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B Safer

Publications and source records attributed to B Safer.

At least 55 records · Page 3Linked to original sources
PubMed1988

Influenza virus regulates protein synthesis during infection by repressing autophosphorylation and activity of the cellular 68,000-Mr protein kinase.

We investigated the mechanisms by which influenza virus prevents shutoff of protein synthesis by a cellular protein kinase normally activated during infection. Earlier work has shown that influenza virus superinfection of cells previously infected by the adenovirus VAI RNA-negative mutant dl331 resulted in selective translation of influenza virus mRNAs and suppression of the elevated protein kinase levels normally found in cells infected by the mutant alone (M. G. Katze, B. M. Detjen, B. Safer, and R. M. Krug, Mol. Cell. Biol. 6:1741-1750, 1986). We elucidated the mechanisms of this kinase repression and can now report that influenza virus encodes a gene product which functions to directly block the autophosphorylation and activity of the interferon-induced, double-stranded-RNA-activated protein kinase, P68. Suppressed P68 activity was found not only in doubly infected cells but also in cells infected by influenza virus alone. Moreover, the decrease in P68 activity correlated with a decrease in the endogenous levels of phosphorylation of the alpha subunit of the eucaryotic initiation factor eIF-2, the natural substrate of the protein kinase. Suppression of P68 activity occurred as early as 2 h after influenza virus infection and required viral gene expression beyond the level of primary mRNA transcription to take place. We confirmed our in vivo observations with in vitro mixing experiments which showed that the influenza virus inhibitor can act in trans to block P68 activity. Combined repression of P68 function and eIF-2 alpha phosphorylation during influenza virus infection is essential for continued catalytic recycling of eIF-2 and efficient mRNA translation.

Animals↗
PubMed1988

DNA affinity labeling of adenovirus type 2 upstream promoter sequence-binding factors identifies two distinct proteins.

A rapid affinity labeling procedure with enhanced specificity was developed to identify DNA-binding proteins. 32P was first introduced at unique phosphodiester bonds within the DNA recognition sequence. UV light-dependent cross-linking of pyrimidines to amino acid residues in direct contact at the binding site, followed by micrococcal nuclease digestion, resulted in the transfer of 32P to only those specific protein(s) which recognized the binding sequence. This method was applied to the detection and characterization of proteins that bound to the upstream promoter sequence (-50 to -66) of the human adenovirus type 2 major late promoter. We detected two distinct proteins with molecular weights of 45,000 and 116,000 that interacted with this promoter element. The two proteins differed significantly in their chromatographic and cross-linking behaviors.

Adenoviruses, Human↗
PubMed1987

Adenovirus VAI RNA complexes with the 68 000 Mr protein kinase to regulate its autophosphorylation and activity.

We have investigated the interaction of VAI RNA with the interferon-induced, double-stranded (ds) RNA-activated protein kinase, P68, both of which regulate protein synthesis in adenovirus-infected cells. Previous work has shown that during infection by the VAI RNA-negative mutant, dl331, both viral and cellular protein synthesis are inhibited due to phosphorylation of the alpha-subunit of the eukaryotic initiation factor, eIF-2, by the P68 protein kinase. Utilizing monoclonal antibodies specific for P68, we demonstrated that the physical levels of P68 in dl331-infected, wild-type Ad2-infected and uninfected cells were all comparable suggesting that the elevated kinase activity detected during mutant infection was not due to increased P68 synthesis. To examine the basis of the increased activity of P68, the protein kinase was purified from infected-cell extracts using the monoclonal antibody. We found that P68 was heavily autophosphorylated during dl331 infection but not during wild-type or mock infection. The extent of autophosphorylation correlated with elevated P68 activity and the loss of the dsRNA requirements to phosphorylate the exogenous substrates, eIF-1 alpha and histones. We also analyzed VAI RNA function in vitro and present evidence that purified VAI RNA can block the autophosphorylation of P68 in the ribosomal salt wash fraction of interferon-treated cells. Finally we suggest VAI RNA functions through a direct interaction with the P68 protein kinase, since we demonstrated that VAI RNA forms a complex with P68 both in vitro and in vivo.

Adenoviruses, Human↗
PubMed1987

The purification and characterization of subunits alpha, beta, and gamma from the rabbit reticulocyte eukaryotic initiation factor 2.

Eukaryotic initiation factor 2 (eIF-2) contains three nonidentical subunits, alpha, beta, and gamma. The simultaneous purification of all three subunits was achieved by reverse-phase HPLC using a 0.1% trifluoroacetic acid-acetonitrile binary solvent system. The order of the eluted subunits, beta, alpha, and gamma, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After hydrolysis in 6 N HCl, picomole level amino acid composition analysis was achieved by the ninhydrin reaction on a Beckman 6300 system. Using second-derivative spectroscopic analysis, Trp was detected in all three subunits. All three subunits were subjected to amino-terminal sequence analysis. The amino-terminal of eIF-2 alpha from amino acid positions 1 to 23 inclusive was determined. The order of eight amino acids from the amino-terminal of eIF-2 gamma was also determined. This characterization and partial determination of the primary sequence of these subunits permit the utilization of molecular biology techniques in order to elucidate the complete primary structure. Additionally, the partial amino acid sequence data permitted the designation of synthetic gene probes as well as the identification of eIF-2 alpha and gamma cDNA and/or genomic clones.

Amino Acid Sequence↗
PubMed1986

Compensatory increase in levels of beta minor globin in murine beta-thalassemia is under translational control.

A 3.7-kilobase pair deletion including the entire beta major globin gene results in beta-thalassemia in a murine model of the disease (Skow, L. C., Burkhart, B. A., Johnson, F. M., Popp, R. A., Popp, D. M., Goldberg, S. Z., Anderson, W. F., Barnett, L. B., and Lewis, S. E. (1983) Cell 34, 1043-1052). There is a compensatory increase in synthesis of beta minor globin, resulting in a beta minor/alpha globin ratio of 0.75 in the homozygous thalassemic mouse, as compared to 0.2 in the normal homozygous diffuse mouse. The results presented here demonstrate that the increase in beta minor globin synthesis occurs at translation rather than at transcription. RNase T1 analysis of reticulocyte mRNA reveals that the beta/alpha ratio of globin mRNA is 0.3, significantly lower than the globin synthetic ratio of 0.7. However, the beta/alpha ratio of mRNA on polysomes is higher than unassociated mRNA, demonstrating that beta minor mRNA is preferentially translated. Elevated synthesis of beta minor globin is maintained during in vitro translation in thalassemic reticulocyte lysate. In this system, partial inhibition of translational elongation by cycloheximide decreases the beta minor/alpha globin synthetic ratio, whereas partial inhibition of initiation by hemin deficiency increases the beta minor/alpha synthetic ratio. This suggests that beta minor mRNA competes with alpha-mRNA for a limiting mRNA binding factor at initiation of translation.

Animals↗
PubMed1986

Adenovirus VAI RNA antagonizes the antiviral action of interferon by preventing activation of the interferon-induced eIF-2 alpha kinase.

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for efficient translation of host cell and viral mRNAs late after infection. The growth of a viral mutant that is unable to produce the RNA is inhibited by interferon, while wild-type virus is not affected. VAI RNA prevents activation of the interferon-induced P1/eIF-2 alpha kinase. This inhibition can be reproduced in extracts of interferon-treated cells where purified VAI RNA prevents activation of latent kinase by double-stranded RNA.

Adenoviruses, Human↗
PubMed1986

An adenovirus mutant unable to express VAI RNA displays different growth responses and sensitivity to interferon in various host cell lines.

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for the efficient translation of host cell and viral mRNAs late after infection. VAI RNA prevented activation of the interferon-induced P1/eIF-2 alpha kinase. In its absence the kinase was activated, eIF-2 alpha was phosphorylated, and translational initiation was inhibited. H5dl331 (dl331), a mutant which cannot express VAI RNA, grew poorly in 293 cells but generated wild-type yields in KB cells. The growth phenotype of the mutant appeared to correlate with the kinetics of kinase induction and activation. Active kinase appeared more rapidly in cell extracts prepared from infected 293 cells, in which dl331 grew poorly, than in extracts of KB cells, in which the mutant grew well. However, when kinase was induced in KB cells by interferon treatment and then activated subsequent to dl331 infection, viral protein synthesis was less severely inhibited than in interferon-treated 293 cells. Thus, activated kinase per se is insufficient to severely inhibit dl331 protein synthesis in KB cells.

Adenoviruses, Human↗
PubMed1986

Translational control by influenza virus: suppression of the kinase that phosphorylates the alpha subunit of initiation factor eIF-2 and selective translation of influenza viral mRNAs.

Selective translation of influenza viral mRNAs occurs after influenza virus superinfection of cells infected with the VAI RNA-negative adenovirus mutant dl331 (M. G. Katze, Y.-T. Chen, and R. M. Krug, Cell 37:483-490, 1984). Cell extracts from these doubly infected cells catalyze the initiation of essentially only influenza viral protein synthesis, reproducing the in vivo situation. This selective translation is correlated with a 5- to 10-fold suppression of the dl331-induced kinase that phosphorylates the alpha subunit of eucaryotic initiation factor eIF-2. This strongly suggests that influenza virus encodes a gene product that, analogous to the adenoviral VAI RNA, prevents the shutdown of overall protein synthesis caused by an eIF-2 alpha kinase turned on by viral infection. Adenoviral mRNA translation was restored to the extract from the doubly infected cells by the addition of the guanine nucleotide exchange factor eIF-2B, which is responsible for the normal recycling of eIF-2 during protein synthesis. This indicates that the residual kinase in the doubly infected cells leads to a limitation in functional (nonsequestered) eIF-2B and hence functional (GTP-containing) eIF-2 and that under these conditions influenza viral mRNAs are selectively translated over adenoviral mRNAs. Addition of double-stranded RNA to the extracts from these cells restored the eIF-2 alpha kinase to a level approaching that seen in extracts from cells infected with dl331 alone and caused the inhibition of influenza viral mRNA translation. This suggests that the putative influenza viral gene product acts against the double-stranded RNA activation of the kinase and indicates that influenza viral mRNA translation is also linked to the level of functional eIF-2. Our results thus indicate that a limitation in functional eIF-2 which causes a nonspecific reduction in the rate of initiation of protein synthesis results in the preferential translation of the better mRNAs (influenza viral mRNAs) at the expense of the poorer mRNAs (adenoviral mRNAs).

Adenoviruses, Human↗
PubMed1985

Adenovirus VAI RNA prevents phosphorylation of the eukaryotic initiation factor 2 alpha subunit subsequent to infection.

The virus-associated VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for efficient translation of mRNAs late after infection. Deletion mutant dl331 fails to produce this RNA and, as a result, grows poorly. Three lines of evidence suggest that VAI RNA facilitates translation by preventing inactivation of the function of eukaryotic initiation factor 2 (eIF-2). First, the mutant's translational defect can be relieved by addition of eIF-2 or eIF-2B (GTP recycling factor). Second, extracts of mutant-infected cells exhibit enhanced protein P1/eIF-2 alpha subunit kinase activity. Third, dl331 can grow with nearly normal kinetics in cells that do not express the kinase.

Adenoviridae Infections↗
PubMed1985

Isolation of stable preinitiation, initiation, and elongation complexes from RNA polymerase II-directed transcription.

Distinct RNA polymerase II transcription preinitiation, initiation, and elongation complexes can be formed in vitro on cloned adenovirus 2 DNA sequences containing the major late promoter. These transcription complexes are stable and can be rapidly isolated by gel filtration of HeLa whole cell extracts. In the absence of exogenous nucleotides and under appropriate salt conditions, a stable but transcriptionally incomplete preinitiation complex is formed. When this complex is incubated in the presence of adenosine or deoxyadenosine triphosphates, the beta-gamma phosphodiester bond is hydrolyzed, and RNA polymerase II joins the complex, thereby converting it into a stable initiation complex capable of forming (but prior to the formation of) the first phosphodiester bond. When this complex is isolated and incubated in the presence of all four nucleoside triphosphates, it is converted into an elongation complex that then permits the synthesis of phosphodiester bonds and the correct run-off transcript. A limiting transcription component is sequestered in the preinitiation complex. This factor is released upon elongation and can reassociate with new DNA templates during subsequent rounds of initiation. Therefore, class II genes do not appear to form activated transcription units stable for multiple rounds of transcription; rather, their transcriptional activity may be controlled in part by regulating the association of transcription factors at each initiation event.

Adenoviridae↗
PubMed1984

The association of eIF-2 with Met-tRNAi or eIF-2B alters the specificity of eIF-2 phosphatase.

In unfractioned reticulocyte lysate, interaction of eukaryotic initiation factor 2 (eIF-2) with other components regulates the accessibility of phosphatases and kinases to phosphorylation sites on its alpha and beta subunits. Upon addition of eIF-2 phosphorylated on both alpha and beta subunits (eIF-2(alpha 32P, beta 32P) to lysate, the alpha subunit is rapidly dephosphorylated, but the beta subunit is not. In contrast, both sites are rapidly dephosphorylated by the purified phosphatase. The basis of this altered specificity appears to be the association of eIF-2 with other translational components rather than an alteration of the phosphatase. Formation of an eIF-2(alpha 32P,beta 32P) Met-tRNAi X GTP ternary complex prevents dephosphorylation of the beta subunit, but has no effect on the rate of alpha dephosphorylation. eIF-2B, a 280,000-dalton polypeptide complex required for GTP:GDP exchange, also protects the beta subunit phosphorylation site from the purified phosphatase. However, the dephosphorylation of eIF-2(alpha 32P) is inhibited by 75% while complexed with eIF-2B. The altered phosphatase specificity upon association of eIF-2 with eIF-2B also affects the access of protein kinases to these phosphorylation sites. In the eIF-2B X eIF-2 complex, the alpha subunit is phosphorylated at 30% the rate of free eIF-2. Under identical conditions, phosphorylation of eIF-2 beta can not be detected. These results illustrate the importance of substrate conformation and/or functional association with other components in determining the overall phosphorylation state allowed by alterations of kinase and phosphatase activities.

Animals↗
PubMed1984

Isolation of an active transcription initiation complex from HeLa cell-free extract.

A two-step procedure has been developed for the formation of RNA polymerase II transcription initiation and elongation complexes. Initiation complexes are rapidly formed in HeLa cell-free extract supplemented with a DNA template containing the adenovirus 2 major late promoter and ATP. Assembly of transcription components required for correct initiation is absolutely dependent on specific eukaryotic promoter sequences. Sarkosyl-sensitive transcription initiation complexes are rapidly converted to Sarkosyl-resistant elongation complexes when supplemented with the remaining nucleoside triphosphates. The 60S initiation complex can be extensively purified by glycerol gradient centrifugation and is easily separated from free RNA polymerase II and free DNA template. Recovery of this stable complex is greater than 90%. Specific transcription cannot be detected if the DNA template is subsequently added to gradient fractions containing HeLa cell-free extract components alone. This suggests that the DNA templates promote the specific assembly of RNA polymerase II and transcription factors required for accurate initiation. Since conversion of purified initiation complexes to elongation complexes can occur without additional HeLa cell components, the presence of transcription components required for initiation and elongation in a single complex is indicated.

Cell-Free System↗
PubMed1984

Homologous globin cell-free transcription system with comparison of heterologous factors.

Mouse erythroleukemia (MEL) cells provide a useful model system to examine the regulation of globin gene expression. MEL cells ordinarily do not express globin genes, but in the presence of inducers, such as dimethyl sulfoxide or hexamethylene bisacetamide, they mimic erythroid differentiation. We have developed a cell-free transcription system from uninduced MEL cells to determine the requirements for mRNA synthesis. The MEL system directs accurate transcription of adenovirus type 2 major late DNA and mouse betamaj-globin with an efficiency comparable to those of HeLa and KB cell extracts. Using the procedure of Matsui et al. (T. Matsui, J. Segall, P.A. Weil, and R.G. Roeder, J. Biol. Chem. 255:11992-11996, 1980), we have isolated three active fractions from both MEL and HeLa cell extracts which are required for accurate transcription and have shown that equivalent fractions from MEL and HeLa cell extracts are interchangeable. Our findings suggest that the components required for initiation of transcription are similar in different cell types, at least to the extent that they can be assayed in these in vitro systems.

Animals↗
PubMed1983

The protein phosphatases involved in cellular regulation. 4. Classification of two homogeneous myosin light chain phosphatases from smooth muscle as protein phosphatase-2A1 and 2C, and a homogeneous protein phosphatase from reticulocytes active on protein synthesis initiation factor eIF-2 as protein phosphatase-2A2.

Two homogeneous protein phosphatases, termed 'smooth muscle phosphatase-I' and 'smooth muscle phosphatase-II', isolated from turkey gizzard as enzymes active against the 20-kDa light chain of smooth muscle myosin, and a third homogeneous protein phosphatase from rabbit reticulocytes, purified as an enzyme active against protein synthesis initiation factor eIF-2, were classified using the criteria defined by Ingebritsen and Cohen [Eur. J. Biochem. (1983) 132, 255-261]. All three enzymes were type-2 protein phosphatases based on their specificity for the alpha-subunit of phosphorylase kinase and insensitivity to inhibitor-1 and inhibitor-2. The substrate specificities of smooth muscle phosphatase-I and the eIF-2 phosphatase were similar to the catalytic subunit of protein phosphatase-2A. Smooth muscle phosphatase-I could be designated as protein phosphatase-2A1 and eIF-2 phosphatase as protein phosphatase-2A2 on the basis of their subunit compositions. The substrate specificity, dependence of activity on Mg2+ and subunit composition of smooth muscle phosphatase-II allowed its assignment as protein phosphatase-2C.

Animals↗
PubMed1983

Double-stranded RNA-dependent phosphorylation of protein P1 and eukaryotic initiation factor 2 alpha does not correlate with protein synthesis inhibition in a cell-free system from interferon-treated mouse L cells.

The double-stranded RNAs (I)n X (C)n and (A)n X (dUfl)n (dUfl is 2'-fluoro-2'-deoxyuridylic acid) have been compared as inhibitors of translation in cell-free systems from interferon-treated mouse L cells and from rabbit reticulocytes. In the interferon-treated mouse L-cell system, both double-stranded RNAs stimulated kinase activity, leading to phosphorylation of protein P1 and eukaryotic initiation factor 2 alpha (eIF-2 alpha), but only (1)n X (C)n activated the (2'-5')-oligoadenylate synthetase. Moreover, in this system, (I)n X (C)n, but not (A)n X (dUfl)n, inhibited translation. Both (A)n X (dUfl)n and (I)n X (C)n also activated the rabbit reticulocyte kinase to phosphorylate protein P1 and eIF-2 alpha, but, in contrast to mouse L-cell systems, both (A)n X (dUfl)n and (I)n X (C)n were potent inhibitors of translation in reticulocyte lysates. These results indicate that protein P1 and eIF-2 alpha phosphorylation are not sufficient to cause inhibition of protein synthesis in interferon-treated mouse L-cell extracts. They further suggest that protein synthesis inhibition by (I)n X (C)n in extracts of interferon-treated L cells correlates better with activation of (2'-5')-oligoadenylate synthetase than with activation of the protein P1 and eIF-2 alpha kinase.

Animals↗
PubMed1983

Identification of the hypusine-containing protein hy+ as translation initiation factor eIF-4D.

A single protein of Mr 17,000-19,000 and pI approximately equal to 5.1, found in all animal cells we have studied to date, undergoes post-translational modification in growing cells to form the unusual amino acid hypusine. Because of the association of this modification with the increasing rate of protein synthesis during lymphocyte growth stimulation, its subcellular distribution, and its widespread occurrence and structural conservation among animal cells, we considered the possibility that this protein might be a translation initiation factor. Purified rabbit reticulocyte factors (eukaryote initiation factors) eIF-4C and eIF-4D were chosen for study because of their Mr (17,000-19,000) and acidic pI. The hypusine-containing protein and purified eIF-4D showed identity of electrophoretic mobility in both isoelectric focusing and NaDodSO4/polyacrylamide gel electrophoresis dimensions, while eIF-4C was clearly nonidentical. Purified eIF-4D contained approximately 1 mol of hypusine per mol of protein. Since only one protein has thus far been observed to contain hypusine, we conclude that eIF-4D is the hypusine-containing protein. On the basis of relative synthesis among lymphocyte proteins and detection by Coomassie blue staining, we also conclude that eIF-4D is a major cell protein. It is possible that the activity of this factor is modulated by It is possible that the activity of this factor is modulated by post-translational hypusine formation, which may play a role in regulation of protein synthesis during lymphocyte growth stimulation.

Animals↗