Biomedical subjects

M B Mathews

Publications and source records attributed to M B Mathews.

At least 37 records · Page 2Linked to original sources

PubMed1998

Regulation of the human proliferating cell nuclear antigen promoter by the adenovirus E1A-associated protein p107.

The adenovirus E1A 243R oncoprotein is capable of transactivating the expression of the human proliferating cell nuclear antigen (PCNA) promoter. Mutational analysis of the E1A 243R protein suggested that both its p300/CBP- and p107-binding regions are required for optimal induction of the PCNA promoter (C. Kannabiran, G. F. Morris, C. Labrie, and M. B. Mathews, J. Virol. 67:425-437, 1993). We show that overexpression of p107 antagonizes the induction of PCNA by E1A 243R in transient expression assays. This inhibition is largely independent of p107's ability to interact with E1A 243R, because p107 mutants unable to bind to E1A 243R retain the ability to repress the E1A-activated PCNA promoter. Electrophoretic mobility shift assays with the PCNA promoter detected the presence of p107 in one of the major DNA-protein complexes, EH1, formed with HeLa cell nuclear extracts. Promoter mutations that disrupt the formation of complex EH1 abrogated p107's ability to reverse E1A 243R-induced PCNA expression. The same mutations characterize a sequence important for the binding of transcription factor RFX1 (C. Labrie, G. F. Morris, and M. B. Mathews, Nucleic Acids Res. 23:3732-3741, 1995), implying that p107 antagonizes E1A 243R-induced PCNA expression through this RFX1-binding site. Our data are suggestive of a novel cooperative mechanism for transactivation of PCNA expression, in which E1A 243R relieves transcriptional repression exerted by p107 on the promoter.

Adenoviridae↗

PubMed1998

Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2alpha kinases PKR and GCN2.

The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit alpha (eIF2alpha) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2alpha kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.

3T3 Cells↗

PubMed1997

Effects of heterologous downstream sequences on the activity of the HIV-1 promoter and its response to Tat.

In HIV-1 infection, Tat acts at least in part to control transcriptional elongation by overcoming premature transcriptional termination. In some other genes this process is governed by DNA elements called attenuators in concert with cellular transcription factors. To understand the action of Tat more fully and explore its role as an anti-attenuator, we examined the ability of several natural and synthetic attenuation sequences to modulate transcription initiated at the HIV LTR. Fragments containing these signals were inserted downstream of the TAR element in an HIV-CAT chimera and their effects on transcription were assessed both in vitro and in vivo. Runoff transcription assays in HeLa cell extracts demonstrated that the attenuators give rise to premature termination of transcripts initiated from the heterologous HIV-LTR promoter in vitro. When transiently expressed following transfection into Cos cells, however, premature transcript termination at the attenuation site was not observed. Nevertheless, many of the inserted sequences exerted marked effects on CAT gene expression and on transactivation by Tat at both the RNA and protein levels. The nature and magnitude of the effects depended upon the identity of the attenuator and its orientation but only one of 16 sequences tested met the criteria for a Tat-suppressible attenuator in vivo. One other sequence, in contrast, severely reduced Tat-activated transcription without inhibiting basal transcription These results indicate that sequences downstream of the HIV LTR can influence its function as a promoter and its response to Tat transactivation, but lend little support to their role as attenuators in vivo.

Animals↗

PubMed1997

Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro.

P-TEFb is a key regulator of the process controlling the processivity of RNA polymerase II and possesses a kinase activity that can phosphorylate the carboxy-terminal domain of the largest subunit of RNA polymerase II. Here we report the cloning of the small subunit of Drosophila P-TEFb and the finding that it encodes a Cdc2-related protein kinase. Sequence comparison suggests that a protein with 72% identity, PITALRE, could be the human homolog of the Drosophila protein. Functional homology was suggested by transcriptional analysis of an RNA polymerase II promoter with HeLa nuclear extract depleted of PITALRE. Because the depleted extract lost the ability to produce long DRB-sensitive transcripts and this loss was reversed by the addition of purified Drosophila P-TEFb, we propose that PITALRE is a component of human P-TEFb. In addition, we found that PITALRE associated with the activation domain of HIV-1 Tat, indicating that P-TEFb is a Tat-associated kinase (TAK). An in vitro transcription assay demonstrates that the effect of Tat on transcription elongation requires P-TEFb and suggests that the enhancement of transcriptional processivity by Tat is attributable to enhanced function of P-TEFb on the HIV-1 LTR.

Amino Acid Sequence↗

PubMed1997

Translation of an uncapped mRNA involves scanning.

tat, an essential gene of human immunodeficiency virus, when placed under the control of the RNA polymerase III promoter from the adenovirus VA RNA1 gene, is transcribed into an uncapped and nonpolyadenylated mRNA. This VA-Tat RNA is translated to produce functional Tat protein in transfected mammalian cells (Gunnery, S., and Mathews, M. B. (1995) Mol. Cell. Biol. 15, 3597-3607). The presence of an upstream open reading frame (ORF) in VA-Tat RNA is inhibitory to the translation of the Tat ORF, suggesting that the RNA is scanned during translation even though it is uncapped. Because the effect of the upstream ORF is relatively small (about 2-fold), we sought more definitive evidence of scanning by introducing secondary structures of varying stabilities into the 5'-untranslated region of VA-Tat RNA. The results of transfection experiments showed that highly stable secondary structure was inhibitory to Tat synthesis, whereas structures of lower stability were not inhibitory, confirming that uncapped mRNA is subject to scanning. Furthermore, translation of the downstream ORF was reduced but not eliminated by mutations that caused the upstream ORF to overlap the Tat ORF. Extending the overlap of the two ORFs further decreased the translation of the downstream ORF. This observation implies that ribosomes reinitiate after termination, possibly after migrating in a 3' to 5' direction through the overlap region of the mRNA. Similar results were obtained with a capped polymerase II transcript, indicating that the translation of polymerase II and polymerase III transcripts occurs through similar mechanisms.

Genes, tat↗

PubMed1997

Transduction of the human immunodeficiency virus type 1 promoter into human chromosomal DNA by adeno-associated virus: effects on promoter activity.

Transcription of the human immunodeficiency virus type 1 (HIV-1) genome takes place after integration of the provirus into human chromosomal DNA. HIV transcription is known to be modulated by viral and cellular factors but the influence of flanking chromosomal sequences on proviral gene expression has not been well defined. To investigate the activity of the integrated HIV promoter, we exploited the ability of recombinant adeno-associated virus (AAV-2) to transfer and stably integrate genes into the human genome at random or site-specifically. Chimeric AAV vectors were constructed containing an HIV-CAT reporter cassette; some vectors also contained the neomycin resistance gene to facilitate the isolation of positive clones. HeLa cells were infected with recombinant AAV, in some instances together with wild-type virus as a source of AAV rep function. We isolated 25 clones of G418-resistant cells which carried the integrated HIV-CAT cassette, generally occupying unique sites that did not correspond to the AAV-specific region of chromosome 19. The HIV promoter was transcriptionally active in most of the clones. Basal promoter activity varied substantially among the clones, and its responsivity to the HIV transactivator Tat was also variable. The integrated HIV promoter was transactivated to comparable degrees by the one-exon form and two-exon form of Tat. These findings provide evidence that the transcriptional activity of the HIV promoter can be greatly influenced by the site of proviral insertion.

Cell Line, Transformed↗

PubMed1997

Transcriptional coactivator cAMP response element binding protein mediates induction of the human proliferating cell nuclear antigen promoter by the adenovirus E1A oncoprotein.

The proliferating cell nuclear antigen (PCNA), a crucial component of eukaryotic cell cycle and DNA replication complexes, is induced by the adenovirus E1A 243R oncoprotein through a cis-acting element termed the PERE (PCNA-E1A responsive element). The PERE contains a sequence homologous to an activating transcription factor (ATF) motif, and ATF-1 is a major component of PERE-protein complexes. We have identified a second PERE-binding protein, the cAMP response element binding protein (CREB) transcription factor, which forms heterodimers with ATF-1 at this site. CREB, but not ATF-1, is able to mediate transactivation of a minimal PCNA-chloramphenicol acetyltransferase reporter by E1A 243R. Further analysis revealed that the transcriptional coactivator, the CREB-binding protein (CBP), associates with PERE-related complexes, and that CBP is able to mediate a strong transactivation response to E1A 243R at the PCNA promoter. Experiments conducted with mutants in the E1A or CREB components support a model whereby E1A 243R transactivates the PCNA promoter via a CBP-CREB-PERE pathway. These findings delineate a paradigm by which E1A 243R can target and transactivate specific DNA promoter sequences.

Adenovirus E1A Proteins↗

PubMed1997

The Tat protein of human immunodeficiency virus type 1 is a substrate and inhibitor of the interferon-induced, virally activated protein kinase, PKR.

We demonstrate that the interferon-induced, double-stranded (ds) RNA-activated kinase, PKR, is able to bind to and phosphorylate the human immunodeficiency virus type 1 (HIV-1) trans-activating protein, Tat. Furthermore, Tat can inhibit the activation and activity of the kinase. Phosphorylation of Tat by PKR is dependent on the prior activation of PKR by dsRNA and occurs on serine and threonine residues adjacent to the basic region important for TAR RNA binding and Tat function. Activated PKR efficiently phosphorylates both the two-exon form of Tat (Tat-86) and the single exon form (Tat-72). Mutagenesis indicates that the interaction between PKR and Tat requires the RNA-binding region of Tat. Tat competes with eukaryotic initiation factor 2, a well-characterized substrate of PKR, for phosphorylation by activated PKR. Tat also inhibits the autophosphorylation of PKR by dsRNA. This biochemical evidence of an intimate relationship between Tat, an important regulator of HIV transcription, and PKR, a pleiotropic cellular regulator, may provide insights into HIV-1 pathogenesis and, more generally, virus/host interactions.

Amino Acid Sequence↗

PubMed1997

Synthesis and purification of single-stranded RNA for use in experiments with PKR and in cell-free translation systems.

The biosynthesis of RNA in vitro using bacteriophage RNA polymerases has opened up many avenues of research. Large amounts of specific RNA species can be readily produced but small amounts of contaminants that are simultaneously generated can interfere with biological assays, PKR, a ribosome-associated and double-stranded (ds) RNA-dependent protein kinase, is an important regulator of the initiation of protein synthesis. It can be activated by very low concentrations of dsRNA and inhibited by small structured RNAs or high concentrations of dsRNA. The best-studied inhibitor of PKR activation is adenovirus VA RNA1. Its gene was cloned into a plasmid under the control of the T7 RNA polymerase promoter, and the optimization of VA RNA transcription is described. A dsRNA by-product of the transcription reaction activates PKR in kinase autophosphorylation assays, and hence a purification protocol that allows the separation and removal of dsRNA contaminants was developed. A scheme to analyze the RNA product with specific nucleases is discussed. In a reticulocyte cell-free translation system the activation of PKR by dsRNA contaminating a synthetic mRNA preparation is likely to lead to shut-off of translation. An assay to directly visualize and measure the level of PKR phosphorylation in the lysate is detailed.

Bacteriophage T7↗

PubMed1997

Induction of CD4 expression and human immunodeficiency virus type 1 replication by mutants of the interferon-inducible protein kinase PKR.

Replication of the human immunodeficiency virus type 1 (HIV-1) is inhibited by interferons (IFNs), and the IFN-inducible protein kinase PKR is thought to mediate this effect by regulating protein synthesis. Here we report that ectopic expression of dominant negative PKR mutants in Jurkat cells induces HIV-1 replication. Specifically, expression of CD4 is upregulated by the PKR mutants, and this correlates with an induction of HIV-1 binding and proviral DNA synthesis upon HIV-1 infection. Moreover, activation of NF-kappaB was induced by an RNA binding-defective mutant of PKR. Thus, it appears that PKR, in addition to translational control, is involved in HIV-1 replication by modulating virus binding through the regulation of CD4 expression and virus gene expression through the activation of NF-kappaB.

CD4 Antigens↗

PubMed1997

Potentiation of human immunodeficiency virus type 1 Tat by human cellular proteins.

The Tat protein is a potent activator of human immunodeficiency virus type 1 transcription. Tat has been shown to act by increasing both transcription initiation and elongation, but a detailed understanding of its interaction with the transcriptional machinery is lacking. With the aim of isolating cellular proteins that interact with Tat and play a role in transactivation, we have reexamined its function in a cell-free transcription assay. Monitoring the appearance of transactivation after addition of purified Tat at intervals to the reaction mix revealed a lag of approximately 10 min before Tat is able to effect transactivation. Incubation of Tat in nuclear or cytoplasmic extracts of human cells was sufficient to eliminate the lag, but nuclear extract from a rodent cell line was inactive. The accelerating effect of the human cell extract could be abrogated by dilution, heat inactivation, or chromatographic depletion. We infer that Tat is potentiated for transactivation through interaction with a protein factor(s) that is specific to human cells.

Animals↗

PubMed1997

Surprising specificity of PKR binding to delta agent genomic RNA.

A direct, ribonuclease T1 protection assay was employed to study the binding of a delta agent genomic RNA transcript containing the conserved domain to the double-stranded RNA- (dsRNA-) dependent protein kinase of mammalian cells, PKR (also known as DAI, p1-elF2, or p68 kinase). In a control reaction, this assay identified a major portion of the same PKR binding site in VA RNA as deduced previously using a footprinting technique (Clarke PA, Mathews MB, 1995, RNA 1:1-20). Although delta agent RNA contains extensive secondary structure throughout the conserved region, we found a remarkable specificity in its PKR binding. The same region was protected by intact PKR and by a 184-amino acid fragment thereof containing the two RNA-binding motifs (dsRBMs) but lacking kinase activity. Two specific opposed, continuous segments of delta agent RNA (extending about 65-70 bases) were obtained reproducibly. Each is more than twice as long as those protected in VA RNA (about 25 bases), suggesting the involvement of PKR dimers in delta RNA binding. The PKR-protected region of delta agent RNA also contains a characteristic tertiary structural element that may be involved in binding specificity.

Base Sequence↗

PubMed1996

Transcriptional activation of the human proliferating-cell nuclear antigen promoter by p53.

Proliferating-cell nuclear antigen (PCNA) is a DNA damage-inducible protein that performs an essential function in DNA replication and repair as an auxiliary factor for DNA polymerases delta and epsilon. Examination of the human PCNA promoter DNA sequence revealed a site with homology to the consensus DNA sequence bound by p53. PCNA promoter fragments with this site intact bound p53 in vitro and were transcriptionally activated by wild-type p53 in transient expression assays in SAOS-2 cells. The resident p53-binding site could be functionally substituted by a previously described p53-binding site from the ribosomal gene cluster. A plasmid expressing a mutated version of p53 derived from a patient with Li-Fraumeni syndrome failed to activate the PCNA promoter in the cotransfection assay. In different cell types, activation of the PCNA promoter by the p53-binding sequence correlated with the status of p53. Activation of the PCNA promoter by wild-type p53 depends upon the level of p53 expression. This concentration dependence and cell type specificity reconciles the observations presented here with prior results indicating that wild-type p53 represses the PCNA promoter. These findings provide a mechanism whereby p53 modulates activation of PCNA expression as a cellular response to DNA damage.

Base Sequence↗

PubMed1996

The regulation of the protein kinase PKR by RNA.

A model is presented for the regulation of the double-stranded RNA (dsRNA)-activated mammalian protein kinase PKR, which is involved in protein synthesis inhibition and the antiviral response in cells. A series of previous findings abut PKROs behavior are reviewed, including its effects on translation; the activation of its protein kinase activity; binding sites for PKR on RNA; PKROs protein domains, which include two double-stranded RNA binding motifs (dsRBMs); and the likelihood of PKR dimer formation. The model which emerges to account for many of these observations includes the suggestion that PKR dimers form which are stabilized and rearranged upon binding to dsRNA regions 60 bp or longer. The hypothesis includes protein conformational changes within each member of a PKR dimer bound to dsRNA which re-position an inhibitory polypeptide domain and thus allow kinase activation. Also considered are ways in which PKR interacts with imperfectly duplexed, highly structured RNA molecules.

Animals↗

PubMed1996

Structure, function, and evolution of adenovirus-associated RNA: a phylogenetic approach.

To explore the structure and function of a small regulatory RNA, we examined the virus-associated (VA) RNA species of all 47 known human adenovirus serotypes and of one simian virus, SA7. The VA RNA gene regions of 43 human adenoviruses were amplified and sequenced, and the structures of 10 representative VA RNAs were probed by nuclease sensitivity analysis. Most human viruses have two VA RNA species, VA RNA, and VA RNAII, but nine viruses (19%) have a single VA RNA gene. Sequence alignments classified the RNAs into eight families, corresponding broadly to the known virus groups, and three superfamilies. One superfamily contains the single VA RNAs of groups A and F and the VA RNAI species of group C; the second contains the VA RNAI species of groups B1, D, and E and the unclassified viruses (adenovirus types 42 to 47), as well as the single VA RNAs of group B2; and the third contains all VA RNAII species. Fourteen regions of homology occur throughout the molecule. The longest of these correspond to transcription signals; most of the others participate in RNA secondary structure. The previously identified tetranucleotide pair, GGGU:ACCC, is nearly invariant, diverging slightly (to GGGU:ACCU) only in the two group F viruses and forming a stem in the central domain that is critical for VA RNA structure and function. Secondary structure models which accommodate the nuclease sensitivity data and sequence variations within each family were generated. The major structural features-the terminal stem, apical stem-loop, and central domain-are conserved in all VA RNAs, but differences exist in the apical stem and central domains, especially of the VA RNAII species. Sequence analysis suggests that an ancestral VA RNA gene underwent duplication during the evolution of viruses containing two VA RNA genes. Although the VA RNAII gene seems to have been lost or inactivated by secondary deletion events in some viruses, the high degree of homology among the VA RNAII species implies that this RNA may play an undiscovered role in virus survival. We speculate that the VA RNA genes originated from cellular sequences containing multiple tRNA genes.

Adenoviridae↗

PubMed1996

Paradoxical interactions between human delta hepatitis agent RNA and the cellular protein kinase PKR.

The genome of the human delta hepatitis agent is a circular, highly structured single-stranded RNA lacking regular runs of RNA-RNA duplex longer than 15 bp. We have tested the ability of delta agent RNA to participate in reactions with a protein containing a motif which confers the ability to bind double-stranded RNA (dsRNA). Surprisingly, highly purified delta agent RNA preparations from which all traces of contaminating dsRNA have been removed activate PKR, the dsRNA-dependent protein kinase activity of mammalian cells (also known as DAI, P1-eIF-2, and p68 kinase). This behavior is in marked contrast to the interaction of PKR with a number of other highly structured viral single-stranded RNAs, which inhibit, rather than stimulate, activation of this kinase. PKR activation leads to inhibition of protein synthesis in the rabbit reticulocyte lysate system. Paradoxically, delta RNA failed to elicit the expected PKR-mediated inhibition of cell-free translation. Instead, delta RNA interfered with PKR activation and the translational block induced by dsRNA. We conclude that the interaction of PKR and delta agent RNA may represent a new category of protein-RNA interactions involving the dsRNA binding motif.

Animals↗

PubMed1996

Autophosphorylation sites participate in the activation of the double-stranded-RNA-activated protein kinase PKR.

The interferon-induced RNA-dependent protein kinase PKR is found in cells in a latent state. In response to the binding of double-stranded RNA, the enzyme becomes activated and autophosphorylated on several serine and threonine residues. Consequently, it has been postulated that autophosphorylation is a prerequisite for activation of the kinase. We report the identification of PKR sites that are autophosphorylated in vitro concomitantly with activation and examine their roles in the activation of PKR. Mutation of one site, threonine 258, results in a kinase that is less efficient in autophosphorylation and in phosphorylating its substrate, the initiation factor eIF2, in vitro. The mutant kinase is also impaired in vivo, displaying reduced ability to inhibit protein synthesis in yeast and mammalian cells and to induce a slow-growth phenotype in Saccharomyces cerevisiae. Mutations at two neighboring sites, serine 242 and threonine 255, exacerbated the effect. Taken together with earlier results (S. B. Lee, S. R. Green, M. B. Mathews, and M. Esteban, Proc. Natl. Acad. Sci. USA 91:10551-10555, 1994), these data suggest that the central part of the PKR molecule, lying between its RNA-binding and catalytic domains, regulates kinase activity via autophosphorylation.

Amino Acid Sequence↗

PubMed1996

Secondary and tertiary structure in the central domain of adenovirus type 2 VA RNA I.

The small (160 nt) adenovirus RNA, VA RNAI, antagonizes the activation of the cellular protein kinase PKR (also known as DAI), a key regulator of gene expression. VA RNA consists of two stems separated by a complex region, the central domain, that is essential for its function. A notable feature of the central domain is a pair of tetranucleotides, GGGU and ACCC, which are mutually complementary and phylogenetically conserved. To investigate their role in the structure and function of VA RNA, we generated three sets of mutations designed to disrupt the putative stem and to restore it with different nucleotides. Substitutions in either of the tetranucleotides abrogated VA RNA function in two independent PKR-based assays, demonstrating the importance of these sequences in vivo. Compensating mutants restored function, indicating that the two tetranucleotides pair in the cell, but all of the compensating mutants were less active than wild-type VA RNA. The effects of the mutations on RNA structure were probed by nuclease sensitivity analysis. Pronounced changes in two loops in the central domain correlated closely with the formation and disruption of the stem, suggesting that the tetranucleotide stem defines a critical element in the structure of the central domain through tertiary interactions with the two loops. A model for the central domain is presented that accommodates these findings and also accounts for the known sites of PKR interaction.

Adenoviruses, Human↗