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A J Shatkin

Publications and source records attributed to A J Shatkin.

At least 19 recordsLinked to original sources
PubMed1996

Regulated, stable expression and nuclear presence of reovirus double-stranded RNA-binding protein sigma3 in HeLa cells.

Reovirus genome segment S4 codes for polypeptide sigma3, a major outer capsid component of virions and a double-stranded RNA (dsRNA)-binding protein implicated in viral cytopathogenesis. We have constructed a stable HeLa cell line (S4tTA) that produces functional sigma3 under tetracycline transactivator control. In the absence of tetracycline, S4tTA cells synthesized stable dsRNA-binding sigma3 that accumulated in the nucleus as well as in the cytoplasm. However, in induced S4tTA cells also expressing reovirus outer shell polypeptide mu1/mu1C, migration of sigma3 into the nucleus was blocked, probably as a result of formation of a complex with mu1/mu1C which was exclusively in the cytoplasm. Mutant analyses indicated a correlation between dsRNA-binding activity and nuclear entry of sigma3, suggesting an additional role(s) for this capsid protein in virus-cell interactions.

Amino Acid Sequence↗
PubMed1995

The Ser36-Ser37 pair in HeLa nuclear protein p21/SIIR mediates Ser/Thr phosphorylation and is essential for Rous sarcoma virus long terminal repeat repression.

Phosphorylation of HeLa SII (or TFIIS)-related nuclear protein p21/SIIR was demonstrated in transfected COS-1 cells. To test for a possible functional link between phosphorylation and the previously described Rous sarcoma virus (RSV) long terminal repeat (LTR) repression (Yeh, C.H., and Shatkin, A.J. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11002-11006), p21/SIIR mutants were constructed and assayed for phosphorylation level and effect on RSV LTR-driven chloramphenicol acetyltransferase (CAT) reporter expression. A major phosphorylation target in p21/SIIR was localized to the Arg/Ser-rich region between amino acids 12 and 49. Deletion of this region impaired the ability of p21/SIIR to down-regulate RSV LTR promoter function. Four serine pairs, all displaying the Arg/Lys-Ser-Ser motif typical of phosphorylation sites, are present in p21/SIIR between positions 31 and 48. Conversion of these individual serine pairs to alanine resulted in decreased phosphorylation in each case. Mutation of the Ser36-Ser37 pair also diminished by severalfold the repression activity of p21/SIIR. The single tyrosine (Tyr155) in p21/SIIR was not detectably phosphorylated in transfected COS-1 cells, suggesting that the Ser36-Ser37 pair mediates Ser/Thr phosphorylation of p21/SIIR and is critical for LTR repression function.

Amino Acid Sequence↗
PubMed1995

A cis-acting element in Rous sarcoma virus long terminal repeat required for promoter repression by HeLa nuclear protein p21.

HeLa cell basic nuclear protein (p21), which represses Rous sarcoma virus long terminal repeat (RSV LTR) promoter activity, diminished v-src expression and the appearance at permissive temperature of the transformed phenotype in tsRSVLA23 Rat-1, a cell line transformed with a temperature-sensitive mutant of RSV. Nuclear run-on analyses using COS-1 cells cotransfected with p21 cDNA and chloramphenicol acetyltransferase reporter indicated that p21 inhibits transcription initiation by targeting a region in the RSV LTR promoter between positions -108 and -85 upstream of the cap site. Insertion of this 24-base pair sequence in place of one of the 72-base pair enhancers in the SV40 early promoter rendered it sensitive to p21 repression. Electrophoretic mobility shift assays using a synthetic oligomer corresponding to the 24-base pair LTR promoter element revealed that p21 altered the pattern of protein.DNA complex formation apparently without binding DNA directly. Complex formation assayed by UV cross-linking and DNA affinity chromatography indicated further that a cellular factor which can interact with this element was decreased in cells transfected with p21 expression plasmid. The results indicate that p21 repression of RSV LTR is mediated by a cis-acting element and may occur by alteration of protein complexes formed on this promoter element.

Animals↗
PubMed1994

Down-regulation of Rous sarcoma virus long terminal repeat promoter activity by a HeLa cell basic protein.

We have previously isolated a HeLa cell cDNA encoding a 21-kDa polypeptide that is 48% similar to transcription factor IIS. To explore the possibility that p21 plays a role in transcriptional regulation in vivo, we tested the effect of p21 expression on the synthesis of reporter chloramphenicol acetyltransferase (CAT) in transfected COS-1 cells. CAT formation under control of the Rous sarcoma virus long terminal repeat (RSV LTR) promoter was decreased nearly 20-fold in cells coexpressing p21. In contrast, CAT production under control of other sequence elements was only slightly reduced (human immunodeficiency virus type 1 LTR, simian virus 40 early promoter), unaffected (human heat shock protein of 70-kDa promoter, adenovirus major late promoter TATA box), or increased (terminal deoxynucleotidyltransferase initiator element, c-fos promoter) by p21 coexpression as compared to cells cotransfected with the parental vector. The abundance of steady-state CAT transcripts from RSV LTR was also decreased by p21 expression in a dose-dependent manner, suggesting that transcription of RSV LTR/CAT is under negative control by p21. Consistent with an effect on transcription, p21 was localized in nuclei of transfected cells. Deletion analysis of p21 indicated that the sequences essential for inhibition of RSV LTR function include the previously identified ARg/Ser-rich region and zinc finger-like motif. Proliferation of chicken embryo fibroblasts transfected with an infectious molecular clone of RSV was diminished by p21 expression, which also resulted in fewer transformed foci.

Animals↗
PubMed1994

A HeLa-cell-encoded p21 is homologous to transcription elongation factor SII.

A 1.2-kb cDNA clone isolated from a HeLa cell library contains an open reading frame encoding a new protein (p21) of 21 kDa that is approx. 48% similar to members of the eukaryotic transcription elongation factor SII (TFIIS) family. The deduced amino-acid sequence of p21 indicates that it is hydrophilic, basic and contains nuclear localization signals, as well as multiple consensus phosphorylation sites.

Amino Acid Sequence↗
PubMed1993

Membrane topology model of Escherichia coli alpha-ketoglutarate permease by phoA fusion analysis.

Escherichia coli alpha-ketoglutarate permease (KgtP) is a 432-amino-acid protein that symports alpha-ketoglutarate and protons. KgtP was predicted to contain 12 membrane-spanning domains on the basis of a calculated hydropathy profile. The membrane topology model of KgtP was analyzed by using kgtP-phoA gene fusions and measuring alkaline phosphatase activities in cells expressing the chimeric proteins. Comparisons of the phosphatase activity levels and the locations of the KgtP-PhoA junctions are consistent with the predicted membrane topology model of KgtP.

Alkaline Phosphatase↗
PubMed1992

Site-directed mutants of Escherichia coli alpha-ketoglutarate permease (KgtP).

To investigate an active site(s) in the Escherichia coli alpha-ketoglutarate premease, 11 point mutants were made in the corresponding structural gene, kgtP, by oligonucleotide-directed mutagenesis and the polymerase chain reaction. On the basis of sequences conserved in KgtP and related members of a transporter superfamily [Henderson P. J. F., & Maiden, M. C. (1990) Philos. Trans. R. Soc. London B 326, 391], Arg76 was replaced with Ala, Asp, or Lys; Asp88 with Asn or Glu; His90 with Ala; Arg92 with Ala or Lys; and Arg198 with Ala, Asp, or Lys. Mutant proteins expressed using the T7 polymerase system were in each case shown to be membrane-associated. However, they differed in transport activity. Mutants H90A and R198K had activities similar to that of wild type, and R76K and R198A retained 10-60% of the wild-type activity. In all other mutants, alpha-ketoglutarate transport was abolished. The results suggest that Arg92, which is highly conserved among other members of the transporter superfamily, is necessary for activity and also that Asp88 is critical for function, as observed for the tetracycline transporter. These data show further that a positive charge is essential at position 76 and is also important, but not absolutely required, at position 198 for alpha-ketoglutarate transport. Unlike lacY permease which was inactivated by deleting the last helix [McKenna, E., Hardy, D., Pastore, J. C., & Kaback, H. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2969], a KgtP truncation mutant missing the last putative membrane-spanning region was relatively stable and also retained 10-50% of the wild-type level of alpha-ketoglutarate transport activity.

Amino Acid Sequence↗
PubMed1992

Escherichia coli alpha-ketoglutarate permease is a constitutively expressed proton symporter.

Escherichia coli kgtP which maps at 56.5 min codes for alpha-ketoglutarate permease (KgtP). This protein, expressed from the cloned gene using the T7 polymerase system and [35S]methionine labeling, fractionated with cell membranes. Right-side-out (RSO) membrane vesicles prepared from a kgtP negative mutant strain did not transport alpha-ketoglutarate, but RSO vesicles from the same strain expressing KgtP from a transforming plasmid transported alpha-ketoglutarate effectively as measured by uptake of the 14C-labeled substrate. E. coli JC7623 strain grown in M9 minimal medium with glucose, glycerol, or alpha-ketoglutarate as carbon source contained a 1.3-kilobase RNA which hybridized to nick-translated kgtP probe. In addition, strain MC1061 cultures grown under these same conditions were all capable of transporting alpha-ketoglutarate, demonstrating that KgtP is constitutively expressed. The Km and Vmax of KgtP assayed in strain MC1061 vesicles were 13-46 microM and 8 nmol/min/mg protein, respectively. Uncouplers that permeabilized the membrane to protons inhibited alpha-ketoglutarate transport into energized vesicles, and the addition of alpha-ketoglutarate to vesicle suspensions under non-energized conditions resulted in an increase in pH. These results indicate that KgtP is an alpha-ketoglutarate-proton symporter.

Biological Transport↗
PubMed1992

Translational effects and sequence comparisons of the three serotypes of the reovirus S4 gene.

Reovirus S4 RNA codes for the dsRNA-binding polypeptide sigma 3, a major virion outer capsid component that also has translational effects in both infected and transfected mammalian cells. To compare the composition and properties of the three different serotypes of sigma 3, a DNA copy of the type 2 gene was cloned and sequenced. The total lengths (1196) and the sequences of leader (33 nucleotides) and trailer (66 nucleotides) regions are highly conserved among the three S4 serotypes. The type 1 and 3 S4 genes are highly related (77 mismatches). However, the type 2 gene contains many mismatches relative to the type 1 and 3 genes (260 and 270 positions, respectively). Most of the mismatches are third position changes, resulting in sigma 3 polypeptides that are 90% or more identical. Transient expression vectors, constructed by replacing the chloramphenicol acetyltransferase (CAT) gene in pRSVCAT with S4 DNA, were used to test the effects of polypeptide sigma 3 on CAT expression in cotransfected COS cells. Transfection with the correctly oriented DNAs resulted in synthesis of the corresponding sigma 3 polypeptides which enhanced CAT expression. The type 2 and type 3 S4 genes were considerably more stimulatory than type 1 when compared to CAT DNA alone. However, with all three serotypes the CAT activity was significantly higher in cells cotransfected with S4 DNA in the correct orientation as compared to the reverse arrangement.

Amino Acid Sequence↗
PubMed1992

Translational stimulation by reovirus polypeptide sigma 3: substitution for VAI RNA and inhibition of phosphorylation of the alpha subunit of eukaryotic initiation factor 2.

COS cells transfected with plasmids that activate DAI depend on expression of virus-associated I (VAI) RNA to prevent the inhibitory effects of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) kinase (DAI) and restore the translation of vector-derived dihydrofolate reductase mRNA. This VAI RNA requirement could be completely replaced by reovirus polypeptide sigma 3, consistent with its double-stranded RNA (dsRNA)-binding activity. S4 gene transfection of 293 cells also partially restored adenovirus protein synthesis after infection with the VAI-negative dl331 mutant. In dl331-infected 293 cells, eIF-2 alpha was present mainly in the acidic, phosphorylated form, and trans complementation with polypeptide sigma 3 or VAI RNA decreased the proportion of eIF-2 alpha (P) from approximately 85 to approximately 30%. Activation of DAI by addition of dsRNA to extracts of S4 DNA-transfected COS cells required 10-fold-higher levels of dsRNA than extracts made from cells that were not producing polypeptide sigma 3. In extracts of reovirus-infected mouse L cells, the concentration of dsRNA needed to activate DAI was dependent on the viral serotype used for the infection. Although the proportion of eIF-2 alpha (P) was greater than that in uninfected cells, most of the factor remained in the unphosphorylated form, even at 16 h after infection, consistent with the partial inhibition of host protein synthesis observed with all three viral serotypes. The results indicate that reovirus polypeptide sigma 3 participates in the regulation of protein synthesis by modulating DAI and eIF-2 alpha phosphorylation.

Animals↗
PubMed1992

Reovirus polypeptide sigma 3 and N-terminal myristoylation of polypeptide mu 1 are required for site-specific cleavage to mu 1C in transfected cells.

N-myristoylated viral polypeptide mu 1 was produced in COS cells transfected with a transient expression vector containing a DNA copy of the reovirus M2 gene. The mu 1 product was specifically cleaved to polypeptide mu 1C in cells that were cotransfected with the reovirus S4 gene and that expressed polypeptide sigma 3. Studies with site-specific mutants of the M2 gene demonstrated that conversion of mu 1 to mu 1C was dependent on myristoylation and the presence of the proteolytic cleavage sequence asparagine 42-proline 43 in mu 1, as well as on the presence of polypeptide sigma 3. The mu 1C product and polypeptide sigma 3 formed complexes that were immunoprecipitated by sigma 3-directed antibody, and a myristoylation-negative M2 double mutant, G2A-N42T, yielded mu 1 that did not undergo cleavage to mu 1C or bind sigma 3. However, the N42T single mutant did form immunoprecipitable complexes with sigma 3, indicating that binding can occur in the absence of cleavage. Polypeptide sigma 3 alternatively can bind double-stranded RNA and in COS cells stimulates translation of reporter chloramphenicol acetyltransferase mRNA translation, presumably by blocking double-stranded RNA-mediated activation of the eukaryotic initiation factor 2 alpha subunit kinase which inhibits the initiation of protein synthesis. Consistent with these observations and with the formation of mu 1C-sigma 3 complexes, coexpression of M2 with S4 DNA prevented the translational stimulatory effect of polypeptide sigma 3.

Capsid↗
PubMed1991

Escherichia coli kgtP encodes an alpha-ketoglutarate transporter.

The witA gene located between pss and rrnG on the Escherichia coli chromosome encodes a 432-amino acid protein. It is homologous to a human hepatoma glucose transporter and to E. coli membrane proteins that transport citrate (CitA), arabinose (AraE), and xylose (XylE), and, like these carrier proteins, WitA also contains 12 highly hydrophobic putative membrane-spanning regions. Gene disruption mutants constructed in two E. coli strains grew slowly or not at all, depending on genetic background, in M9 minimal medium containing alpha-ketoglutarate. Growth on alpha-ketoglutarate and uptake of alpha-[14C]ketoglutarate were restored by transformation with plasmids containing witA. These complementation studies indicate that WitA is an alpha-ketoglutarate transporter and should be renamed kgtP(alpha-ketoglutarate permease).

Amino Acid Sequence↗
PubMed1990

Active site localization in a viral mRNA capping enzyme.

Capping of reovirus mRNAs is catalyzed by a guanylyltransferase that corresponds to virion structural polypeptide lambda 2. It forms a phosphoamide linked enzyme-pG covalent complex as an intermediate in the capping reaction. The nucleotide attachment site on lambda 2 was localized to a region between amino acids 213 and 269 by incubating virus particles with [alpha-32P]GTP followed by proteolytic cleavage and analysis of the resulting fragments using sequence-directed antibodies as probes. The 213-269 region contains as potential GMP acceptors a single lysine, 1 arginine, and 4 histidine residues, as deduced from the nucleotide sequence of the L2 gene encoding lambda 2. Digestion of 32P-labeled capping intermediate with alkali after oxidation and beta-elimination yielded phospholysine as the only phosphoamino acid, localizing the active site to a region in lambda 2 that includes the lysine at position 226.

Amino Acid Sequence↗
PubMed1990

Expression of the two reovirus S1 gene products in transfected mammalian cells.

Reovirus genomic segment S1, which has been implicated in the viral inhibition of cellular DNA synthesis, is transcribed into a single mRNA that encodes two proteins, the approximately 49-kDa hemagglutinin, sigma 1, and the apparently nonstructural protein, p14. These two polypeptides have been expressed in mammalian cells, together or independently, in order to assess their role in the shutdown of host DNA replication. Results obtained with transient and stable expression systems demonstrate that production of serotype 3 sigma 1 and p14 together or individually is not sufficient to change the kinetics of DNA replication in uninfected cells. However, inhibition of DNA synthesis by reovirus type 1 infection was enhanced in cells producing type 3 sigma 1 and p14 but not sigma 1 by itself. In addition, expression of p14 alone led to increased cytopathic effects following infection by either type 1 or type 3 virus. The results suggest that interactions with other viral components are required to elicit the effects of the S1-specified polypeptides on cellular DNA synthesis.

Animals↗
PubMed1990

Translation of bicistronic viral mRNA in transfected cells: regulation at the level of elongation.

The S1 species of mammalian reovirus mRNA, like a number of other viral but not cellular mRNAs, codes for two dissimilar polypeptides by initiation of translation at two 5'-proximal, out-of-frame AUG codons. To determine if uninfected cells can utilize bicistronic genes, a bovine papilloma virus-based vector system was used to select mouse C127 cell lines containing multiple integrated copies of the reovirus S1 gene. These cell lines produced both reovirus polypeptides from a single mRNA. In addition, studies of COS cells transfected with the S1 gene containing small changes around the first AUG suggest that bicistronic mRNA translation is regulated at the level of elongation. A model is proposed in which ribosomes engaged in translation of one reading frame interfere with movement of ribosomes in the other frame because of differences in codon usage. Expression of bicistronic genes may be similarly regulated in virus-infected cells.

Animals↗