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M B Mathews

Publications and source records attributed to M B Mathews.

At least 19 recordsLinked to original sources
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↗
PubMed1990

Control of translation in adenovirus-infected cells.

The initiation of protein synthesis in adenovirus-infected cells is regulated during the late phase in two ways, which may be related. The overall translation rate is maintained by a small viral RNA, VA RNAI, which prevents the phosphorylation of initiation factor eIF-2 by a double-stranded RNA-activated protein kinase, DAI. In addition, the relative efficiency of translation of host cell and viral mRNA populations is regulated in the infected cell during the late phase such that viral mRNAs are selectively utilized. Three viral elements have been implicated in this process: the 5' leader present on most late viral mRNAs; the late protein, 100K; and VA RNA. This article reviews the mechanisms underlying these translational control phenomena.

Adenoviridae↗
PubMed1989

Identification of a 90-kDa polypeptide which associates with adenovirus VA RNAI and is phosphorylated by the double-stranded RNA-dependent protein kinase.

Interferon treatment of mammalian cells induces a double-stranded (ds) RNA-dependent protein kinase known as DAI. When activated, DAI phosphorylates the alpha-subunit of eukaryotic initiation factor eIF-2, impairing its ability to be recycled and leading to the inhibition of protein synthesis. We have identified a novel DAI substrate in the ribosomal salt wash of rabbit reticulocyte lysates. This substrate is a 90-kDa polypeptide which has been purified to apparent homogeneity. It can be cross-linked by ultraviolet irradiation to adenovirus VA RNAI, a small RNA polymerase III transcript RNA which acts as an inhibitor of DAI. As assayed by a nitrocellulose filter binding assay, the 90-kDa polypeptide is also able to associate with authentic double-stranded RNA, but not single-stranded RNA, made in vitro. Thus, this newly identified substrate of DAI appears to have affinity for dsRNA structures and may be involved in dsRNA-regulated processes in the reticulocyte. Polyclonal and monoclonal antibodies directed against the 90-kDa polypeptide co-precipitate DAI, suggesting that these two proteins may exist as a complex.

Adenoviruses, Human↗
PubMed1989

HIV-1 Tat protein increases transcriptional initiation and stabilizes elongation.

We studied regulation of human immunodeficiency virus-1 (HIV-1) transcription by Tat and, for comparative purposes, by the adenovirus E1A protein. These two trans-activators exerted different effects. Two classes of HIV-1-promoted cytoplasmic RNA were detected, one class corresponding to full-length transcripts and the other to transcripts ending 55 and 59 nucleotides from the transcription start. Tat increased the level of the full-length class only, whereas E1A increased the levels of both classes of RNA. We also measured the effects of Tat and E1A on RNA synthesis rates. Without trans-activators, HIV-1-directed transcription was relatively weak and exhibited a marked polarity. Both Tat and E1A dramatically increased promoter-proximal transcription, while only Tat suppressed transcriptional polarity. Mutations in the TAR element did not influence basal transcription rates or the response to E1A, but eliminated trans-activation by Tat. We propose that Tat acts through TAR to increase initiation complex formation on the HIV-1 promoter and to stabilize complexes during elongation.

Adenovirus Early Proteins↗
PubMed1989

Regulation of proliferating cell nuclear antigen during the cell cycle.

The proliferating cell nuclear antigen (PCNA), also known as cyclin and DNA polymerase delta auxiliary factor, is present in reduced amounts in nongrowing cells and is synthesized at a greater rate in the S phase of growing cells. The recently discovered involvement of PCNA in DNA replication suggested that this pattern of expression functions to regulate DNA synthesis. We have investigated this possibility further by examining the synthesis, stability, and accumulation of PCNA in HeLa cells fractionated by centrifugal elutriation into nearly synchronous populations of cells at various positions in the cell cycle. In these fractionated cells we found that there is an increase in the rate of PCNA synthesis with a peak in early S phase of the cell cycle, but the magnitude of the increase is only 2-3-fold. This change reflects similar changes in the amount of PCNA mRNA. The fluctuating synthesis of PCNA maintains this protein at a roughly constant proportion of the total cell protein, although the amount doubles/cell in the cell cycle. Consistent with this observation, the stability of PCNA does not differ significantly from that of total cellular protein in synchronized HeLa cells. We also observed that a maximum of one-third of the total PCNA is tightly associated with the nucleus, presumably in replication complexes, at the peak of S phase. We conclude that the cyclic synthesis of PCNA in cycling HeLa cells maintains PCNA in excess of the amount involved directly in DNA replication and the amount of the protein neither fluctuates significantly with the cell cycle nor is limiting for DNA synthesis.

Antigens, Neoplasm↗
PubMed1989

Modification of protein synthesis initiation factors and the shut-off of host protein synthesis in adenovirus-infected cells.

A substantial body of data, largely derived from study of cell extracts, indicates that protein synthesis in adenovirus-infected cells requires VA RNAI at late times of infection to prevent the activation of a protein kinase known as DAI, and the consequent phosphorylation of the alpha-subunit of initiation factor eIF-2. To verify this conclusion, we have measured the steady-state levels of eIF-2 alpha phosphorylation in cells infected with wild-type virus (Ad2) and a mutant that produces no VA RNAI (Ad5dl331). Consistent with the proposed mechanism, the alpha-subunit was very highly phosphorylated (approximately 90%) at late times of infection with Ad5dl331. Surprisingly, eIF-2 alpha phosphorylation also increased (to approximately 30%) at late times of infection with Ad2, suggesting that VA RNA and DAI might be involved in the selective translation of viral mRNA and the shut-off of host cell protein synthesis during the late phase. In agreement with this model, host protein synthesis shut-off is defective in cells expressing low levels of DAI.

Adenoviruses, Human↗