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C A Rosen

Publications and source records attributed to C A Rosen.

At least 19 recordsLinked to original sources
PubMed1992

Identification of a naturally occurring transforming variant of the p65 subunit of NF-kappa B.

Transcription factor NF-kappa B comprises two proteins, p50 and p65, that have sequence similarity to the v-rel oncogene. In primary hematopoietic cell populations an alternatively spliced form of NF-kappa B p65 mRNA was observed that encoded a protein designated p65 delta. Expression of the p65 delta cDNA in Rat-1 fibroblasts resulted in focus formation, anchorage-independent growth in soft agar, and tumor formation in athymic nude mice, effects not obtained with expression of p65 or a p65 delta mutant that contains a disruption within the transcriptional activation domain. Thus, p65 delta, which associated weakly and interfered with DNA binding by p65, may sequester an essential limiting regulatory factor or factors required for NF-kappa B function.

Animals↗
PubMed1992

HIV regulatory proteins: potential targets for therapeutic intervention.

With the incidence of HIV infection on the rise worldwide, it is obvious that new approaches must be taken to halt the spread of disease. Unfortunately, this is no easy task; of all retroviruses studied to date HIV remains the most complex in terms of genomic organization, regulation of gene expression, and replication. However, as the mechanism of action of the unique viral regulatory proteins is deciphered, new windows of opportunity for attacking the virus like cycle are opened. The essential regulatory function served by both Tat and Rev transacting regulatory proteins makes them attractive targets for prophylactic and therapeutic intervention. This review will focus on our current understanding of Tat and Rev function.

Animals↗
PubMed1992

I kappa B interacts with the nuclear localization sequences of the subunits of NF-kappa B: a mechanism for cytoplasmic retention.

NF-kappa B is an inducible transcription factor comprised of a 50-kD (p50) and a 65-kD (p65) subunit. Induction of NF-kappa B activity, which is a critical event in many signal transduction pathways, involves release from a cytoplasmic inhibitory protein, I kappa B, followed by translocation of the active transcription factor complex into the nucleus. Earlier studies suggested that I kappa B targets the p65 subunit of NF-kappa B. However, we demonstrate by in vitro and in vivo methods that the recently cloned I kappa B/MAD-3 interacts with both the p50 and p65 subunits of NF-kappa B, as well as c-Rel. Furthermore, an alternatively spliced, dimerization-deficient transforming variant of p65 (p65 delta) interacts extremely weakly with I kappa B/MAD-3, suggesting that dimerization is important for interaction. We demonstrate that the conserved nuclear localization sequences (NLSs) of NF-kappa B and c-Rel are the targets for I kappa B/MAD-3 interaction. Indirect immunofluorescence experiments demonstrate that I kappa B/MAD-3 expression retains both p65 and p50 in the cytoplasm. Furthermore, and most important, a p65 that contains an SV40 large T antigen NLS in addition to its own NLS is no longer retained in the cytoplasm in the presence of I kappa B/MAD-3. We propose that I kappa B/MAD-3 masks the NLSs of NF-kappa B and c-Rel and that this constitutes the mechanism for cytoplasmic retention of these proteins.

Alternative Splicing↗
PubMed1992

HIV-1 Tat acts as a processivity factor in vitro in conjunction with cellular elongation factors.

The HIV-1 trans-activator Tat increases the rate of transcription from the HIV-1 LTR promoter through the stem-loop-containing TAR RNA. To analyze the mechanisms of Tat action, a cell-free trans-activation system with no preincubation has been developed. Recombinant Tat specifically increased the level of a long runoff transcript but not a promoter-proximal transcript in a TAR-dependent fashion. These observations and the result of pulse-chase experiments support strongly the hypothesis that Tat enhances the ability of RNA polymerase to elongate over longer distances. Increased levels of the purified cellular factor TFIIF, essential for initiation and also implicated in elongation of transcription, obviated trans-activation by Tat by increasing the basal (Tat-independent) activity. However, another elongation factor, ATN/TFIIS, showed synergistic activation with Tat. An antiserum against a recombinant form of the large subunit of TFIIF (RAP 74) preferentially suppressed the activated level of transcription exerted by Tat. We propose the hypothesis that Tat acts as a processivity factor on RNA polymerase II in an analogous manner to TFIIF.

Amino Acid Sequence↗
PubMed1992

I-Rel: a novel rel-related protein that inhibits NF-kappa B transcriptional activity.

The NF-kappa B transcription factor complex is comprised of two subunits, p50 and p65, that share significant homology to the rel oncogene. We have isolated a cDNA encoding a novel 66-kD rel-related protein, designated I-Rel. Unlike other rel-related proteins, I-Rel does not interact with DNA. I-Rel forms heterodimers with p50, however, and greatly attenuates its DNA-binding activity--an effect probably resulting from the presence of a domain inhibitory to DNA binding present within the 121 amino-terminal residues of I-Rel. In contrast, I-Rel does not associate with p65. Transfection experiments demonstrate that I-Rel suppresses NF-kappa B-induced transcription, probably through its association with p50. Expression of I-Rel mRNA is induced by mitogenic stimulation and accumulates after the appearance of p50 transcripts. Our findings suggest that p50 and I-Rel are components of a feedback pathway where expression of I-Rel may modulate indirectly the expression of genes responsive to the NF-kappa B transcription factor complex.

Amino Acid Sequence↗
PubMed1992

Contribution of the TATA motif to Tat-mediated transcriptional activation of human immunodeficiency virus gene expression.

Tat-mediated transcriptional activation of human immunodeficiency virus (HIV) gene expression requires the presence of the cis-acting Tat-responsive element, TAR, and a functional enhancer-promoter element. The ability of Tat to function with heterologous enhancer sequences led us to examine the role of the minimal basal promoter for trans activation. Substitution of HIV TATA sequences (nucleotides -20 to -35) with TATA elements derived from other promoters had little effect on the basal level of transcription or the ability to activate the HIV long terminal repeat upon stimulation through upstream activation sequences. In contrast, minimal alterations within the TATA motif had a profound effect on trans activation, as demonstrated by the 3- to 10-fold reduction in activation following expression of Tat. Our findings suggest that minor changes in the TATA motif affect the composition of the initiation-elongation complex and that the composition of this complex is critical for Tat-dependent activation of gene expression.

Animals↗
PubMed1992

Selection of optimal kappa B/Rel DNA-binding motifs: interaction of both subunits of NF-kappa B with DNA is required for transcriptional activation.

Analysis of the p50 and p65 subunits of the NF-kappa B transcription factor complex has revealed that both proteins can interact with related DNA sequences through either homo- or heterodimer formation. In addition, the product of the proto-oncogene c-rel can bind to similar DNA motifs by itself or as a heterodimer with p50 or p65. However, these studies have used a limited number of known kappa B DNA motifs, and the question of the optimal DNA sequences preferred by each homodimer has not been addressed. Using purified recombinant p50, p65, and c-Rel proteins, optimal DNA-binding motifs were selected from a pool of random oligonucleotides. Alignment of the selected sequences allowed us to predict a consensus sequence for binding of the individual homodimeric Rel-related proteins, and DNA-protein binding analysis of the selected DNA sequences revealed sequence specificity of the proteins. Contrary to previous assumptions, we observed that p65 homodimers can interact with a subset of DNA sequences not recognized by p50 homodimers. Differential binding affinities were also obtained with p50- and c-Rel-selected sequences. Using either a p50- or p65-selected kappa B motif, which displayed differential binding with respect to the other protein, little to no binding was observed with the heterodimeric NF-kappa B complex. Similarly, in transfection experiments in which the selective kappa B binding sites were used to drive the expression of a chloramphenicol acetyltransferase reporter construct, the p65- and p50-selected motifs were activated only in the presence of p65 and p50/65 (a chimeric protein with the p50 DNA binding domain and p65 activation domain) expression vectors, respectively, and neither demonstrated a significant response to stimuli that induce NF-kappa B activity. These findings demonstrate that interaction of both subunits of the heterodimeric NF-kappa B complex with DNA is required for DNA binding and transcriptional activation and suggest that transcriptional activation mediated by the individual rel-related proteins will differ dramatically, depending on the specific kappa B motifs present.

Base Sequence↗
PubMed1992

Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative.

The NF-kappa B transcription factor complex is composed of two proteins, designated p50 and p65, both having considerable homology to the product of the rel oncogene. We present evidence that the p65 subunit is a potent transcriptional activator in the apparent absence of the p50 subunit, consistent with in vitro results demonstrating that p65 can interact with DNA on its own. To identify the minimal activation domain, chimeric fusion proteins between the DNA binding domain of the yeast transcriptional activator protein GAL4 and regions of the carboxy terminus of p65 were constructed, and their transcriptional activity was assessed by using a GAL4 upstream activation sequence-driven promoter-chloramphenicol acetyltransferase fusion. This analysis suggests that the boundaries of the activation domain lie between amino acids 415 and 550. Moreover, single amino acid changes within residues 435 to 459 greatly diminished activation. Similar to other activation domains, this region contains a leucine zipper-like motif as well as an overall net negative charge. To identify those residues essential for DNA binding, we made use of a naturally occurring derivative of p65, lacking residues 222 to 231 (hereafter referred to as p65 delta), and produced via an alternative splice site. Gel mobility shift analysis using bacterially expressed p65, p65 delta, and various mutants indicates that residues 222 to 231 are important for binding to kappa B DNA. Coimmunoprecipitation analysis suggests that these residues likely contribute to the multimerization function required for homomeric complex formation or heteromeric complex formation with p50 in that no association of p65 delta with itself or with p50 was evident. However, p65 delta was able to form weak heteromeric complexes with p65 that were greatly reduced in their ability to bind DNA. On the basis of these findings, we suggest that subtle changes within the proposed multimerization domain can elicit different effects with the individual Rel-related proteins and that a potential role of p65 delta may be to negatively regulate NF-kappa B function through formation of nonfunctional heteromeric complexes.

Amino Acid Sequence↗
PubMed1992

HIV-1 LTR activation model: evaluation of various agents in skin of transgenic mice.

Mice containing the HIV-1 long terminal repeat (LTR) regulating the expression of firefly luciferase reporter gene were investigated for their use as a model for activation of the LTR. As a limited test of this model, a number of different factors were screened for their ability to affect reporter gene activities in the skin. Reporter gene levels were increased in the skin by topical treatment of dimethylsulfoxide, retinoic acid, phorbol ester, ultraviolet light, and hydrogen peroxide, all of which have previously been shown to cause increased HIV production in cultured human cells. Topically applied arachidonic acid, histamine, ethanol, acetone, and methanol did not increase reporter gene activities. A lack of published reports on activation of HIV-1 in human cells by these agents suggests that they do not activate viral expression in human cells, which corroborates with the findings of this report. Minor forms of skin wounding and intraperitoneally administered psoralen plus ultraviolet light also increased reporter gene activities in skin. Control and test treatments could be performed on the same mouse and repetitive samples could be obtained from each treatment area. These transgenic mice might be useful as predictive models for regulation of the LTR in epidermal or dendritic cells.

Administration, Topical↗
PubMed1991

Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B.

The sequence reported in our 22 March 1991 report "Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NFkappaB" [Science 251, 1490 (1991)], contained some errors. Resequencing under strong denaturing conditions revealed three insertions at nucleotide positions 1194, 1212, and 1220, which changed the AA sequence from RSAR-PRLGP to QISQASALAP (residues 372 to 380), thus accounting for some of the divergence in this region. A corrected sequence has been sent to GenBank.

Amino Acid Sequence↗
PubMed1991

The number of positively charged amino acids in the basic domain of Tat is critical for trans-activation and complex formation with TAR RNA.

The basic domain of Tat is required for trans-activation of viral gene expression. We have performed scanning peptide studies to demonstrate that only this domain is capable of binding to the TAR RNA stem-loop. Strikingly, the basic domain of the other human immunodeficiency virus trans-acting factor, Rev, but no other region, is also capable of binding to TAR. Peptide derivatives of Tat do not require the highly conserved glutamine residue at position 54 for TAR binding, since it may be substituted or deleted. In addition, the two lysine residues may be replaced by arginines. Analysis of binding and trans-activation demonstrated that homopolymers of arginine can completely substitute for the basic domain. Such homopolymers have high affinity for wild-type TAR RNA and lower affinity for mutant TAR. Homopolymers of six to nine arginines substituting for the basic domain of Tat enable full trans-activation in vivo. Homopolymers of at least seven arginines are required for detectable in vitro complex formation, although approximately 30% trans-activation is achieved with a mutant Tat containing only five arginines.

Amino Acid Sequence↗
PubMed1991

Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B.

A DNA probe that spanned a domain conserved among the proto-oncogene c-rel, the Drosophila morphogen dorsal, and the p50 DNA binding subunit of NF-kappa B was generated from Jurkat T cell complementary DNA with the polymerase chain reaction (PCR) and degenerate oligonucleotides. This probe was used to identify a rel-related complementary DNA that hybridized to a 2.6-kilobase messenger RNA present in human T and B lymphocytes. In vitro transcription and translation of the complementary DNA resulted in the synthesis of a protein with an apparent molecular size of 65 kilodaltons (kD). The translated protein showed weak DNA binding with a specificity for the kappa B binding motif. This protein-DNA complex comigrated with the complex obtained with the purified human p65 NF-kappa B subunit and binding was inhibited by I kappa B-alpha and -beta proteins. In addition, the 65-kD protein associated with the p50 subunit of NF-kappa B and the kappa B probe to form a complex with the same electrophoretic mobility as the NF-kappa B-DNA complex. Therefore the rel-related 65-kD protein may represent the p65 subunit of the active NF-kappa B transcription factor complex.

Amino Acid Sequence↗
PubMed1991

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.

Base Sequence↗
PubMed1991

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).

Diphtheria Toxin↗
PubMed1991

Structural and functional analysis of the human immunodeficiency virus type 2 Rev protein.

The Rev proteins of the human immunodeficiency virus (HIV) are necessary for expression of viral structural gene products. Site-directed mutations were made within the HIV-2 rev gene to identify functional domains. We observed that similar to HIV-1 Rev, the HIV-2 Rev protein was phosphorylated, albeit to a much lesser extent than was HIV-1 Rev. We also found that like HIV-1 Rev, HIV-2 Rev localized to the nucleus, with a marked accumulation in the nucleolus. Mutations within a stretch of basic residues prevented both nuclear and nucleolar localization. Furthermore, mutant Rev proteins able to localize in the nucleus but unable to localize in the nucleolus were nonfunctional.

Amino Acid Sequence↗
PubMed1991

Identification and characterization of intragenic sequences which repress human immunodeficiency virus structural gene expression.

Examination of the life cycle of the human immunodeficiency virus (HIV) has shown that multiple levels of regulation exist, including some which require the virus-encoded Rev protein. In the absence of Rev, mRNAs encoding the structural proteins remain untranslated, a phenomenon which appears, in part, to be caused by nuclear entrapment of these RNA species. To examine the basis for repression of structural gene mRNA expression, a heterologous assay system was utilized to determine whether regions present within gag and pol contain elements capable of suppressing gene expression when present in cis. Both genes were found to contain cis-acting repressor sequences (CRS) that block gene expression when present within the 3' untranslated portion of a heterologous gene transcript. The element within pol was found to have the strongest repressive effect. While Rev alone was unable to reverse the repression observed with the pol sequence, addition of the env Rev-responsive element (RRE) in cis and Rev in trans did cause reversal of inhibition. Deletion mutagenesis defined a 260-bp element within the 3' portion of pol that contains a potent CRS which functions when present in the sense orientation. The corresponding region in HIV-2 pol was found to contain a functionally similar CRS element. To examine the mechanism of repression, the effects of the CRS elements on both the abundance and subcellular distribution of the mRNAs were examined. Neither was dramatically altered when examined in the context of a heterologous reporter (chloramphenicol acetyltransferase) mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Construction of a replication-competent murine retrovirus vector expressing the human immunodeficiency virus type 1 tat transactivator protein.

A replication-competent Akv murine leukemia virus-based vector encoding the human immunodeficiency virus tat cDNA under control of the simian virus 40 early promoter sequences was constructed. The simian virus 40 tat sequences were placed within the U3 region of the 3' long terminal repeat. The resulting virus, derived by transfection, replicated efficiently in mouse NIH 3T3 cells and maintained the tat cDNA insert. It has been suggested that Tat function requires the presence of a human-specific cofactor, which is absent in murine cells. However, infection of murine cells with the Akv virus encoding tat resulted in significant transactivation of a human immunodeficiency virus long terminal repeat-driven reporter gene, indicating that human cofactors are not always required for Tat function. The vector system described may be useful for introduction of foreign genes in vivo and in whole animals when virus spread is required for efficient infection and levels of gene expression.

Animals↗