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Biomedical subjects

C E Samuel

Publications and source records attributed to C E Samuel.

At least 91 records · Page 5Linked to original sources
PubMed1987

Biosynthesis of reovirus-specified polypeptides. Efficiency of expression of cDNAs of the reovirus S1 and S4 genes in transfected animal cells differs at the level of translation.

Full-length cDNAs of the reovirus serotype 1 Lang strain S1 and S4 genes were cloned in Escherichia coli using bacteriophage M13 and expressed in monkey COS cells under the control of the SV40 late promoter using the eukaryotic expression vector pJC119. The s1-encoded sigma 1 and s4-encoded sigma 3 gene products were expressed in transfected COS cells and were indistinguishable from the authentic sigma 1 and sigma 3 polypeptides synthesized in reovirion-infected COS cells. The relative translational efficiencies of the s1 and s4 mRNAs in transfected COS cells were similar to the efficiencies observed in virion-infected cells; the s4 mRNA was translated approximately five times more efficiently than the s1 mRNA. Our results suggest that the differential translation of the reovirus s1 and s4 mRNAs in vivo may be attributed to intrinsic structural properties of the individual mRNAs and is independent of competition with other viral mRNAs.

Animals↗
PubMed1987

Progress toward the understanding of the molecular mechanisms of interferon action.

Results of cellular and molecular analyses of type I (IFN-alpha and IFN-beta) and type II (IFN-gamma) human interferons demonstrate that these proteins differentially regulate the expression of certain cellular genes and induce mechanistically distinct antiviral states in human cells. Although none of the human genes whose expression is regulated by IFN have yet been localized to human chromosome 21, the localization of the receptor for the type I IFNs to chromosome 21 may have important consequences with regard to the functions of the interferon system in Down Syndrome individuals.

Animals↗
PubMed1986

Mechanism of interferon action. Expression of vesicular stomatitis virus G gene in transfected COS cells is inhibited by interferon at the level of protein synthesis.

The effect of interferon on the expression of the vesicular stomatitis virus glycoprotein G gene was examined in simian COS cells transfected with the expression vector pSVGL containing the G gene under the control of the SV40 late promoter. When COS cells were treated with interferon 24 h after transfection, the synthesis of vesicular stomatitis virus G protein was inhibited by about 80% as compared to that in untreated controls. By contrast, under the same conditions, neither the plasmid copy number nor the G gene mRNA levels were detectably affected by interferon treatment. Likewise, the synthesis of simian virus 40 large T-antigen was not inhibited by interferon treatment of transfected COS cells even though the synthesis of vesicular stomatitis virus G protein was markedly inhibited. The residual G protein synthesized in transfected, interferon-treated COS cells appeared to be normally glycosylated.

Animals↗
PubMed1986

Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain bicistronic s1 mRNA which encodes the minor capsid polypeptide sigma 1a and the nonstructural polypeptide sigma 1bNS.

Human reovirus serotype 1 Lang strain s1 mRNA, which encodes the minor capsid cell attachment protein sigma 1a and the nonstructural protein sigma 1bNS, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. The Lang strain s1 mRNA is 1462 nucleotides in length and possesses two open reading frames. The first begins at nt 14 and has a coding capacity of 418 amino acids, sufficient to account for sigma 1a; the second begins at nt 75 and has a coding capacity of 119 amino acids, sufficient to account for sigma 1bNS. Comparison of the Lang serotype s1 sequence derived from cDNA clones of s1 mRNA with the Lang S1 sequence derived from cDNA clones of the S1 dsRNA genome segment definitively establishes that reovirus plus-strand mRNA is structurally equivalent to the plus-strand of the dsRNA genome segment.

Base Sequence↗
PubMed1986

Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s3 mRNA which encodes the nonstructural RNA-binding protein sigma NS.

Human reovirus serotype 1 Lang strain s3 mRNA, which encodes the nonstructural RNA-binding polypeptide sigma NS, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. A complete consensus nucleotide sequence was determined. The Lang strain s3 mRNA is 1198 nucleotides in length and possesses an open reading frame with a coding capacity of 366 amino acids, sufficient to account for a sigma NS polypeptide of 41,179 daltons. Comparison of the serotype 1 (Lang) s3 sequence with the serotype 3 (Dearing) s3 sequence reveals 86.8 percent homology at the nucleotide level. The predicted sigma NS polypeptides of the Lang and Dearing strains display 97 percent homology at the amino acid level.

Amino Acid Sequence↗
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

Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s4 mRNA which encodes the major capsid surface polypeptide sigma 3.

Serotype 1 Lang strain s4 mRNA, which encodes the major capsid surface polypeptide sigma 3 of reovirions, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. A complete consensus nucleotide sequence for s4 mRNA has been determined from cDNA clones. The Lang strain s4 mRNA is 1196 nucleotides in length and possesses an open reading frame with a coding capacity of 365 amino acids, sufficient to account for a sigma 3 polypeptide of 41,212 daltons. Comparison of the serotype 1 (Lang) s4 sequence with the serotype 3 (Dearing) s4 sequence reveals 94% homology at the nucleotide level; the predicted sigma 3 polypeptides of the Lang and Dearing strains display 96% homology at the amino acid level. Two third base C codons (leu:CUC and ser:AGC) are used about one-tenth as frequently in the reovirus s4 mRNAs as compared to mammalian cellular mRNAs.

Amino Acid Sequence↗
PubMed1985

Mechanism of interferon action. Production and characterization of monoclonal and polyclonal antibodies to the interferon-induced phosphoprotein P1.

Monoclonal and polyclonal antibodies to the interferon-induced phosphoprotein P1 were prepared using protein P1 purified from human amnion U cells as the immunogen. Rabbit antiserum to protein P1 recognized with comparable efficiency P1 both from human U cells and from mouse L929 cells. Immunoprecipitates that contained protein P1 also possessed a protein kinase activity that catalyzed the phosphorylation of protein P1 and the alpha subunit of initiation factor eIF-2. Three BALB/C mouse monoclonal antibodies efficiently recognized human protein P1, but either did not recognize or recognized very poorly P1 from mouse cells. A fourth monoclonal antibody against human P1 recognized mouse P1 with nearly equal efficiency. Immunoprecipitation of human P1 with different sequential combinations of the monoclonal antibodies suggest that two antigenic classes of protein P1 may exist.

Animals↗
PubMed1985

Mechanism of interferon action. Purification and substrate specificities of the double-stranded RNA-dependent protein kinase from untreated and interferon-treated mouse fibroblasts.

The double-stranded RNA (dsRNA)-dependent protein kinase which catalyzes the phosphorylation of ribosome-associated protein P1 and the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2) was purified and characterized from mouse fibroblast L929 cells treated with either natural or recombinant interferon and from untreated cells. The dsRNA-dependent P1/eIF-2 alpha kinase was purified at least 1,500-fold from interferon-treated cells; the kinase activity that catalyzed the phosphorylation of eIF-2 alpha copurified with protein P1. The yield of P1/eIF-2 alpha protein kinase activity obtained following purification from cells treated with interferon was about 5-10 times greater than the yield from an equivalent number of untreated cells. The purified protein kinase remained dsRNA dependent. When P1 kinase was activated by dsRNA, a major phosphopeptide designated Xds was phosphorylated; Xds was not phosphorylated from P1 which had not been activated by dsRNA. The apparent native molecular weight of the purified mouse L929 dsRNA-dependent kinase as determined by sedimentation analysis was about 62,000, comparable to the molecular weight of 67,000 determined for denatured L929 phosphoprotein P1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase was highly selective for the alpha subunit of protein synthesis initiation factor eIF-2 and endogenous protein P1. Kinase activity was dependent upon Mg2+, and the Km for ATP was determined to be 5 X 10(-6) M. Histones (H1, H2A-B, H3, and H4) and protein synthesis initiation factors other than eIF-2 (eIF-3, eIF-4A, eIF-4B, and eIF-5) were not substrates or were very poor substrates for the purified dsRNA-dependent protein kinase. N-Ethylmaleimide, ethylenediaminetetraacetic acid, AMP, pyrophosphate, spermine, spermidine, and high concentrations of potassium inhibited both P1 and eIF-2 alpha phosphorylation by the purified kinase, whereas ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phenanthroline did not significantly affect the phosphorylation of either protein P1 or eIF-2 alpha.

Amnion↗
PubMed1985

Mechanism of interferon action. The interferon-induced phosphoprotein P1 possesses a double-stranded RNA-dependent ATP-binding site.

Protein P1, the interferon-induced protein phosphorylated in the presence of dsRNA in human amnion U-cells, was covalently labeled with [alpha-32P]ATP following ultraviolet irradiation. The photoaffinity labeling of protein P1 was dependent upon double-stranded RNA. Antibody prepared against phosphorylated protein P1 immunoprecipitated the double-stranded RNA-dependent photoaffinity-labeled product. The extent of photoaffinity labeling was significantly decreased by the addition of unlabeled ATP, GTP, or AMP; adenosine had little effect on the photoaffinity labeling of protein P1. These results suggest that protein P1 possesses a site capable of binding an adenine nucleotide in a double-stranded RNA-dependent manner.

Adenosine Triphosphate↗
PubMed1985

Mechanism of interferon action: inhibition of vesicular stomatitis virus replication in human amnion U cells by cloned human gamma-interferon. I. Effect on early and late stages of the viral multiplication cycle.

The molecular basis of the inhibition of vesicular stomatitis virus (VSV) replication by pure recombinant gamma-interferon (IFN-gamma) in human amnion U cells was examined. A saturating concentration of IFN-gamma induced, at maximum, about a two log10 reduction in infectious VSV yield. The kinetics of induction of the antiviral activity by IFN-gamma were first order over the period of about 6-18 h, following a lag of about 3 h, after treatment with a saturating concentration of IFN-gamma. The relationship of the inhibition in VSV infectivity to the early and late events of the VSV multiplication cycle was investigated. IFN-gamma treatment had no detectable effect on the adsorption and penetration of VSV virions or on their uncoating to yield viral nucleocapsids. The polypeptides of adsorbed or uncoated VSV particles were neither preferentially degraded nor detectably altered in IFN-gamma-treated U cells, as compared to untreated U cells. Progeny virions isolated from IFN-gamma-treated U cells, although greatly reduced in number, were found to be equally as infectious as those isolated from untreated U cells. Progeny virions from IFN-gamma-treated cells also possessed the same composition of viral proteins as was observed for virions from untreated cells. These results suggest that conditions of IFN-gamma treatment sufficient to reduce the yield of infectious VSV progeny 100-fold do not detectably affect either the early or the late stages of the VSV multiplication cycle.

Amnion↗
PubMed1985

Mechanism of interferon action: inhibition of vesicular stomatitis virus replication in human amnion U cells by cloned human gamma-interferon. II. Effect on viral macromolecular synthesis.

The effects of recombinant human gamma-interferon (IFN-gamma) on vesicular stomatitis virus (VSV) macromolecular synthesis in human amnion U cells were examined. Saturating concentrations of IFN-gamma caused only a 3 to 5-fold reduction of viral protein synthesis in wild-type VSV-infected cells, an extent insufficient to account for the 100-fold inhibition of viral infectivity. By use of the VSV mutant tsG41, which is competent in RNA transcription but defective in RNA replication at 40 degrees C, it was shown that the apparent IFN-induced inhibition of viral protein synthesis was likely due to a reduction in the synthesis of primary transcripts in IFN-gamma-treated U cells. Dot blot hybridization analysis revealed that saturating concentrations of IFN-gamma reduced both primary (measured with mutant tsG41-infected U cells) and total (measured with wild-type-infected U cells) viral RNA synthesis by about 4-fold, an extent of inhibition comparable to the observed reduction in viral protein synthesis. Analysis of RNA, fractionated by agarose gel electrophoresis after denaturation with glyoxal, with cDNA probes to individual VSV mRNAs did not reveal any detectable difference in the structural integrity of VSV mRNA isolated from IFN-gamma treated as compared to untreated U cells. These results suggest that IFN-gamma treatment causes a small reduction in the efficiency of transcript formation catalyzed by input parental virions. However, the results also indicate that the principal cause of the IFN-gamma-induced inhibition of VSV replication in U cells is the alteration of a step in replication other than viral macromolecular synthesis. This implies that the molecular mechanism of viral inhibition by IFN-gamma is fundamentally different from that of IFN-alpha in human amnion U cells.

Amnion↗
PubMed1985

Biosynthesis of reovirus-specified polypeptides: the reovirus s1 mRNA encodes two primary translation products.

Reovirus serotypes 1 (Lang strain) and 3 (Dearing strain) code for a hitherto unrecognized low-molecular-weight polypeptide of Mr approximately 12,000. This polypeptide (p12) was synthesized in vitro in L-cell-free protein synthesizing systems programmed with either reovirus serotype 1 mRNA, reovirus serotype 3 mRNA, or with denatured reovirus genome double-stranded RNA, and in vivo in L-cell cultures infected with either reovirus serotype. The synthesis of p12 in vivo was insensitive to actinomycin D, and occurred at similar times after infection as the previously identified reovirus encoded lambda, mu, and sigma polypeptides. Pulse-chase experiments in vivo, and the relative kinetics of synthesis of p12 in vitro, indicate that it is a primary translation product. Fractionation of reovirus mRNAs by velocity sedimentation and translation of separated mRNAs in vitro suggests that p12 is coded for by the s1 mRNA, which also codes for the previously recognized sigma 1 polypeptide. Synthesis of both p12 and sigma 1 in vitro in L-cell-free protein synthesizing systems programmed with denatured reovirus genome double-stranded RNA also suggests that these two polypeptides can be coded by the same mRNA species. The Mr approximately 12,000 polypeptide was not a detectable structural component of purified virions, and antiserum prepared against purified reovirions did not immunoprecipitate p12. It is proposed that the Mr approximately 12,000 polypeptide encoded by the S1 genome segment be designated sigma 1bNS, and that the polypeptide previously designated sigma 1 be renamed sigma 1a.

Animals↗
PubMed1985

Biosynthesis of reovirus-specified polypeptides. The s1 mRNA synthesized in vivo is structurally and functionally indistinguishable from in vitro-synthesized s1 mRNA and encodes two polypeptides, sigma 1a and sigma 1bNS.

The structural and functional properties of the reovirus serotype 1 (Lang strain) s1 mRNA were examined. Reovirus s-class mRNAs, synthesized either in vivo within infected mouse L cells or in vitro by chymotrypsin-derived cores of purified virions, were purified by filter-hybridization using cDNA clones of the S-class genome segments. S1 cDNA-selected mRNA encoded the synthesis of the Mr approximately 12,000 nonstructural polypeptide designated sigma 1bNS in addition to the well-established structural polypeptide sigma 1, now designated sigma 1a. The coding properties of in vivo- and in vitro-synthesized s1 mRNA were equivalent: both encoded sigma 1a and sigma 1bNS. Primer extension analysis of s1 mRNA revealed a single major 5' terminus for both in vivo- and in vitro-synthesized s1 mRNA. These results suggest that there is a single transcript of the reovirus S1 genome segment which is functionally dicistronic, and likely encodes both sigma 1a and sigma 1bNS.

Animals↗
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↗