Dopamine stimulates expression of the human immunodeficiency virus type 1 via NF-kappaB in cells of the immune system.

Recent studies have reported that lymphocytes produce, transport and bind dopamine present in plasma. However, the action of dopamine on HIV-1 gene expression in cells of the immune system has not yet been examined. Here, we have investigated the regulation of HIV-1 expression by dopamine in Jurkat T cells and in primary blood mononuclear cells (PBMC). HIV-1 replication was increased by dopamine, which correlated with the increased levels of HIV-1 transactivation. Our transient expression data revealed that dopamine stimulated transcription through the NF-kappaB element present in the long terminal repeat. The importance of NF-kappaB sites was confirmed by using vectors containing wild-type or mutant kappaB sites in a heterologous promoter. Consistent with the role of NF-kappaB in mediating dopamine responsiveness, the proteasome inhibitor MG132 abolished dopamine-induced transcriptional activation. We further explored the effect of dopamine in the presence of phorbol esters or tumor necrosis factor-alpha (TNF-alpha) known to activate NF-kappaB. The combination of dopamine and TNF-alpha led to a stimulation of HIV-1 transcription and replication. However, in contrast with TNF-alpha, dopamine treatment did not affect NF-kappaB DNA binding activity nor the concentrations of p50, p65 and IkappaB-alpha proteins, which suggests a distinct NF-kappaB activation mechanism. These results reveal a new link between the dopamine system, cytokine signaling pathway and regulation of gene expression via the involvement of NF-kappaB in T cells and PBMC.

A novel candidate oncogene, MCT-1, is involved in cell cycle progression.

Using the arbitrarily primed-PCR (AP-PCR) assay to detect genetic abnormalities that occur in a panel of lymphoid cell lines, we identified an amplified stretch of genomic DNA that contained a putative open reading frame. Northern blot analysis with this genomic clone revealed widespread low level expression in normal human tissue. The full cDNA sequence was obtained with no significant homology to any known genes in the genome database. We termed this novel gene with multiple copies in a T-cell malignancy as MCT-1. MCT-1 was localized to the long arm of chromosome Xq22-24 by flourescence in situ hybridization analysis. Although there was no significant homology at the primary sequence level, there was a limited degree of amino acid homology with a domain of cyclin H that appears to specify protein-protein complexes. This relationship between MCT-1 and cyclin H implied a potential role for MCT-1 in cell cycle regulation. Overexpression of MCT-1 increased the proliferative rate of cells by decreasing the length of the G1 phase without a reciprocal increase in the S and G2-M phases. Recent work has established the role of cell cycle regulatory molecules in the development of certain human malignancies. Therefore, we investigated the transforming ability of MCT-1 overexpression using soft agar growth assays and demonstrated that only MCT-1-overexpressing cells were able to establish colonies. Taken together, MCT-1 is a novel candidate oncogene with homology to a protein-protein binding domain of cyclin H.

Retinoic acid modulates a bimodal effect on cell cycle progression in human adult T-cell leukemia cells.

Retinoids, the analogues of vitamin A, have a broad range of effects on different cell types. One biologically active form of vitamin A is all-trans-retinoic acid (ATRA), which binds to retinoic acid receptors, as does its intracellular metabolite, 9-cis-RA. Earlier studies have documented G1 cell cycle arrest and the induction of apoptosis in human adult T-cell leukemia cells after ATRA treatment. Previous work exploring the growth-inhibitory activity of ATRA in human malignancies has implicated several mechanisms that can arrest cells in the G1 phase of the cell cycle, including activation of p21Waf1 and inhibition of cyclin D1 expression. Therefore, we decided to examine the effects of ATRA exposure on G1 cell cycle components in human adult T-cell leukemia cells. Our data demonstrate a correlation between cyclin/cyclin-dependent kinase activity and subunit complex formation with duration of drug exposure. We also observed an increase in p53 protein levels that were not associated with an increase in p21Waf1 levels. Furthermore, we observed a differential effect on cell cycle progression that was temporally related to length of ATRA exposure. These observations, consistent with a bimodal effect of ATRA on cell cycle progression, may have important implications for the clinical application of ATRA.

Regulation of TNFalpha and TGFbeta-1 gene transcription by HIV-1 Tat in CNS cells.

Tat is a transcription transactivator produced by the human immunodeficiency virus type 1 (HIV-1) at the early phase of infection and plays a critical role in the expression and replication of the viral genome. This 86 amino acid protein, which can be secreted from the infected cells, has the ability to enter uninfected cells and exert its activity upon the responsive genes. Earlier results indicated that in addition to the HIV-1 promoter, Tat has the capacity to induce transcription of a variety of cellular genes. In this study, we demonstrate that exposure of cells from the central nervous system (U-87MG and SK-N-MC) and the lymphoid T cells (Jurkat) to highly purified Tat increases transcriptional activity of the reporter constructs containing the promoters from the transforming growth factor beta-1 (TGFbeta-1), the tumor necrosis factor alpha (TNFalpha), and the HIV-1 LTR. In addition, Tat treatment results in increased levels of TGFbeta-1 and TNFalpha mRNAs in these cells. Activation of the TGFbeta-1 and TNFalpha promoter constructs by Tat in U-87MG and SK-N-MC cells required amino acid residues 2 to 36 which spans the acidic and the cysteine-rich domains of Tat. In both CNS and lymphoid cells, the level of endogenous TGFbeta-1 mRNA was increased by mutant Tat protein containing amino acids 1 to 48 but not with a mutant Tat protein with a deletion between residues 2 to 36. TNFalpha mRNA level was increased by mutant Tat spanning residues 1 to 48 in U-87MG cells, but not in SK-N-MC and Jurkat cells. These observations suggest that activation of cellular and viral genes by Tat in various cells may be mediated by different pathways as evidenced by the requirements of the different regions of Tat. Activation of the TGFbeta-1 and TNFalpha promoters by wild-type Tat was severely affected by the mutant peptides spanning residues 2 to 36 and 1 to 48 suggesting that both truncated Tat peptides may function as dominant negative mutants over TNFalpha and TGFbeta-1 gene transcription. The importance of these findings in Tat-induced regulation of viral and cellular genes in various cell types is discussed.

Functional interaction between cyclin T1/cdk9 and Puralpha determines the level of TNFalpha promoter activation by Tat in glial cells.

In addition to its stimulatory effect on transcription of the HIV-1 LTR, the early protein of HIV-1, Tat, exhibits detrimental effects on the CNS by deregulating the expression of several cytokines and immunomodulators including TNFalpha. Activation of the viral promoter by Tat requires several cellular proteins including cyclin T1 and its partner, cdk9, which upon association with the TAR sequence of the LTR, forms a complex that enhances the activity of RNA polymerase II. Here, we examined the involvement of cyclin T1/cdk9 in Tat-mediated transcriptional activation of the TNFalpha promoter which has no TAR sequence. Results from transfection of human astrocytic cells revealed that both cyclin T1 and cdk9 stimulate the basal promoter activity of TNFalpha, although the level of such activation is decreased in the presence of Tat. Ectopic expression of Puralpha, a brain-derived regulatory protein which binds to Tat, enhanced the basal level of TNFalpha transcription, yet exerted a negative effect on the level of Tat activation of the TNFalpha promoter. The antagonistic effect of Puralpha and Tat upon the TNFalpha promoter was diminished in the presence of cyclin T1 and cdk9, suggesting cooperativity of Puralpha with cyclin T1 and cdk9 in Tat activation of the TNFalpha promoter. Results from protein-protein binding studies showed the interaction of Puralpha with both cyclin T1 and cdk9 through distinct domains of Puralpha which are in juxtaposition with each other. Interestingly, the site for cyclin T1 binding within Puralpha is adjacent to the region which is important for Tat/Puralpha association. In light of these observations, we propose a model which ascribes a bridging role for Puralpha in assembling Tat, cyclin T1, and cdk9 around the promoter region of TAR-negative genes such as TNFalpha, which is responsive to Tat activation.

p21(WAF1) gene promoter is epigenetically silenced by CTIP2 and SUV39H1.

Mainly regulated at the transcriptional level, the cellular cyclin-dependent kinase inhibitor, CDKN1A/p21(WAF1) (p21), is a major cell cycle regulator of the response to DNA damage, senescence and tumor suppression. Here, we report that COUP-TF-interacting protein 2 (CTIP2), recruited to the p21 gene promoter, silenced p21 gene transcription through interactions with histone deacetylases and methyltransferases. Importantly, treatment with the specific SUV39H1 inhibitor, chaetocin, repressed histone H3 lysine 9 trimethylation at the p21 gene promoter, stimulated p21 gene expression and induced cell cycle arrest. In addition, CTIP2 and SUV39H1 were recruited to the silenced p21 gene promoter to cooperatively inhibit p21 gene transcription. Induction of p21(WAF1) gene upon human immunodeficiency virus 1 (HIV-1) infection benefits viral expression in macrophages. Here, we report that CTIP2 further abolishes Vpr-mediated stimulation of p21, thereby indirectly contributing to HIV-1 latency. Altogether, our results suggest that CTIP2 is a constitutive p21 gene suppressor that cooperates with SUV39H1 and histone methylation to silence the p21 gene transcription.

Molecular mimicry in inducing DNA damage between HIV-1 Vpr and the anticancer agent, cisplatin.

The human immunodeficiency virus type 1 (HIV-1) viral protein R (vpr) gene is an evolutionarily conserved gene among the primate lentiviruses. Several functions are attributed to Vpr including the ability to cause cell death, cell cycle arrest, apoptosis and DNA damage. The Vpr domain responsible for DNA damage as well as the mechanism(s) through which Vpr induces this damage is unknown. Using site-directed mutagenesis, we identified the helical domain II within Vpr (aa 37-50) as the region responsible for causing DNA damage. Interestingly, Vpr Delta(37-50) failed to cause cell cycle arrest or apoptosis, to induce Ku70 or Ku80 and to suppress tumor growth, but maintained its capability to activate the HIV-1 LTR, to localize to the nucleus and to promote nonhomologous end-joining. In addition, our cytogenetic data indicated that helical domain II induced chromosomal aberrations, which mimicked those induced by cisplatin, an anticancer agent. This novel molecular mimicry function of Vpr might lead to its potential therapeutic use as a tumor suppressor.

p27(SJ), a novel protein in St John's Wort, that suppresses expression of HIV-1 genome.

Transcription of the HIV-1 genome is controlled by the cooperation of viral regulatory proteins and several host factors which bind to specific DNA sequences within the viral promoter spanning the long terminal repeat, (LTR). Here, we describe the identification of a novel protein, p27(SJ), present in a laboratory callus culture of Hypericum perforatum (St John's Wort) that suppresses transcription of the HIV-1 genome in several human cell types including primary culture of microglia and astrocytes. p27(SJ) associates with C/EBPbeta, a transcription factor that regulates expression of the HIV-1 genome in macrophages and monocytic cells, and the viral transactivator, Tat. The association of p27(SJ) with C/EBPbeta and Tat alters their subcellular localization, causing their accumulation in the perinuclear cytoplasmic compartment of the cells. Fusion of a nuclear localization signal to p27(SJ) forces its entry into the nucleus and diminishes the capacity of p27(SJ) to suppress Tat activity, but does not alter its ability to suppress C/EBPbeta activation of the LTR. Results from binding assays showed the inhibitory effect of p27(SJ) on C/EBPbeta interaction with DNA. Finally, our results demonstrate that expression of p27(SJ) decreases the level of viral replication in HIV-1-infected cells. These observations suggest the potential for the development of a therapeutic advance based on p27(SJ) protein to control HIV-1 transcription and replication in cells associated with HIV-1 infection in the brain.

p73 modulates HIV-1 Tat transcriptional and apoptotic activities in human astrocytes.

HIV-1 Tat is a potent transcriptional activator of the viral promoter with the ability to modulate a number of cellular regulatory circuits including apoptosis. Tat exerts its effects through interaction with viral as well as cellular proteins. Here, we studied the influence of p73, a protein that is implicated in apoptosis and cell cycle control, on Tat apoptotic function in the central nervous system. We recently demonstrated the ability of Tat to associate with p73, and that this association modulates Tat transcriptional activity (Amini et al., Mol Cell Biol 2005; 18: 8126-8138). We demonstrated that p73 interferes with Tat-mediated apoptosis by preventing the up-regulation of Bax and down-regulation of Bcl-2 proteins in astrocytes. Thus, the interplay between Tat and p73 may affect Tat contribution to apoptotic events in the brain, limiting its involvement in the neuropathology often observed in the brains of HIV-1 patients.

Transcription of the human transferrin gene in neuronal cells.

We have recently shown that a combination of three transcription factors governs the expression of the human transferrin gene in different brain cell types, oligodendrocytes, choroid plexus cells and neuronal cells. It was essential to elucidate the role of each factor in the regulation of transferrin gene transcription. Site-directed mutagenesis and co-transfection experiments in neuronal cells revealed that chicken ovalbumin upstream promoter transcription factor (COUP-TF), which binds to the promoter region I, acts as a repressor. Overexpression of the CCAAT/enhancer binding protein (C/EBP-alpha), which binds to the promoter region II, transactivates the -164/+1 promoter, even enables the -125/+1 region to promote transcription, and synergistically activates transcription in the presence of CREB. The C/EBP-alpha-mediated activation is antagonized by COUP-TF. The positive action of the cAMP response element-binding protein called CRI-BP is revealed by mutations of the central region I site which repress transcription. Moreover addition of dibutyryl cyclic AMP or overexpression of the catalytic subunit of protein kinase A increase transcription from the wild-type and not from the CRI mutant promoter, which shows that CRI-BP is responsible for mediating cAMP stimulation of Tf gene transcription.

Distinct positive and negative regulatory elements control neuronal and hepatic transcription of the human transferrin gene.

Transferrin (Tf), the iron-transport protein, plays an essential role in the central nervous system development, plasticity, and aging. As a first step toward elucidating the role of each transcription factor involved in the regulation of Tf gene expression, we have recently shown that similar promoter elements direct cell-type specific transcription in oligodendrocytes, epithelial choroid plexus cells, and in the neuronal cell line B103. Here we have analyzed the regulatory elements that control the level of expression of the Tf gene in neuronal cells. Transient expression experiments in B103 cells revealed that the -164/+1 promoter region is stimulated by a position-dependent -1140/-1000 upstream region. DNase I footprinting, gel retardation assays, and antibody reactivity data allowed us to characterize the nuclear factors interacting with this region. The upstream region I-binding protein (URI-BP) belongs to the steroid/retinoid receptor family, while URII-BP is a member of the nuclear factor I (NF-I) family. Interestingly, no enhancer nor silencer activity is detected in B103 cells. This contrasts with our findings in hepatoma cells, where the activity of the -125/+1 promoter can be repressed by a -1000/-819 upstream negative-acting region and stimulated by the -3600/-3300 enhancer. We demonstrate that the negative-acting region presents the characteristics of a silencer that interacts with a nuclear protein present in liver and absent in B103 cells. Similarly, B103 cells lack a nuclear protein able to bind to an essential site of the enhancer. This shows that in B103 cells, the inactivity of the silencer and the enhancer regions results from the absence of at least one essential nuclear protein.

Regulation of human immunodeficiency virus type 1 gene transcription by nuclear receptors in human brain cells.

Infection of cells of the central nervous system by the human immunodeficiency virus type-1 (HIV-1) leads to HIV-1-associated neuropathology. Recent studies have demonstrated the importance of long terminal repeat (LTR) binding sites in determining the pathogenicity of HIV. Here we have investigated the presence and the functional role of transcription factors that have the potential to interact, directly or indirectly, with the nuclear receptor-responsive element in the LTR of HIV-1, in different human cell lines of the brain. Cotransfection experiments showed that in oligodendroglioma TC-620 cells, the retinoic acid receptor and the retinoid X receptor activate LTR-driven transcription in the absence of ligand. Addition of all-trans- or 9-cis-retinoic acid reverses this effect. In contrast, in astrocytoma, neuronal, and microglial cells, no significant effect of the retinoid acid pathway was detected. This retinoid response is mediated by distinct molecular interactions in the lymphotropic LAI and the neurotropic JR-CSF HIV-1 strains. Moreover, retinoid receptors were found to antagonize the chicken ovalbumin upstream promoter transcription factor- as well as the c-JUN-mediated LTR transactivation. Our findings demonstrate the importance of the retinoic acid signaling pathway and of cross-coupling interactions in the repression of HIV-1 LTR gene expression.

Chicken ovalbumin upstream promoter transcription factor, a transcriptional activator of HIV-1 gene expression in human brain cells.

Viral infection of the central nervous system by the human immunodeficiency virus type 1 leads to a wide range of neuropathological disorders. However, the molecular mechanisms governing transcription of the human immunodeficiency virus type 1 genome in brain remain unclear. We have recently established that in brain cells, proteins belonging to the steroid/thyroid/retinoic acid receptor family bind to the -352 to -320 region of the long terminal repeat (LTR). Here, by supershift experiments, we have identified chicken ovalbumin upstream promoter transcription factor (COUP-TF), an orphan member of this nuclear receptor family, as one of the major proteins interacting with this LTR site. Cotransfection studies revealed that COUP-TF is able to dramatically activate LTR-directed gene transcription in human oligodendroglioma but not in astrocytoma cells. This activation occurs through two mechanisms, depending on the LTR sequence. Moreover, in neuronal cells COUP-TF and dopamine, a catecholamine neurotransmitter, enhance LTR-directed transcription by acting on the proximal LTR region. These results reveal the importance of COUP-TF and the dopamine signaling pathway as activators of human immunodeficiency virus type 1 gene expression in brain.

Transcription of the human immunodeficiency virus type 1 (HIV-1) promoter in central nervous system cells: effect of YB-1 on expression of the HIV-1 long terminal repeat.

Transcription of human immunodeficiency virus type 1 (HIV-1) is regulated by a series of host DNA-binding proteins which interact with the upstream viral promoter sequences, and the viral regulatory protein Tat which associates with TAR RNA sequences located in the leader of the viral transcript. Here we have examined the ability of the cellular protein YB-1 to modulate transcription of the HIV-1 promoter in a human astrocytic cell line (U-87MG), a neuronal cell line (SK-N-MC) and lymphoid cells (Jurkat) by transfection assay. Ectopic expression of YB-1 in U-87MG and SK-N-MC augments basal transcriptional activity of the viral sequence located between -80 and -43, which encompasses the GC-rich motif. In accord with the previous report, in Jurkat cells YB-1-mediated activation of the HIV-1 promoter required sequences which are located further upstream from the GC-rich motif. Combined overexpression of YB-1 and the GC-rich binding protein, SP1, in the transfected cells decreased the level of activation of the viral promoter, suggesting that YB-1 and SP1 may exert negative effects on each other's function. Results from band shift assay with purified YB-1 and SP1 indicated that SP1 and YB-1 bind to the GC-rich DNA sequence in the double-stranded and single-stranded configurations, respectively. However, efficient binding of SP1 to the double-stranded GC-rich motif corresponding to the HIV-1 long terminal repeat (LTR) is diminished in the presence of YB-1. Similarly, in the presence of SP1, YB-1 loses its ability to become associated with its target single-stranded DNA probe. No evidence for direct association of YB-1 and SP1 either in the presence or in the absence of DNA was observed. These data suggest that while YB-1 stimulates expression of the LTR in central nervous system cells, the level of activity of other cellular proteins, such as SP1, may dictate binding of YB-1 to its target sequence, and therefore affect the regulatory function of this protein.

Cooperative actions of HIV-1 Vpr and p53 modulate viral gene transcription.

Transcription of the human immunodeficiency virus type-1 (HIV-1) genome is controlled by cooperative interaction of viral encoded proteins and host regulatory proteins. In this study, we have examined the capacity of the viral auxiliary protein, Vpr, to modulate transcriptional activity of the HIV-1 promoter sequence located within the long terminal repeat (LTR). We demonstrate that ectopic expression of Vpr in human astrocytic cells, U-87MG, enhances the basal activity of the viral promoter in transfected cells and that the GC-rich sequences, spanning nucleotides -80 to -43, are important for this activity. Since this region serves as the target for p53-induced suppression of LTR activity and interacts with the ubiquitous transcription factor, Sp1, we examined the cooperative activity of Vpr, p53, and Sp1 upon LTR transcription. Results from co-transfection studies indicated that overexpression of wild type p53, but not mutant p53, decreases the level of activation of the LTR by Vpr. Transcriptional activation of the LTR by Vpr required the presence of Sp1 since overexpression of Vpr in cells with no endogenous Sp1 failed to augment LTR activity. Results from protein-protein interaction studies indicated that Vpr is associated with both p53 and Sp1 in cells with ectopic expression of these proteins. Moreover, it was evident that p53 and Sp1 interact with each other in these cells. These functional and structural studies provided a working model on the cooperative interaction of Vpr with cellular proteins Sp1 and p53 and control of viral gene transcription at immediate early stage of infection prior to the participation of other viral regulatory proteins.

Cooperative interaction between HIV-1 regulatory proteins Tat and Vpr modulates transcription of the viral genome.

The virion-associated protein of human immunodeficiency virus, type 1 (HIV-1), Vpr, is a small protein with 96 amino acid residues that has the ability to modulate transcription of HIV-1 long terminal repeat (LTR) promoter activity and affects several cellular functions. In this study we have employed molecular approaches to further investigate the mechanism by which Vpr exerts its regulatory effect upon the LTR. We show that by structural and functional interaction with Tat, a potent viral regulatory protein, Vpr synergistically enhances the transcriptional activity of the HIV-1 LTR. Because Tat utilizes cyclin T1 and its partner, CDK9 to elevate the level of transcription from the LTR, we examined the cooperativity between Vpr, Tat, and cyclin T1/CDK9 on viral gene transcription. Results from co-transfection studies indicated superactivation of LTR by Tat and cyclin T1/CDK9 in the presence of wild type Vpr. This activation was not observed with the R73S mutant of Vpr, which contains arginine to serine transition at residue 73. Interestingly, expression of R73S mutant in cells exerts a negative effect on the observed superactivation of the LTR by Tat, cyclin T1/CDK9, and wild type Vpr. Results from protein-protein interaction studies indicated that Vpr is associated with both Tat and cyclin T1 in cells expressing these proteins. Use of deletion mutant proteins in binding studies revealed that the binding sites for Tat and Vpr within cyclin T1 are distinct and that association of these two viral proteins with cyclin T1 is independent from each other. These observations suggest a working model on the cooperative interaction of Vpr with viral and cellular proteins and its involvement in control of viral gene transcription and replication. Moreover identification of R73S mutant of Vpr provides a new therapeutic avenue for controlling HIV-1 gene transcription and replication in the infected cells.

Identification of a cellular protein that binds to Tat-responsive element of TGF beta-1 promoter in glial cells.

Tat is a transcriptional transactivator produced by the human immunodeficiency virus type 1 (HIV-1) and plays a pivotal role in enhancing expression of the viral genome in the infected cells. Although initial studies have suggested that interaction of Tat with the transactivation responsive element (TAR); located within the LTR, is essential for Tat function, subsequent studies indicated that Tat has the ability to augment transcription of viral and cellular genes by a TAR-independent mechanism. In early studies we demonstrated that HIV-1 Tat stimulates transcription of the transforming growth factor, TGF beta-1, gene in glial cells. In this study, we have identified a cellular protein that interacts with the Tat-responsive region located between nucleotides -323 to -453 of the regulatory sequence of the TGF beta-1 promoter. Results from footprinting analysis revealed association of cellular proteins with the 130 nucleotide sequence located in the Tat-responsive region. Analysis of the associated protein by UV-crosslinking suggested the involvement of a protein between 40-45 kDa in size which preferentially interacts with the GC/GA rich sequence of the TGF beta-1 Tat-responsive sequence in a single-stranded configuration. The ability of the previously identified 40 kDa protein, named Pur alpha to bind to the GC/GA sequence in the single-stranded configuration, similar to those from TGF beta-1 promoter prompted us to investigate its binding capacity to the TGF beta-1 sequence and its transcriptional activity on the TGF beta-1 promoter. Results from band shift studies indicated the association of the bacterially produced Pur alpha to the TGF beta-1 DNA sequences positioned within the Tat-responsive region. Overexpression of Pur alpha in glial cells constitutively producing Tat augmented transcription of the TGF beta-1 gene. These results are consistent with previous reports on the cooperative action of Pur alpha and Tat in modulating other eukaryotic promoters. The importance of these findings with regard to deregulation of other cellular genes by HIV-1 Tat is discussed.

Transdominant activity of human immunodeficiency virus type 1 Vpr with a mutation at residue R73.

The 96-amino-acid-long human immunodeficiency virus type 1 virion-encoded accessory protein Vpr is of particular interest, as this protein, which is found in association with viral particles, can exert a regulatory effect on both virus replication and host cell function. Evidently, Vpr, through interaction with several host regulatory proteins, can modulate transcription from the viral long terminal repeat promoter. Expression of Vpr in cells results in deregulation of cell proliferation during the cell cycle pathway at the G(2) stage. Vpr has unique structural features consisting of multiple functional domains. In this study, we have focused on the leucine/isoleucine-rich domain near the carboxyl terminus of Vpr at residue 73 (arginine) and have demonstrated that alterations at this residue result in ablation of transcriptional activity of Vpr and its ability to block cell cycle events at the G(2) stage. Interestingly, substitution mutations at R73 have resulted in a peptide with dominant negative activities on wild-type functions in transcription and host proliferation events. Results from in vitro and in vivo protein-protein interaction studies have revealed that functionally inactive mutant Vpr can be associated with wild-type protein, presumably through the N-terminal regions of the protein which have been shown to be important for Vpr oligomerization. Thus, it is likely that complexation of the mutant Vpr with wild-type protein functionally inactivates Vpr. The importance of these findings in light of the development of therapeutic strategies is discussed.

Brain-specific expression of the human transferrin gene. Similar elements govern transcription in oligodendrocytes and in a neuronal cell line.

We have identified the regulatory sequences that govern the expression of the human transferrin gene in cultured brain cells and compared them with the data obtained with the neuronal cell line B103. Oligodendrocytes and epithelial choroid plexus cells from rat brain were cultured and used for transient expression experiments. Deletion analysis of 1.8 kilobase pairs of the 5' regulatory sequences revealed a -1530/-1140 positive-acting region in oligodendrocytes. The -164/+1 promoter region was sufficient to confer cell type-specific transcription in oligodendrocytes, epithelial choroid plexus cells, and B103 cells. DNase I footprinting experiments revealed three protected sequences, the proximal regions I and II, and the central region I. Gel retardation and antibody reactivity data allowed us to identify most of the nuclear factors present in oligodendrocytes interacting with the promoter sequences. Chicken ovalbumin upstream promoter transcription factor, a CAAT/enhancer-binding protein, and a cAMP response element-binding protein called CRI-BP interact with the proximal regions I and II and central region I sites, respectively. These data confirm the results obtained with the neuronal cell line and emphasize the importance of the three promoter elements for the transferrin gene-specific expression in the central nervous system compared with only two elements required for liver- and testis-specific expression.

Suppression of HIV-1 transcription and replication by a Vpr mutant.

Vpr, the 96 amino acid long protein represents one of the auxiliary proteins of human immunodeficiency virus type-1 (HIV-1), which exhibits the ability to increase the rate of replication of the virus in T cells. Structurally, this protein is composed of several regions such as the acidic domain with alpha helix at the amino terminus, leucine-isoleucine-rich domain (LR) near the carboxyl terminus and an arginine-rich domain at the C-terminus. Here, we evaluated the ability of wild-type and a spectrum of Vpr mutants with altered amino acid residues within the three major domains of Vpr to regulate of transcription of the HIV-1 LTR. Our results revealed that alterations of amino acids within the LR domain at position 73 from arginine to serine, renders Vpr defective in stimulating transcription of the viral promoter in human T-lymphocytic and astrocytic cells. Mutations within the N- and C-terminal domains had little or no effect on the transcriptional activity of Vpr. Of interest, ectopic expression of this mutant protein exerts a negative effect on the ability of wild-type Vpr, as well as the viral transactivator, Tat, in augmenting viral gene transcription. Production of the mutant Vpr interferes with the replication of the wild-type and delta Vpr virus in the cells. Accordingly, a Vpr mutant virus containing the transition of arginine to serine at position 73 exhibited an inhibitory effect on the replication of wild-type virus. Our results provide a new avenue for the utilization of the variant of the HIV-1 regulatory protein, Vpr, in suppressing replication of the viral genome in infected cells.