infected individuals. In addition, many of the Tat and Rev epitopes were shown to overlap with regions having functional activity or with regions previously identified as T-cell epitopes.

U1 small nuclear RNA chimeric ribozymes with substrate specificity for the Rev pre-mRNA of human immunodeficiency virus.

The in vivo effectiveness of ribozymes strongly depends on the correct choice of the vector molecule. High levels of expression, stability, active conformation, and correct cellular localization are the most important features for a ribozyme vector. We have exploited the utilization of the U1 small nuclear RNA (snRNA) as a vector for specifically targeting a ribozyme into the nucleus. The Rev pre-mRNA of human immunodeficiency virus type 1 was chosen as target for testing the activity of the Ul-ribozyme. The catalytic core of the hammerhead motif, plus the recognition sequences, substituted the stem-loop III of the U1 snRNA. The resulting construct displays efficient cleavage activity in vitro. In addition, in the in vivo system of Xenopus laevis oocytes, the Ul-chimeric ribozyme accumulates in large amounts in the nucleus and produces a considerable reduction of Rev pre-mRNA levels. The Rev-specific ribozyme was also inserted in a derivative of the Ul snRNA mutated in the region of pairing with the 5' splice site, such as to match it with the suboptimal splice junction of the Rev precursor. This construct shows more efficient reduction of Rev pre-mRNA in vivo than the wild-type U1 vector.

Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.

We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.

Binding of intracellular anti-Rev single chain variable fragments to different epitopes of human immunodeficiency virus type 1 rev: variations in viral inhibition.

Intracellular immunization to target the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev has been explored as a genetic therapy for AIDS. Efficient intracellular expression of rearranged immunoglobulin heavy and light chain variable regions of anti-Rev monoclonal antibodies, with various vectors, and subsequent inhibition of HIV-1 replication have been previously reported by our laboratories. To further understand the molecular mechanisms and effects that intracellular anti-Rev single chain variable fragments (SFvs) have against HIV-1, via blocking of Rev function, two anti-Rev SFvs which specifically bind to differing epitopes of the Rev protein have been cloned. One SFv binds to the Rev activation domain, and the second SFv binds to the distal C terminus of Rev in the nonactivation region. Further studies now demonstrate that both anti-Rev SFvs lead to variable resistance to HIV-1 infection. Although binding affinity assays demonstrated that the SFv which specifically recognizes the Rev activation domain (D8) had an extracellular binding affinity significantly lower than that of the SFv specific to the nonactivation region (D1O), the SFv D8 demonstrated more potent activity in inhibiting virus production in human T-cell lines and peripheral blood mononuclear cells than did SFv D10. Thus, extracellular binding affinities of an SFv for a target viral protein cannot be used to directly predict its activity as an intracellular immunization moiety. These data demonstrate potential approaches for intracellular immunization against HIV-1 infection, by efficiently blocking specific motifs of Rev to after the function of this retroviral regulatory protein. These studies extend the understanding of the effects, on a molecular level, of SFvs binding to critical epitopes of Rev and further suggest that rational design of SFvs, with interactions involving specific viral moieties which mediate HIV-1 expression, may hold promise for the clinical application of genetic therapies to combat AIDS.

Two species of Rev proteins, with distinct N termini, are expressed by caprine arthritis encephalitis virus.

Several cDNA clones representing alternatively spliced Rev-specific transcripts were isolated from a cDNA library prepared from Himalayan tahr cells infected with caprine arthritis encephalitis virus (CAEV). We previously characterized two rev-like cDNA species, d1 and d2, and a tat e1 cDNA containing the rev coding sequence downstream to the tat. In these cDNAs, the rev coding domain derives its amino terminus from the N terminus of env, which is spliced to the 3' open reading frame encoding the putative Rev protein. In this study, we report the genetic structure of a fourth rev-like cDNA (designated g1), which lacks the 5' env-derived sequences. All of these rev transcripts, including cDNA g1, increased the level of chloramphenicol acetyltransferase expression when cotransfected with a reporter plasmid containing the CAEV Rev-response element-spanning region downstream of the cat coding sequences. Western blot (immunoblot) analysis showed that each transfected cDNA species gave rise to a 16-kDa protein lacking env-encoded amino-terminal epitopes. In contrast, CAEV-infected Himalayan tahr cells expressed only a 20-kDa protein, whose N terminus, in contrast, is derived from the env. Moreover, only the 20-kDa protein was also detected in the mature CAEV virions. These observations suggest that the transcripts d1, d2, and e1 can potentially, in appropriate cellular context, encode two Rev isoforms differing in their N termini, whereas the g1 transcript encodes only the 16-kDa species. Elucidation of the significance of the 16-kDa Rev protein in CAEV biology must await further studies.

Expression of a protective gene-prolongs survival of T cells in human immunodeficiency virus-infected patients.

The resistance of acquired immunodeficiency syndrome (AIDS) to traditional drug therapy has prompted a search for alternative treatments for this disease. One potential approach is to provide genetic resistance to viral replication to prolong latency. This strategy requires the definition of effective antiviral genes that extend the survival of T cells in human immunodeficiency virus (HIV)-infected individuals. We report the results of a human study designed to determine whether a genetic intervention can prolong the survival of T cells in HIV-infected individuals. Gene transfer was performed in enriched CD4+ cells with plasmid expression vectors encoding an inhibitory Rev protein, Rev M10, or a deletion mutant control, deltaRev M10, delivered by gold microparticles. Autologous cells separately transfected with each of the vectors were returned to each patient, and toxicity, gene expression, and survival of genetically modified cells were assessed. Cells that expressed Rev M10 were more resistant to HIV infection than those with deltaRev M10 in vitro. In HIV-infected subjects, Rev M10-transduced cells showed preferential survival compared to deltaRev M10 controls. Rev M10 can therefore act as a specific intracellular inhibitor that can prolong T-cell survival in HIV-1-infected individuals and potentially serve as a molecular genetic intervention which can contribute to the treatment of AIDS.