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Biomedical subjects

M B Mathews

Publications and source records attributed to M B Mathews.

At least 73 records · Page 4Linked to original sources
PubMed1993

The adenovirus E1A 12S product displays functional redundancy in activating the human proliferating cell nuclear antigen promoter.

The adenovirus E1A 243R oncoprotein stimulates expression from the promoter of the human proliferating cell nuclear antigen (PCNA). To gain insight into the mechanism of activation, we analyzed deletion and point mutations of the 243R protein for their abilities to activate PCNA promoter-directed reporter gene expression upon cotransfection into HeLa cells. Large deletions that in combination span the entire protein severely impaired the ability of E1A 243R to induce PCNA expression. Smaller deletions and specific point mutations that target specific E1A-binding proteins were less deleterious to PCNA induction. The data suggest that E1A activates transcription of the PCNA gene by multiple mechanisms and that, of the known 243R-associated proteins, p300 and p107-cyclin A can mediate the response while p105-RB does not appear to participate. Presumably, the functional redundancy ensures that 243R can activate expression of this essential DNA replication protein regardless of cell type and physiological conditions.

Adenovirus E1A Proteins↗
PubMed1993

Comparative analysis of the structure and function of adenovirus virus-associated RNAs.

The protein kinase DAI is an important component of the interferon-induced cellular defense mechanism. In cells infected by adenovirus type 2 (Ad2), activation of the kinase is prevented by the synthesis of a small, highly ordered virus-associated (VA) RNA, VA RNAI. The inhibitory function of this RNA depends on its structure, which has been partially elucidated by a combination of mutagenesis and RNase sensitivity analysis. To gain further insight into the structure and function of this regulatory RNA, we have compared the primary sequences, secondary structures, and functions of seven VA RNA species from five human and animal adenoviruses. The sequences exhibit variable degrees of homology, with a particularly close relationship between the VA RNAII species of Ad2 and Ad7 and notably divergent sequence for the avian (CELO) virus VA RNA. Apart from two pairs of mutually complementary tetranucleotides which are highly conserved, homologies are limited to transcription signals located within the RNA sequence and at its termini. Secondary structure analysis indicated that all seven RNAs conform to the model in which VA RNA possesses three main structural regions, a terminal stem, an apical stem-loop, and a central domain, although these elements vary in size and other details. The apical stem is implicated in binding to DAI, and the central domain is essential for inhibition of DAI activation. One of the pairs of conserved tetranucleotides (CCGG:C/UCGG) provides further evidence for the existence of the apical stem, but the other conserved pair (GGGU:ACCC) strongly suggests a revised structure for the central domain. In two functional assays conducted in vivo, the VA RNAI species of Ad2 and Ad7 were the most active, their corresponding VA RNAII species displayed little activity, and the single VA RNAs of Ad12 and simian adenovirus type 7 exhibited intermediate activity. Correlation of the structural and functional data suggests that the VA RNAII species adopt a structure different from those of the other VA RNA species and may play a different role in the life cycle of the virus.

Adenoviruses, Human↗
PubMed1993

Mutational analysis of the central domain of adenovirus virus-associated RNA mandates a revision of the proposed secondary structure.

Protein synthesis in adenovirus-infected cells is regulated during the late phase of infection. The rate of initiation is maintained by a small viral RNA, virus-associated (VA) RNAI, which prevents the phosphorylation of eukaryotic initiation factor eIF-2 by a double-stranded RNA-activated protein kinase, DAI. On the basis of nuclease sensitivity analysis, a secondary-structure model was proposed for VA RNA. The model predicts a complex stem-loop structure in the central part of the molecule, the central domain, joining two duplexed stems. The central domain is required for the inhibition of DAI activation and participates in the binding of VA RNA to DAI. To assess the significance of the postulated stem-loop structure in the central domain, we generated compensating, deletion, and substitution mutations. A substitution mutation which disrupts the structure in the central domain abolishes VA RNA function in vitro and in vivo. Base-compensating mutations failed to restore the function or structure of the mutant, implying that the stem-loop structure may not exist. To confirm this observation, we tested mutants with alterations in the hypothetical loop and short stem that constitute the main features of the wild-type model structure. The upper part of the hypothetical loop could be deleted without abolishing the ability of the RNA to block DAI activation in vitro, whereas other loop mutations were deleterious for function and caused major rearrangements in the molecule. Base-compensating mutations in the stem did not restore the expected base pairing, even though the mutant RNAs were still functional in vitro. Surprisingly, a mutant with a noncompensating substitution mutation in the stem was more effective than wild-type VA RNAI in DAI inhibition assays but was ineffective in vivo. The structural and functional consequences of these mutations do not support the proposed model structure for the central domain, and we therefore suggest an alternative structure in which tertiary interactions may play a significant role in shaping the specificity of VA RNA function in the infected cell. Discrepancies between the functionality of mutant forms of VA RNA in vivo and in vitro are consistent with the existence of additional roles for VA RNA in the cell.

Adenoviruses, Human↗
PubMed1993

A complex promoter element mediates transactivation of the human proliferating cell nuclear antigen promoter by the 243-residue adenovirus E1A oncoprotein.

The adenovirus E1A oncoproteins interfere with the normal regulation of cellular proliferation through interactions with cell cycle regulatory proteins. In view of the essential role of proliferating-cell nuclear antigen (PCNA) in DNA replication, we performed a mutational analysis of the minimal human PCNA promoter (nucleotides -87 to +62) to define sequence elements which mediate transactivation by the 243-residue E1A protein (E1A 243R). Linker-scanning and site-directed mutants were examined for basal and E1A-induced expression of chloramphenicol acetyltransferase (CAT) from PCNA promoter-CAT reporter constructs transiently expressed in HeLa cells. The results define the cis-acting element required for induction of PCNA by E1A 243R as a region between -59 and -45 relative to the transcription initiation site. This PCNA E1A-responsive element (PERE), which is protected from DNase I digestion by nuclear extracts from 293 cells, includes the sequence AGCGTGG immediately upstream of the ATF binding site previously shown to be important for activation of PCNA by E1A 243R (G. F. Morris and M. B. Mathews, J. Virol. 65:6397-6406, 1991). Mutation of either the upstream component or the ATF site within the PERE diminishes basal promoter activity and abrogates transactivation by E1A 243R. This novel cis-acting element is also essential for both basal and E1A-induced expression in the context of the full-length PCNA promoter.

Adenovirus E1A Proteins↗
PubMed1993

Organization of the double-stranded RNA-activated protein kinase DAI and virus-associated VA RNAI in adenovirus-2-infected HeLa cells.

We have examined the cellular distribution of the double-stranded RNA-activated protein kinase DAI in adenovirus 2 (Ad2)-infected and uninfected HeLa cells. In uninfected cells DAI was found to be concentrated in the cytoplasm. In addition, DAI was localized in the nucleoli and diffusely distributed throughout the nucleoplasm. Cells treated with alpha-interferon displayed a similar pattern of distribution for DAI. When RNA polymerase I activity was inhibited by the drug actinomycin D, nucleoli segregated and DAI was found to colocalize with the dense fibrillar region of the nucleoli. During mitosis, the distribution of DAI paralleled that of rRNA. In adenovirus-infected cells the localization of DAI was similar to that in uninfected interphase cells. VA RNAI was detected in Ad2-infected cells by 10-14 hours post-infection as fine dots in the nucleoplasm. By 18-24 hours post-infection, VA RNAI appeared in bigger and more abundant dots in the nucleoplasm and the cytoplasm was intensively labeled. Transient expression of the VA RNAI gene in uninfected cells resulted in a similar localization of the RNA. Our results are consistent with a role for DAI and VA RNAI in protein synthesis and suggest that DAI may play an early role in ribosome biogenesis in the nucleolus in addition to its cytoplasmic role in translation.

Adenoviruses, Human↗
PubMed1992

Tat-responsive region RNA of human immunodeficiency virus type 1 stimulates protein synthesis in vivo and in vitro: relationship between structure and function.

The Tat-responsive region (TAR) sequence is present at the 5' end of human immunodeficiency virus 1 mRNAs and as a cytoplasmic form of 58-66 nucleotides. TAR RNA blocks the activation and autophosphorylation of the double-stranded RNA-activated protein kinase in vitro. We show here that TAR RNA also prevents the double-stranded RNA-mediated inhibition of translation in a cell-free system. Mutagenic and structural analyses of TAR RNA indicate that a stem of at least 14 base pairs is required for this activity, whereas the loop and bulge required for transactivation by Tat are dispensable. Truncation of the RNA to 68 nucleotides results in the loss of translational rescue ability, suggesting that the short cytoplasmic TAR RNA produced by viral transcription in vivo may not have the capability to suppress activation of the kinase. However, because longer TAR transcripts stimulate expression in a transient assay in vivo, the TAR structure at the 5' end of viral mRNAs could still exert this function in cis.

Base Sequence↗
PubMed1992

Two RNA-binding motifs in the double-stranded RNA-activated protein kinase, DAI.

The protein kinase DAI, the double-stranded RNA-activated inhibitor of translation, is an essential component of the interferon-induced cellular antiviral response. The enzyme is regulated by the binding of activator and inhibitor RNAs. We synthesized DAI in vitro and located its RNA-binding domain within the amino-terminal 171 residues. This domain contains two copies of an RNA-binding motif characterized by a high density of basic amino acids, by the presence of conserved residues, and by a probable alpha-helical structure. Deletion of either of the two motifs prevents the binding of dsRNA, but their relative positions can be exchanged, suggesting that they cooperate to interact with dsRNA. Clustered point mutations within the RNA-binding motifs and duplications of the individual motifs indicate that the first copy of the motif plays the more important role. Mutations that impair binding have similar effects on the binding of double-stranded RNAs of various lengths and of adenovirus VA RNAI, implying that discrimination between activator and inhibitory RNAs takes place subsequent to RNA binding.

Amino Acid Sequence↗
PubMed1992

Role of the apical stem in maintaining the structure and function of adenovirus virus-associated RNA.

Adenovirus virus-associated (VA) RNAI maintains efficient protein synthesis during the late phase of infection by preventing the activation of the double-stranded-RNA-dependent protein kinase, DAI. A secondary structure model for VA RNAI predicts the existence of two stems joined by a complex stem-loop structure, the central domain. The structural consequences of mutations and compensating mutations introduced into the apical stem lend support to this model. In transient expression assays for VA RNA function, foreign sequences inserted into the apical stem were fully tolerated provided that the stem remained intact. Mutants in which the base of the apical stem was disrupted retained partial activity, but truncation of the apical stem abolished the ability of the RNA to block DAI activation in vitro, suggesting that the length and position of the stem are both important for VA RNA function. These results imply that VA RNAI activity depends on secondary structure at the top of the apical stem as well as in the central domain and are consistent with a two-step mechanism involving DAI interactions with both the apical stem and the central domain.

Adenoviruses, Human↗
PubMed1992

Premature termination and processing of human immunodeficiency virus type 1-promoted transcripts.

We have used transient expression assays to study transcription directed by the human immunodeficiency virus (HIV) type 1 promoter. A plasmid containing an HIV-reporter gene fusion and a simian virus 40 origin of DNA replication was transfected into COS-1 cells in the presence or absence of a Tat expression vector. HIV-promoted RNA was analyzed by in vivo labeling, by RNase protection mapping, and in run-on transcription assays. As observed previously, two populations of HIV RNA accumulate in vivo: short, attenuated transcripts and long, polyadenylated mRNA. The short transcripts labeled in vivo were longer and more heterogeneous than expected from RNase protection assays. Moreover, comparison of transcripts labeled in vivo with run-on transcription products revealed that similar, if not identical, short RNAs accumulate in vitro. Utilizing the run-on assay, we show that following transcriptional termination, the attenuated transcripts undergo processing to generate one species of RNA. We also provide evidence that Tat does not act as an antiterminator to relieve a discrete elongation block but instead modifies transcriptional complexes, enabling them to overcome putative pause sites and continue transcription of the template.

Base Sequence↗
PubMed1992

Interactions between double-stranded RNA regulators and the protein kinase DAI.

The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The activity of DAI is controlled by RNA regulators, including dsRNA activators and highly structured single-stranded RNAs which block activation by dsRNA. To elucidate the mechanism of activation, we studied the interaction of DAI with RNA duplexes of discrete sizes. Molecules shorter than 30 bp fail to bind stably and do not activate the enzyme, but at high concentrations they prevent activation by long dsRNA. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. These dsRNAs fail to activate at high concentrations and also prevent activation by long dsRNA. Analysis of complexes between dsRNA and DAI suggests that at maximal packing the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation the binding site protects about 80 bp of duplex. When the RNA-binding site is fully occupied with an RNA activator, the complex appears to undergo a conformational change.

Binding Sites↗
PubMed1991

Tat transactivation of the human immunodeficiency virus type 1 promoter is influenced by basal promoter activity and the simian virus 40 origin of DNA replication.

We examined the activation of transcription from the human immunodeficiency virus type 1 (HIV-1) promoter by the viral Tat protein in a transient expression system. Plasmids contained a HIV-reporter gene cassette and a simian virus 40 origin of DNA replication. Run-on assays of transcription complex distribution and analysis of cytoplasmic RNA accumulation confirmed that Tat is able to activate transcription by two mechanisms: by increasing the rate of transcriptional initiation and the efficiency of transcriptional elongation. The degree to which Tat stimulated initiation is determined by the basal level of HIV-directed transcription, which is influenced by the presence [corrected] of the simian virus 40 replication origin. Tat functions primarily to increase the efficiency of elongation when the origin is present and the basal level of transcription is high [corrected]. On the other hand, Tat functions primarily to increase the rate of initiation when the origin is absent [corrected] and the basal level of transcription is 10-fold lower. These studies suggest that the site of integration of the virus into the cellular genome may significantly affect the level of expression from the HIV promoter and consequently the pathobiology of the virus.

Animals↗
PubMed1991

The adenovirus E1A transforming protein activates the proliferating cell nuclear antigen promoter via an activating transcription factor site.

The transforming region of adenovirus (E1) stimulates expression of a reporter construct linked to the promoter for the human proliferating cell nuclear antigen (PCNA) gene in a cotransfection assay (G. F. Morris and M. B. Mathews, J. Biol. Chem. 264:13856-13864, 1989). The major products of the E1 region were assessed individually for their contribution to transactivation of the PCNA promoter. The E1A 13S and 12S products and the E1B 19-kDa product elevated expression from the PCNA promoter, whereas the E1B 55-kDa product did not. Induction of the PCNA promoter by E1A differed from transcriptional activation of the adenovirus E3 promoter in that the PCNA promoter is activated by the E1A 12S product whereas the E3 promoter is repressed; furthermore, the PCNA promoter is activated upon E1A overexpression, whereas the E3 promoter responds less well to high amounts of E1A. A site for the activating transcription factor ATF located approximately 50 nucleotides upstream from the transcription initiation site in the PCNA promoter mediates a positive response to the E1A 12S and 13S products.

Adenovirus Early Proteins↗
PubMed1990

Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNAI from a T7 vector.

Bacteriophage RNA polymerases are widely used to synthesize defined RNAs on a large scale in vitro. Unfortunately, the RNA product contains a small proportion of contaminating RNAs, including complementary species, which can lead to errors of interpretation. We cloned the gene encoding Ad2 VA RNAI into a vector containing a T7 RNA polymerase promoter in order to generate large quantities of VA RNA for the study of its interaction with the dsRNA-dependent protein kinase DAI. Exact copies of VA RNAI were synthesized efficiently, but were contaminated with small amounts of dsRNA which activated DAI and confounded interpretation of kinase assays. We therefore developed a method to remove the dsRNA contaminants, allowing VA RNAI and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable.

Adenoviruses, Human↗
PubMed1990

Analysis of the proliferating cell nuclear antigen promoter and its response to adenovirus early region 1.

The levels of the mRNA for the proliferating cell nuclear antigen (PCNA), a DNA replication factor, increase upon growth stimulation of quiescent cells. To study the transcriptional aspect of this response, we have cloned a PCNA gene fragment from size-fractionated human placental DNA. This fragment contains 1269 nucleotides upstream from the PCNA transcriptional start site and includes an Alu sequence that is transcribed in vitro. The PCNA genomic DNA promotes transcription of a linked heterologous reporter gene in HeLa and 293 cells. Transient expression assays and in vitro transcription analyses showed that 249 nucleotides of upstream sequence are sufficient for full promoter activity in HeLa cells, whereas only 172 nucleotides are needed in 293 cells. Co-transfection with a plasmid expressing the adenovirus E1 gene transactivates the PCNA promoter in HeLa cells. An E1-responsive element maps in the 85-nucleotide region immediately upstream of the site of transcription initiation.

Adenovirus Early Proteins↗
PubMed1990

Interaction of adenovirus VA RNAl with the protein kinase DAI: nonequivalence of binding and function.

Adenovirus VA RNAL maintains protein synthesis by preventing activation of the double-stranded RNA (dsRNA)-dependent protein kinase DAI. There appears to be a single binding site for dsRNA on DAI, and this site is blocked by VA RNAl. VA RNAl binds to purified DAI and can be cross-linked to the enzyme by UV irradiation. To determine the relationship between DAI binding and VA RNAl structure and function, we examined the binding abilities of wild-type and mutant VA RNAs. In several cases, the ability to bind DAI efficiently in vitro did not correlate with function in vivo. Secondary structure analysis suggested that efficient binding requires an apical stem-loop structure, whereas inhibition of DAI activation requires the central domain of the VA RNA molecule. We propose that the duplex stem permits VA RNA to interact with the dsRNA binding site on DAI and inhibits activation by juxtaposing the central domain of the RNA with the enzyme's active site.

Adenoviruses, Human↗
PubMed1990

Tat-responsive region RNA of human immunodeficiency virus 1 can prevent activation of the double-stranded-RNA-activated protein kinase.

Transcription from the human immunodeficiency virus type 1 promoter gives rise to short cytoplasmic transcripts of approximately 60 nucleotides as well as to longer mRNAs. These RNAs contain the Tat-responsive region sequence, which is capable of assuming a stem-loop structure and has been implicated in the regulation of both transcription and translation. It has been reported that Tat-responsive region RNA inhibits translation in vitro through activation of an interferon-induced protein kinase, the double-stranded-RNA-activated inhibitor, which phosphorylates eukaryotic initiation factor 2. We show that the activation property is due to double-stranded RNA that often contaminates RNA synthesized in vitro using bacteriophage RNA polymerases. After purification, high concentrations of Tat-responsive region RNA inhibit the activation of double-stranded RNA-activated inhibitor, suggesting that it may serve to protect human immunodeficiency virus type 1 infection from a cellular defense mechanism.

Cloning, Molecular↗
PubMed1990

Synergy between HIV-1 Tat and adenovirus E1A is principally due to stabilization of transcriptional elongation.

We studied the combined effects of Tat and general trans-activators, such as E1A and phorbol esters, on human immunodeficiency virus-1 (HIV-1) gene expression. Interaction between these two types of trans-activators may be involved in the transition from transcriptional quiesence during viral latency to active gene expression during productive infection. E1A cooperated with Tat to produce a fourfold greater increase in accumulation of full-length, cytoplasmic HIV-1-directed RNA than is expected if they were acting additively to increase RNA accumulation. Similarly, phorbol 12-myristate 13-acetate (PMA) also cooperated with Tat to elevate HIV RNA levels synergistically. Analysis of transcription rates across the HIV-1-directed transcription unit indicated, unexpectedly, that synergy between Tat and E1A could not be accounted for by increased promoter proximal transcription rates that were merely additive. However, Tat and E1A produced a greater than additive increase in transcription rates in the 3' end of the gene. These findings imply that synergy between Tat and E1A (or other general transcriptional activators) is due principally to stabilization of transcriptional elongation. Furthermore, the observation that Tat elicits only a small increase in promoter proximal transcription in the presence of E1A suggests that the magnitude of the effect of Tat on initiation is decreased when the basal level of transcription is increased. These findings underscore the importance of the ability of Tat to stabilize elongation, as well as to stimulate initiation, in an HIV-1-directed transcription unit.

Adenoviridae↗