Biomedical subjects

K Khalili

Publications and source records attributed to K Khalili.

At least 145 records · Page 8Linked to original sources

PubMed1994

Activation of HIV-1 transcription by Tat in cells derived from the CNS: evidence for the participation of NF-kappa B--a review.

Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of acquired immune deficiency syndrome (AIDS). The Tat protein of HIV-1 is a potent activator of transcription directed by the viral long terminal repeat. It has been widely reported that this activation requires a specific interaction between Tat and a RNA target termed TAR in the 5'-leader sequence of HIV-1 mRNAs. In this report we present data and describe results which illustrate that under appropriate conditions activation of transcription by Tat occurs independent of the TAR element. The ability to mediate TAR-independent transactivation by Tat is constitutive in some central nervous system cells and requires prior activation in others such as T lymphocytes. Evidence implicating a specific transcription factor in mediating Tat activation is also presented. Studies with site-directed mutants demonstrate that the RNA-binding domain of Tat is dispensable for TAR-independent activation of HIV-1. In contrast, the requirement for specific components of the Tat activation domain suggests that common targets exist for this viral activation factor to exert its activity in TAR-independent and TAR-dependent transactivation pathways of HIV-1 transcriptional activation. A working model of TAR-independent transactivation, which we believe may be responsible for the activation of cellular genes which contribute to AIDS pathology, is presented.

Base Sequence↗

PubMed1994

Transcription of a human neurotropic virus promoter in glial cells: effect of YB-1 on expression of the JC virus late gene.

We have isolated a partial recombinant cDNA clone from a HeLa expression library which encodes a protein capable of binding to the central region of the human neurotropic JC virus (JCV) enhancer/promoter, termed the B region. Sequence analysis revealed a complete homology of the partial cDNA clone to the N-terminal region, of a previously described DNA-binding protein, termed YB-1. Band shift analyses have indicated that the bacterially produced YB-1 interacts specifically with the double-stranded B oligonucleotide as well as the corresponding single-stranded DNA fragment representing the early promoter sequence. Further analysis indicated that the YB-1 protein binds specifically to the C/T-rich sequence of the B domain, which is located in close proximity to the TATA box within the virus enhancer/promoter. Results from cotransfection experiments demonstrated that the full-length (YB-1) but not the partial cDNA enhances expression of the JCV late (JCVL) promoter in glial cells. Cointroduction into glial cells of a recombinant expressing the YB-1 and JCVL deletion mutants indicated that removal of the C/T-rich sequence of the B domain reduces the level of activation of the virus promoter by YB-1. Further cotransfection experiments revealed that the virus transactivating protein T antigen appears to diminish the ability of YB-1 to activate JCVL gene expression. RNA studies indicated that YB-1 is expressed in several cell types and tissues. Examination of YB-1 RNA from mouse brain at various stages of development revealed high levels of YB-1 RNA at early stages of development and lower levels at all subsequent developmental stages.

Antigens, Viral, Tumor↗

PubMed1994

Central nervous system-derived cells express a kappa B-binding activity that enhances human immunodeficiency virus type 1 transcription in vitro and facilitates TAR-independent transactivation by Tat.

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent activator of long terminal repeat-directed transcription. While in most cell types, activation requires interaction of Tat with the unusual transcription element TAR, astrocytic glial cells support TAR-independent transactivation of HIV-1 transcription by Tat. This alternative pathway of Tat activation is mediated by the viral enhancer, a kappa B domain capable of binding the prototypical form of the transcription factor nuclear factor kappa B (NF-kappa B) present in many cell types, including T lymphocytes. Tat transactivation mediated by the kappa B domain is sufficient to allow replication of TAR-deleted mutant HIV-1 in astrocytes. The present study demonstrates the existence of kappa B-specific binding factors present in human glial astrocytes that differ from prototypical NF-kappa B. The novel astrocyte-derived kappa B-binding activity is retained on an HIV-1 Tat affinity column, while prototypical NF-kappa B from Jurkat T cells is not. In vitro transcription studies demonstrate that astrocyte-derived kappa B-binding factors activate transcription of the HIV-1 long terminal repeat and that this activation is dependent on the kappa B domain. Moreover, TAR-independent transactivation of HIV-1 transcription is reproduced in vitro in an astrocyte factor-dependent manner which correlates with kappa B-binding activity. The importance of the central nervous system-enriched kappa B transcription factor in the regulation of HIV-1 expression is discussed.

Astrocytes↗

PubMed1994

The tumor suppressor protein p53 strongly alters human immunodeficiency virus type 1 replication.

The p53 tumor suppressor gene product, a sequence-specific DNA-binding protein, has been shown to act as a transcriptional activator and repressor both in vitro and in vivo. Consistent with its role in regulating transcription are recent observations that the N-terminal acidic domain of p53 binds directly to the TATA box-binding protein subunit of the general transcription factor, TF IID. It is now demonstrated that wild-type p53 (wt-p53) inhibits human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-directed chloramphenicol acetyltransferase activity in a cotransfection assay system. Importantly, this effect of wt-p53 on the HIV-1 LTR was also demonstrated by in vitro transcription assays. In addition, the Sp1 sites and the TATA box of the HIV-1 LTR are demonstrated to be the primary sites involved with p53-induced effects on this viral promoter. The upstream elements of the HIV-1 LTR, including the nuclear factor kappa B (NF-kappa B) binding sites, decrease the p53-induced inhibitory effects on viral transcription. In the presence of the HIV-1 TAR sequence and Tat protein, the HIV-1 LTR also becomes less sensitive to wt-p53-induced inhibition. By using a retroviral vector delivery system, mutant forms of p53 genes were expressed in two HIV-1 latently infected cell lines, ACH-2 and U1. In the ACH-2 cell line, which is now demonstrated to contain an endogenous mutant form of p53 (amino acid 248, Arg to Gln), additional mutant p53 proteins did not alter HIV-1 replication. In U1 cells, which completely lack endogenous p53, overexpression of mutant p53 led to an increase in HIV-1 replication. Thus, these data indicate a possible functional role for wt-p53 and mutant p53 proteins in the control of HIV-1 replication patterns and proviral latency.

Base Sequence↗

PubMed1994

Identification and characterization of a novel GGA/C-binding protein, GBP-i, that is rapidly inducible by cytokines.

Immunosuppressive states with accompanying alterations in cytokine profiles have been postulated to play a vital role in the reactivation of viruses from latency. Cytokines regulate gene expression by activating transcription factors via well-characterized signal transduction pathways. In this study, we report the identification of a novel inducible protein, GBP-i, that binds to a double-stranded GGA/C-rich region of the transcriptional control region of the human papovavirus JC virus (JCV), specifically within the origin of viral DNA replication. GBP-i is distinct from previously characterized GC-box-binding proteins with respect to both its sequence specificity and its electrophoretic mobility on native and denaturing gels. GBP-i responds within 90 min to phorbol myristate acetate stimulation; however, unlike typical phorbol myristate acetate-inducible factors, this rapid induction is regulated primarily at the transcriptional level. Further, the induction of GBP-i appears to be widespread and mediated by many inflammatory cytokines, including interleukin-1 beta, tumor necrosis factor alpha, gamma interferon, and transforming growth factor beta. Interestingly, the induced protein acts as a transcriptional repressor in its native context in the JCVL promoter. However, when its binding sequence is transposed to a heterologous promoter, GBP-i appears to function as a transcriptional activator. The data presented here suggest a role for GBP-i in cytokine-mediated induction of viral and cellular genes.

Base Sequence↗

PubMed1993

The transcriptional enhancer element, kappa B, regulates promoter activity of the human neurotropic virus, JCV, in cells derived from the CNS.

Studies on the regulation of the human neurotropic virus (JCV) promoter, have been focused primarily on the 98 bp tandem repeat sequence which confers glial-specificity to viral gene expression. We demonstrate that a distinct regulatory element outside of the 98 bp region, which spans a stretch of 10 nucleotides (nt) (5'-GGGAATTTCC-3') increases transcriptional activity of JCV late (JCVL), and early (JCVE) promoters in glial cells. Sequence analysis of this motif reveals extensive homology to the kappa B sequence of HIV-1 (5'-GGGACTTTCC-3'). A DNA fragment corresponding to the 10 nt sequence of JCV exhibits transcriptional activity when placed upstream of the test promoter in glial cells. The induction mediated by this regulatory motif is moderately enhanced in response to phorbol 12-myristate 13-acetate (PMA) in glial cells. Band-shift and UV-crosslinking experiments suggest that glial cells constitutively produce proteins that specifically interact with the JCV kappa B, but not the HIV-1 kappa B motif. Treatment of cells with PMA results in formation of new complexes that are sensitive to the kappa B sequences derived from the JCV and HIV-1 genomes. These results suggest that the kappa B sequence located in the JCV genome may play a role in transcriptional regulation of JCV gene expression by interacting with inducible and uninducible nuclear proteins from glial cells.

Base Sequence↗

PubMed1993

TAR-independent activation of HIV-1 requires the activation domain but not the RNA-binding domain of Tat.

The Tat protein of HIV-1 is a potent activator of transcription directed by the viral long terminal repeat. In most cell types this activation requires a specific interaction between Tat and an RNA target termed TAR in the 5'-leader sequence of HIV-1 mRNAs. We have previously reported that in astrocytic cells Tat is capable of activating transcription in a TAR-independent manner through an alternative Tat-responsive element in the LTR (J. P. Taylor et al., EMBO J. 11(9), 3395-3403, 1992). In this report we demonstrate that TAR-independent activation by Tat can effectively bypass competition by decoy TAR RNA molecules. Studies with site-directed mutants demonstrate that the RNA-binding domain of Tat is dispensable for TAR-independent activation of HIV-1. In contrast, the requirement for specific components of the Tat activation domain suggests that common targets exist for this viral trans-activator to exert its activity in TAR-independent and TAR-dependent transactivation pathways of HIV-1 transcriptional activation.

Amino Acid Sequence↗

PubMed1993

Inhibition of human neurotropic virus (JCV) DNA replication in glial cells by camptothecin.

Progressive multifocal leukoencephalopathy is a subacute demyelinating disease of the central nervous system (CNS) resulting from opportunistic infections in immunocompromised patients infected with a common polyomavirus, JC virus (JCV). Unlike other polyomaviruses, JCV exhibits an unusually narrow tissue tropism by primarily infecting glial cells of the CNS. JCV DNA replication is similar to that of the well-characterized papovavirus, SV40, which requires the viral early protein T-antigen and host-replication factors including DNA polymerases and DNA topoisomerase I. In this study we have been able to effectively block replication of viral DNA in glial cells using camptothecin, a drug which inhibits DNA topoisomerase activity. Pulse-treatment of cells with non-toxic levels of camptothecin specifically blocks viral DNA replication with no inhibitory effect on host transcription and translation processes as examined by viral gene expression in the transfected cells. Furthermore, drug treatment of the cells exhibits no significant effect on DNA topoisomerase I gene transcription. We further demonstrate that repeated pulse-treatment of cells with the drug is required for complete blockage of viral DNA replication. The importance of these findings in the treatment of AIDS encephalopathy is discussed.

Antineoplastic Agents↗

PubMed1993

A developmentally regulated DNA-binding protein from mouse brain stimulates myelin basic protein gene expression.

Transcription of the myelin basic protein (MBP) gene is regulated in a cell-type-specific and developmental stage-specific manner during myelin formation in the murine central nervous system. The 5'-flanking region of the MBP gene contains several regulatory elements that differentially contribute to the cell-type-specific transcription of MBP in cells derived from the central nervous system. The proximal element, termed MB1, which is located between nucleotides -14 and -50 with respect to the RNA start site, has previously been shown to have characteristics of a cell-type-specific enhancer element. In this study, we used band shift and UV cross-linking assays to identify DNA-binding proteins in mouse brain nuclear extract which interact with the MB1 element. Fractionation of these extracts has allowed the identification of a 38- to 41-kDa nuclear protein, derived from mouse brain tissue at the peak of myelination, which specifically binds the MB1 DNA sequence. Fractions enriched in the MB1-binding protein have been shown to stimulate transcription of the MBP promoter in extract derived from HeLa cells. MB1 binding protein activity is expressed in a tissue-specific and development stage-specific pattern which coincides with the pattern of MBP transcription, suggesting that this protein may be a biologically relevant transcription factor for the MBP gene in vivo.

Aging↗

PubMed1993

Evidence for stimulation of the transforming growth factor beta 1 promoter by HIV-1 Tat in cells derived from CNS.

Infection by human immunodeficiency virus type 1 (HIV-1), the etiologic agent of the acquired immunodeficiency syndrome (AIDS), is often complicated with a high incidence of neurologic disorders. It is believed that HIV-1, in addition to infecting both macroglial and microglial cells, may influence the expression of several strategic genes of uninfected neighboring or latently infected brain cells. It is suspected that the viral-encoded transregulatory protein, Tat, facilitates cross-communications between these cells. In support of this concept, earlier studies demonstrated that Tat is released from the infected cells, and has the capacity to be taken up by the uninfected cells and exert its biological activity on the responsive gene. Recent studies in several laboratories suggest the involvement of Tat in altering the expression of a limited number of cellular regulatory factors which, in turn, may mediate the altered physiology of the cells. In this communication, we demonstrate the ability of the HIV-1 Tat protein to increase expression of transforming growth factor beta 1 (TGF-beta 1), a cytokine with potent immunosuppressive activity, in human astrocytic glial cells. Implications of the Tat-mediated induction of TGF-beta 1 expression and cytokine involvement in the regulation of immune response and central nervous system (CNS) pathology are discussed.

Astrocytes↗

PubMed1993

GA/GC-rich sequence confers Tat responsiveness to human neurotropic virus promoter, JCVL, in cells derived from central nervous system.

The human immunodeficiency virus type I (HIV-1) nuclear protein, Tat, is a potent transactivating factor that stimulates the rate of transcription of responsive promoters. Evidently, to exert its activity, Tat requires to be localized in close proximity of the transcription initiation site. Previous studies in our laboratory have demonstrated that Tat has the capacity to increase transcriptional activity of the late gene of a human neurotropic virus JC (JCV) in glial cells. In the present study, using deletion mutation analysis, we have identified a region upstream of the JCV late RNA start sites, termed upstream target (upTAR), that positively responds to Tat activation in glial cells. Using synthetic oligonucleotides spanning the upTAR sequence linked to a heterologous promoter, we have identified a GA/GC-rich region (GGAGGCGGAGGC) that confers TAT responsiveness preferentially on glial cell lines. Using gel mobility-shift and UV cross-linking assays, we have demonstrated that four major complexes (a-d) from glial and HeLa (non-glial) cells interact with the upTAR sequence. Whereas the molecular weights of the participant proteins in these complexes are similar in both glial and non-glial extracts, glial cells are enriched in proteins that form a major c complex. Interestingly, the participant proteins in complex c are developmentally regulated during brain development. The possible role of these proteins in increasing local concentrations of Tat in the vicinity of the JCV late RNA start sites is discussed.

Animals↗

PubMed1992

Activation of expression of genes coding for extracellular matrix proteins in Tat-producing glioblastoma cells.

The Tat protein of human immunodeficiency virus type 1 has been increasingly implicated in directly contributing to the disease AIDS by altering the expression of strategic cellular genes. In this study we demonstrate that the presence of the human immunodeficiency virus type 1 regulatory protein Tat is associated with a significant induction in the expression of certain protein components of the extracellular matrix in glial-derived cells. Northern blot analysis reveals that in cells expressing Tat there is a marked elevation in the steady-state RNA levels for fibronectin and types I and III collagen. Metabolic labeling of the Tat-producing cells demonstrates that this induction is also reflected at the level of protein synthesis. Transient transfection experiments indicate that the presence of Tat results in increased transcription of fibronectin and alpha I type I collagen promoters. Possible mechanisms for this phenomenon and their significance with regard to AIDS are discussed.

Collagen↗

PubMed1992

Analysis of the proximal transcriptional element of the myelin basic protein gene.

The gene encoding myelin basic protein (MBP) contains multiple activator sequences spanning upstream of its transcriptional initiation site which differentially promote transcription in glial cells. The proximal activator sequence, designated MB1, activates transcription in a glial cell type specific manner. This sequence resides between -14 to -50 with respect to the RNA initiation site of the MBP gene. We have identified within the MB1 sequence a 10-nucleotide domain, 5'-ACCTTCAAAG-3', that increases transcription of a test promoter in glial and Schwann cells. This proximal motif functions in both orientations and specifically interacts with a nuclear protein derived from glial cells. Results of in vivo competition experiments indicate that this 10-nucleotide motif positively contributes to the overall transcriptional activity obtained from the entire MBP promoter in glial cells.

Base Sequence↗

PubMed1992

TAR-independent transactivation by Tat in cells derived from the CNS: a novel mechanism of HIV-1 gene regulation.

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is essential for productive infection and is a potential target for antiviral therapy. Tat, a potent activator of HIV-1 gene expression, serves to greatly increase the rate of transcription directed by the viral promoter. This induction, which seems to be an important component in the progression of acquired immune deficiency syndrome (AIDS), may be due to increased transcriptional initiation, increased transcriptional elongation, or a combination of these processes. Much attention has been focused on the interaction of Tat with a specific RNA target termed TAR (transactivation responsive) which is present in the leader sequence of all HIV-1 mRNAs. This interaction is believed to be an important component of the mechanism of transactivation. In this report we demonstrate that in certain CNS-derived cells Tat is capable of activating HIV-1 through a TAR-independent pathway. A Tat-responsive element is found upstream within the viral promoter that in glial-derived cell lines allows transactivation in the absence of TAR. Deletion mapping and hybrid promoter constructs demonstrate that the newly identified Tat-responsive element corresponds to a sequence within the viral long terminal repeat (LTR) previously identified as the HIV-1 enhancer, or NF-kappa B domain. DNA band-shift analysis reveals NF-kappa B binding activity in glial cells that differs from that present in T lymphoid cells. Further, we observe that TAR-deleted mutants of HIV-1 demonstrate normal late gene expression in glial cells as evidenced by syncytia formation and production of viral p24 antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

A novel sequence-specific DNA-binding protein, LCP-1, interacts with single-stranded DNA and differentially regulates early gene expression of the human neurotropic JC virus.

We have identified a novel brain-derived single-stranded-DNA-binding protein that interacts with a region of the human neurotropic JC virus enhancer designated the lytic control element (LCE). This nuclear factor, LCP-1 (for lytic control element-binding protein 1), specifically recognizes the LCE, as determined by gel retardation assays. Alkylation interference showed that specific nucleotides within the LCE were contacted by LCP-1. Subsequent experiments revealed that point mutations within the LCE differentially affected LCP-1 binding. UV cross-linking and competition analysis suggested that the LCP-1 DNA-protein complexes were 50 to 52 and 100 to 120 kDa in size. Promoter mutations that affected LCP-1 binding reduced early mRNA transcription during the early phase of the lytic cycle. However, upon DNA replication in the presence of JC virus T antigen, when early mRNA initiation shifts to new locations indicative of the late phase, the LCP-1 mutations had no effect. We suggest that the JC virus early transcription unit is differentially regulated by LCP-1 prior to but not after DNA replication, suggesting a novel mechanism by which DNA structure regulates eukaryotic gene expression.

Base Sequence↗

PubMed1992

Evidence that a sequence similar to TAR is important for induction of the JC virus late promoter by human immunodeficiency virus type 1 Tat.

A specific RNA sequence located in the leader of all human immunodeficiency virus type 1 (HIV-1) mRNAs termed the transactivation response element, or TAR, is a primary target for induction of HIV-1 long terminal repeat activity by the HIV-1-derived trans-regulatory protein, Tat. Human neurotropic virus, JC virus (JCV), a causative agent of the degenerative demyelinating disease progressive multifocal leukoencephalopathy, contains sequences in the 5' end of the late RNA species with an extensive homology to HIV-1 TAR. In this study, we examined the possible role of the JCV-derived TAR-homologous sequence in Tat-mediated activation of the JCV late promoter (Tada et al., Proc. Natl. Acad. Sci. USA 87:3479-3483, 1990). Results from site-directed mutagenesis revealed that critical G residues required for the function of HIV-1 TAR that are conserved in the JCV TAR homolog play an important role in Tat activation of the JCV promoter. In addition, in vivo competition studies suggest that shared regulatory components mediate Tat activation of the JCV late and HIV-1 long terminal repeat promoters. Furthermore, we showed that the JCV-derived TAR sequence behaves in the same way as HIV-1 TAR in response to two distinct Tat mutants, one of which that has no ability to bind to HIV-1 TAR and another that lacks transcriptional activity on a responsive promoter. These results suggest that the TAR homolog of the JCV late promoter is responsive to HIV-1 Tat induction and thus may participate in the overall activation of the JCV late promoter mediated by this transactivation.

Base Sequence↗

PubMed1992

TAR-independent replication of human immunodeficiency virus type 1 in glial cells.

The molecular mechanisms involved in the replication of human immunodeficiency virus type 1 (HIV-1) may differ in various cell types and with various exogenous stimuli. Astrocytic glial cells, which can support HIV-1 replication in cell cultures and may be infected in vivo, are demonstrated to provide a cellular milieu in which TAR mutant HIV-1 viruses may replicate. Using transfections of various TAR mutant HIV-1 proviral constructs, we demonstrate TAR-independent replication in unstimulated astrocytic cells. We further demonstrate, using viral constructs with mutations in the tat gene and in the nuclear factor kappa B (NF-kappa B)-binding sites (enhancer) of the HIV-1 long terminal repeat, that TAR-independent HIV-1 replication in astrocytic cells requires both intact NF-kappa B moiety-binding motifs in the HIV-1 long terminal repeat and Tat expression. We measured HIV-1 p24 antigen production, syncytium formation, and levels and patterns of viral RNA expression by Northern (RNA) blotting to characterize TAR-independent HIV-1 expression in astrocytic glial cells. This alternative regulatory pathway of TAR-independent, Tat-responsive viral production may be important in certain cell types for therapies which seek to perturb Tat-TAR binding as a strategy to interrupt the viral lytic cycle.

Astrocytes↗

PubMed1991

A recombinant cDNA derived from human brain encodes a DNA binding protein that stimulates transcription of the human neurotropic virus JCV.

The human neurotropic virus JCV contains a 98-base pair repeat enhancer/promoter sequence that confers glial-specific transcription to the viral early and late promoters. The central region of this repeat, designated the B-domain, binds to a glial-derived nuclear protein that stimulates transcription of the viral promotor in vitro. We now report the isolation of a recombinant cDNA clone, termed glial factor-1 (GF1), from a brain expression library that encodes a novel protein which interacts with the JCV B-domain. Results from RNA studies indicate that the GF1 transcript is more abundant in brain than in other tissues and that the level of GF1 RNAs increases progressively during brain development. Cotransfection of the recombinant GF1 expressor plasmid with JCV promoters indicates that GF1 stimulates transcription of the JCV late promoter and to a lesser extent the JCV early promoter predominantly in cells of human glial origin. Thus, GF1 is a sequence-specific DNA binding protein that may play a role in determining the glial-specific expression of JCV.

Amino Acid Sequence↗