Characterization of the electrophoretic mobility mutation in the N protein of the tsD1 mutant of vesicular stomatitis virus New Jersey serotype

1982 ◽  
Vol 60 (11) ◽  
pp. 1065-1076 ◽  
Author(s):  
Earl Brown ◽  
Ludvik Prevec
Keyword(s):  
New Jersey ◽  
Electrophoretic Mobility ◽  
Vesicular Stomatitis Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Specific Chemical ◽  
N Protein ◽  
Sds Page ◽  
Vesicular Stomatitis

Some isolates of the temperature sensitive mutant tsD1 of complementation group D of vesicular stomatitis virus of New Jersey serotype have a nucleocapsid (N) protein which shows an increased electrophoretic mobility on sodium dodecyl sulfate –polyacrylamide gel electrophoresis (SDS–PAGE) when compared with wild type. Utilizing techniques involving specific chemical cleavage at tryptophan or methionine residues, as well as enzymatic cleavage with carboxypeptidases A and B, we have determined that residues near the carboxyterminus are responsible for the electrophoretic difference of the mutant protein. We have further shown that there are no differences in the tryptic peptides of the mutant compared with the wild type or a non-ts revertant in this region of the protein. We have identified a tryptic peptide located outside the relevant carboxyterminal region which is distinct in mutant and revertant. We conclude that the mutation producing the aberrant electrophoretic mobility of N protein of the tsD1 mutant is a missense point mutation located at least 40 amino acid residues from the carboxyterminus and which interacts with a more proximal carboxyregion so as to influence electrophoretic mobility on SDS–PAGE.

scholarly journals Moving the Glycoprotein Gene of Vesicular Stomatitis Virus to Promoter-Proximal Positions Accelerates and Enhances the Protective Immune Response

2000 ◽  
Vol 74 (17) ◽  
pp. 7895-7902 ◽  
Author(s):  
E. Brian Flanagan ◽  
L. Andrew Ball ◽  
Gail W. Wertz
Keyword(s):  
Immune Response ◽  
Protein Expression ◽  
Vesicular Stomatitis Virus ◽  
Wild Type Virus ◽  
Wild Type ◽  
Gene Encoding ◽  
N Protein ◽  
Glycoprotein G ◽  
Vesicular Stomatitis ◽  
Negative Sense

ABSTRACT Vesicular stomatitis virus (VSV) is the prototype of the Rhabdoviridae and contains nonsegmented negative-sense RNA as its genome. The 11-kb genome encodes five genes in the order 3′-N-P-M-G-L-5′, and transcription is obligatorily sequential from the single 3′ promoter. As a result, genes at promoter-proximal positions are transcribed at higher levels than those at promoter-distal positions. Previous work demonstrated that moving the gene encoding the nucleocapsid protein N to successively more promoter-distal positions resulted in stepwise attenuation of replication and lethality for mice. In the present study we investigated whether moving the gene for the attachment glycoprotein G, which encodes the major neutralizing epitopes, from its fourth position up to first in the gene order would increase G protein expression in cells and alter the immune response in inoculated animals. In addition to moving the G gene alone, we also constructed viruses having both the G and N genes rearranged. This produced three variant viruses having the orders 3′-G-N-P-M-L-5′ (G1N2), 3′-P-M-G-N-L-5′ (G3N4), and 3′-G-P-M-N-L-5′ (G1N4), respectively. These viruses differed from one another and from wild-type virus in their levels of gene expression and replication in cell culture. The viruses also differed in their pathogenesis, immunogenicity, and level of protection of mice against challenge with wild-type VSV. Translocation of the G gene altered the kinetics and level of the antibody response in mice, and simultaneous reduction of N protein expression reduced replication and lethality for animals. These studies demonstrate that gene rearrangement can be exploited to design nonsegmented negative-sense RNA viruses that have characteristics desirable in candidates for live attenuated vaccines.

Glutathione synthetase from the fission yeast. Purification and its unique heteromeric subunit structure

1993 ◽  
Vol 71 (9-10) ◽  
pp. 447-453 ◽  
Author(s):  
Chiaki W. Nakagawa ◽  
Norihiro Mutoh ◽  
Yukimasa Hayashi
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Fission Yeast ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Glutathione Synthetase ◽  
Subunit Structure ◽  
Wild Type ◽  
Native Page ◽  
Sds Page

Glutathione (GSH) synthetase (EC 6.3.2.3) was purified from the fission yeast Schizosaccharomyces pombe L972h− and from the GSH synthetase deficient mutant MN101/pYS41, which harbors a plasmid containing the GSH synthetase gene of the fission yeast. GSH synthetase is expressed at 10 times higher the amount in MN101/pYS41 than in wild-type L972 h−. The purified enzyme gave a single band on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (native PAGE). The molecular weight of this enzyme was determined to be 1.2 × 105 by Sepharose CL-6B gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that this enzyme was composed of two kinds of subunits, A (Mr = 33 × 103) and B (Mr = 26 × 103), and existed as a heterotetramer (A2B2). The enzyme purified from the wild-type fission yeast, which did not harbor the plasmid, showed the same electrophoretic mobilities on both native PAGE and SDS–PAGE and similar catalytic properties under standard conditions. This enzyme is most active at 45 °C and pH 8.0–8.5 with 20 mM Mg2+ + 10 mM ATP and 50 mM K+. The strict requirement for the monovalent cation is rather specific for the enzymes from yeasts. The presence of sugar components in the enzyme is also observed, similar to that in the rat kidney enzyme.Key words: Schizosaccharomyces pombe, glutathione synthetase, heteromeric subunit structure.

scholarly journals Construction and Characterization of Haemophilus ducreyi Lipooligosaccharide (LOS) Mutants Defective in Expression of Heptosyltransferase III and β1,4-Glucosyltransferase: Identification of LOS Glycoforms Containing Lactosamine Repeats

2000 ◽  
Vol 68 (6) ◽  
pp. 3352-3361 ◽  
Author(s):  
Melanie J. Filiatrault ◽  
Bradford W. Gibson ◽  
Birgit Schilling ◽  
Shuhua Sun ◽  
Robert S. Munson ◽  
...  
Keyword(s):  
Lipid A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Haemophilus Ducreyi ◽  
Ionization Mass ◽  
Wild Type ◽  
Reading Frame ◽  
Plasmid Library ◽  
Sds Page ◽  
Isogenic Mutants

ABSTRACT To begin to understand the role of the lipooligosaccharide (LOS) molecule in chancroid infections, we constructed mutants defective in expression of glycosyltransferase genes. Pyocin lysis and immunoscreening was used to identify a LOS mutant of Haemophilus ducreyi 35000. This mutant, HD35000R, produced a LOS molecule that lacked the monoclonal antibody 3F11 epitope and migrated with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Structural studies indicated that the principal LOS glycoform contains lipid A, Kdo, and two of the three core heptose residues. HD35000R was transformed with a plasmid library of H. ducreyi 35000 DNA, and a clone producing the wild-type LOS was identified. Sequence analysis of the plasmid insert revealed one open reading frame (ORF) that encodes a protein with homology to the WaaQ (heptosyltransferase III) ofEscherichia coli. A second ORF had homology to the LgtF (glucosyltransferase) of Neisseria meningitidis. Individual isogenic mutants lacking expression of the putative H. ducreyi heptosyltransferase III, the putative glucosyltransferase, and both glycosyltransferases were constructed and characterized. Each mutant was complemented with the representative wild-type genes in trans to restore expression of parental LOS and confirm the function of each enzyme. Matrix-assisted laser desorption ionization mass spectrometry and SDS-PAGE analysis identified several unique LOS glycoforms containing di-, tri-, and poly-N-acetyllactosamine repeats added to the terminal region of the main LOS branch synthesized by the heptosyltransferase III mutant. These novel H. ducreyi mutants provide important tools for studying the regulation of LOS assembly and biosynthesis.

scholarly journals Involvement of the Matrix Protein in Attachment of Porcine Reproductive and Respiratory Syndrome Virus to a Heparinlike Receptor on Porcine Alveolar Macrophages

2002 ◽  
Vol 76 (9) ◽  
pp. 4312-4320 ◽  
Author(s):  
P. L. Delputte ◽  
N. Vanderheijden ◽  
H. J. Nauwynck ◽  
M. B. Pensaert
Keyword(s):  
Alveolar Macrophages ◽  
Matrix Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Respiratory Syndrome Virus ◽  
Structural Protein ◽  
N Protein ◽  
Differentiated Cells ◽  
Sds Page ◽  
The Matrix

ABSTRACT The porcine reproductive and respiratory syndrome virus (PRRSV) has a very restricted tropism for well-differentiated cells of the monocyte-macrophage lineage, which is probably determined by specific receptors on these cells. In this study, the importance of heparinlike molecules on porcine alveolar macrophages (PAM) for PRRSV infection was determined. Heparin interacted with the virus and reduced infection of PAM up to 92 or 88% for the American and European types of PRRSV, respectively. Other glycosaminoglycans, similar to heparin, had no significant effect on infection while heparinase treatment of PAM resulted in a significant reduction of the infection. Analysis of infection kinetics showed that PRRSV attachment to heparan sulfate occurs early in infection. A heparin-sensitive binding step was observed which converted completely into a heparin-resistant binding after 120 min at 4°C. Using heparin-affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it was observed that the structural matrix (M) and nucleocapsid (N) proteins attached to heparin. Nonreducing SDS-PAGE revealed that M bound to heparin mainly as a complex with glycoprotein GP5 and that the N protein bound to heparin as a homodimer. GP3, which was identified as a minor structural protein of European types of PRRSV, did not bind to heparin. Since the N protein is not exposed on the virion surface, it was concluded that the structural M protein and the M-GP5 complex contribute to PRRSV attachment on a heparinlike receptor on PAM. This is the first report that identifies a PRRSV ligand for a cell surface heparinlike receptor on PAM.

scholarly journals Dual Lipid Modification of the Yeast Gγ Subunit Ste18p Determines Membrane Localization of Gβγ

1999 ◽  
Vol 19 (11) ◽  
pp. 7705-7711 ◽  
Author(s):  
Jodi E. Hirschman ◽  
Duane D. Jenness
Keyword(s):  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cysteine Residue ◽  
Wild Type ◽  
Molecular Weights ◽  
Sds Page ◽  
Membrane Attachment ◽  
The Stability ◽  
Mutant Forms

ABSTRACT The pheromone response in the yeast Saccharomyces cerevisiae is mediated by a heterotrimeric G protein. The Gβγ subunit (a complex of Ste4p and Ste18p) is associated with both internal and plasma membranes, and a portion is not stably associated with either membrane fraction. Like Ras, Ste18p contains a farnesyl-directing CaaX box motif (C-terminal residues 107 to 110) and a cysteine residue (Cys 106) that is a potential site for palmitoylation. Mutant Ste18p containing serine at position 106 (mutation ste18-C106S) migrated more rapidly than wild-type Ste18p during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic mobility of wild-type Ste18p (but not the mutant Ste18p) was sensitive to hydroxylamine treatment, consistent with palmitoyl modification at Cys 106. Furthermore, immunoprecipitation of the Gβγ complex from cells cultured in the presence of [3H]palmitic acid resulted in two radioactive species on nonreducing SDS-PAGE gels, with molecular weights corresponding to Gγ and Gβγ. Substitution of serine for either Cys 107 or Cys 106 resulted in the failure of Gβγ to associate with membranes. The Cys 107 substitution also resulted in reduced steady-state accumulation of Ste18p, suggesting that the stability of Ste18p requires modification at Cys 107. All of the mutant forms of Ste18p formed complexes with Ste4p, as assessed by coimmunoprecipitation. We conclude that tight membrane attachment of the wild-type Gβγ depends on palmitoylation at Cys 106 and prenylation at Cys 107 of Ste18p.

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