scholarly journals Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers.

1992 ◽  
Vol 12 (1) ◽  
pp. 89-102 ◽  
Author(s):  
S W Wang ◽  
N A Speck
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Newborn Mice ◽  
The Core ◽  
Core Site ◽  
Disease Specificity ◽  
Murine Leukemia

The Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major genetic determinant of the thymic disease specificity of the Moloney virus genetically maps to two protein binding sites in the Moloney virus enhancer, the leukemia virus factor b site and the adjacent core site. Point mutations introduced into either of these sites significantly shifts the disease specificity of the Moloney virus from thymic leukemia to erythroleukemia (N. A. Speck, B. Renjifo, E. Golemis, T. Frederickson, J. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We have purified several polypeptides that bind to the core site in the Moloney virus enhancer. These proteins were purified from calf thymus nuclear extracts by selective pH denaturation, followed by chromatography on heparin-Sepharose, nonspecific double-stranded DNA-cellulose, and core oligonucleotide-coupled affinity columns. We have achieved greater than 13,000-fold purification of the core-binding factors (CBFs), with an overall yield of approximately 19%. Analysis of purified protein fractions by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals more than 10 polypeptides. Each of the polypeptides was recovered from an SDS-polyacrylamide gel, and those in the molecular size range of 19 to 35 kDa were demonstrated to have core-binding activity. The purified CBFs were shown by DNase I footprint analyses to bind the core site in the Moloney virus enhancer specifically, and also to core motifs in the enhancers from a simian immunodeficiency virus, the immunoglobulin mu chain, and T-cell receptor gamma-chain genes.

Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers

1992 ◽  
Vol 12 (1) ◽  
pp. 89-102
Author(s):  
S W Wang ◽  
N A Speck
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Newborn Mice ◽  
The Core ◽  
Core Site ◽  
Disease Specificity ◽  
Murine Leukemia

The Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major genetic determinant of the thymic disease specificity of the Moloney virus genetically maps to two protein binding sites in the Moloney virus enhancer, the leukemia virus factor b site and the adjacent core site. Point mutations introduced into either of these sites significantly shifts the disease specificity of the Moloney virus from thymic leukemia to erythroleukemia (N. A. Speck, B. Renjifo, E. Golemis, T. Frederickson, J. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We have purified several polypeptides that bind to the core site in the Moloney virus enhancer. These proteins were purified from calf thymus nuclear extracts by selective pH denaturation, followed by chromatography on heparin-Sepharose, nonspecific double-stranded DNA-cellulose, and core oligonucleotide-coupled affinity columns. We have achieved greater than 13,000-fold purification of the core-binding factors (CBFs), with an overall yield of approximately 19%. Analysis of purified protein fractions by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals more than 10 polypeptides. Each of the polypeptides was recovered from an SDS-polyacrylamide gel, and those in the molecular size range of 19 to 35 kDa were demonstrated to have core-binding activity. The purified CBFs were shown by DNase I footprint analyses to bind the core site in the Moloney virus enhancer specifically, and also to core motifs in the enhancers from a simian immunodeficiency virus, the immunoglobulin mu chain, and T-cell receptor gamma-chain genes.

Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor

1993 ◽  
Vol 13 (6) ◽  
pp. 3324-3339
Author(s):  
S Wang ◽  
Q Wang ◽  
B E Crute ◽  
I N Melnikova ◽  
S R Keller ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Cell Receptor ◽  
Cdna Clones ◽  
The Core ◽  
Core Site ◽  
Disease Specificity ◽  
Murine Leukemia

Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.

scholarly journals Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor.

1993 ◽  
Vol 13 (6) ◽  
pp. 3324-3339 ◽  
Author(s):  
S Wang ◽  
Q Wang ◽  
B E Crute ◽  
I N Melnikova ◽  
S R Keller ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Cell Receptor ◽  
Cdna Clones ◽  
The Core ◽  
Core Site ◽  
Disease Specificity ◽  
Murine Leukemia

Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.

scholarly journals Core-Binding Factor Influences the Disease Specificity of Moloney Murine Leukemia Virus

1999 ◽  
Vol 73 (7) ◽  
pp. 5535-5547 ◽  
Author(s):  
Amy F. Lewis ◽  
Terryl Stacy ◽  
William R. Green ◽  
Lekidelu Taddesse-Heath ◽  
Janet W. Hartley ◽  
...  
Keyword(s):  
T Cell ◽  
Latent Period ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Cell Disease ◽  
The Core ◽  
Core Site ◽  
Disease Specificity ◽  
Murine Leukemia

ABSTRACT The core site in the Moloney murine leukemia virus (Moloney MLV) enhancer was previously shown to be an important determinant of the T-cell disease specificity of the virus. Mutation of the core site resulted in a significant shift in disease specificity of the Moloney virus from T-cell leukemia to erythroleukemia. We and others have since determined that a protein that binds the core site, one of the core-binding factors (CBF) is highly expressed in thymus and is essential for hematopoiesis. Here we test the hypothesis that CBF plays a critical role in mediating pathogenesis of Moloney MLV in vivo. We measured the affinity of CBF for most core sites found in MLV enhancers, introduced sites with different affinities for CBF into the Moloney MLV genome, and determined the effects of these sites on viral pathogenesis. We found a correlation between CBF affinity and the latent period of disease onset, in that Moloney MLVs with high-affinity CBF binding sites induced leukemia following a shorter latent period than viruses with lower-affinity sites. The T-cell disease specificity of Moloney MLV also appeared to correlate with the affinity of CBF for its binding site. The data support a role for CBF in determining the pathogenic properties of Moloney MLV.

scholarly journals Expression of murine leukemia virus envelope glycoprotein gp69/71 on mouse thymocytes. Evidence for two structural variants distinguished by presence vs. absence of GIX antigen.

1975 ◽  
Vol 142 (2) ◽  
pp. 518-523 ◽  
Author(s):  
J S Tung ◽  
E Fleissner ◽  
E S Vitetta ◽  
E A Boyse
Keyword(s):  
Murine Leukemia Virus ◽  
Inbred Mice ◽  
Leukemia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Antigen ◽  
Mouse Strains ◽  
Structural Variants ◽  
Virus Envelope ◽  
Murine Leukemia

Thymocytes of several mouse strains were tested for expression of the gp69/71 envelope component of murine leukemia virus by surface iodination, followed by immunoprecipitation and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Theses strains included two congenic lines differing from their partner stocks with respect to expression of GIX antigen demonstrable in the cytoxicity assay. We conclude that:(a) two structural variants of gp69/71 can be expressed on mouse thymocytes, (b) these are distinguishable by a small difference in mobility in SDS gels, (c) one carries GIX antigen and the other not, (d) they are coded, or their expression is regulated, by different chromosomal loci that are not closely linked, and (e) both can be expressed together on the thymocytes of inbred mice. In the intact thymocyte plasma membrane, the sites of group-specific antigen shared by the two gp69/71 variants, unlike the GIX type specificity carried by only one of them, are probably inaccessible to antibody.

scholarly journals Stabilization of TM Trimer Interactions during Activation of Moloney Murine Leukemia Virus Env

2007 ◽  
Vol 82 (5) ◽  
pp. 2358-2366 ◽  
Author(s):  
Mathilda Sjöberg ◽  
Birgitta Lindqvist ◽  
Henrik Garoff
Keyword(s):  
Cell Membrane ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Moloney Murine Leukemia Virus ◽  
Structural Conversion ◽  
Murine Leukemia

ABSTRACT The transmembrane subunit (TM) of the trimeric retrovirus Env complex is thought to direct virus-cell membrane fusion by refolding into a cell membrane-interacting, extended form that subsequently folds back on itself into a very stable trimer of hairpin-like TM polypeptides. However, so far there is only limited evidence for the formation of a stable TM trimer during Env activation. Here we have studied the oligomer composition and stability of an intermediate and the fully activated form of Moloney murine leukemia virus (Mo-MLV) Env. Activation of Mo-MLV Env is controlled by isomerization of its intersubunit disulfide. This results in surface subunit (SU) dissociation and TM refolding. If activation is done in the presence of an alkylator, this will modify the isomerization-active thiol in the SU of Env and arrest Env at an intermediate stage, the isomerization-arrested state (IAS) of its activation pathway. We generated IAS and fully activated Envs in vitro and in vivo and studied their states of oligomerization by two-dimensional blue native polyacrylamide gel electrophoresis (PAGE) and nonreducing sodium dodecyl sulfate (SDS)-PAGE. The IAS Env was composed of trimers of SU-TM complexes, whereas the activated Env consisted of SU monomers and TM trimers. When the oligomers were subjected to mild SDS treatment the TM trimer was found to be 3.5 times more resistant than the IAS oligomer. Thus, this demonstrates that a structural conversion of TM takes place during activation, which results in the formation of a stable TM trimer.

scholarly journals Mutation of the core or adjacent LVb elements of the Moloney murine leukemia virus enhancer alters disease specificity.

1990 ◽  
Vol 4 (2) ◽  
pp. 233-242 ◽  
Author(s):  
N A Speck ◽  
B Renjifo ◽  
E Golemis ◽  
T N Fredrickson ◽  
J W Hartley ◽  
...  
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
The Core ◽  
Disease Specificity ◽  
Murine Leukemia

scholarly journals Palmitoylation of the Intracytoplasmic R Peptide of the Transmembrane Envelope Protein in Moloney Murine Leukemia Virus

1999 ◽  
Vol 73 (11) ◽  
pp. 8975-8981 ◽  
Author(s):  
Katharina E. P. Olsen ◽  
Klaus B. Andersen
Keyword(s):  
Murine Leukemia Virus ◽  
Transmembrane Protein ◽  
Mechanistic Model ◽  
Leukemia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Sensitivity ◽  
Moloney Murine Leukemia Virus ◽  
Nih 3T3 ◽  
Murine Leukemia

ABSTRACT Previously it was reported that the 16-amino-acid (aa) C-terminal cytoplasmic tail of Moloney murine leukemia virus (MoMLV) transmembrane protein Pr15E is cleaved off during virus synthesis, yielding the mature, fusion active transmembrane protein p15E and the 16-aa peptide (R peptide or p2E). It remains to be elucidated how the R peptide impairs fusion activity of the uncleaved Pr15E. The R peptide from MoMLV was analyzed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostained with antiserum against the synthetic 16-aa R peptide. The R peptide resolved with an apparent molecular mass of 7 kDa and not the 4 kDa seen with the corresponding synthetic peptide. The 7-kDa R peptide was found to be membrane bound in MoMLV-infected NIH 3T3 cells, showing that cleavage of the 7-kDa R-peptide tail must occur before or during budding of progeny virions, in which only small amounts of the 7-kDa R peptide were found. The 7-kDa R peptide was palmitoylated since it could be labeled with [3H]palmitic acid, which explains its membrane association, slower migration on gels, and high sensitivity in immunoblotting. The present results are in contrast to previous findings showing equimolar amounts of R peptide and p15E in virions. The discrepancy, however, can be explained by the presence of nonpalmitoylated R peptide in virions, which were poorly detected by immunoblotting. A mechanistic model is proposed. The uncleaved R peptide can, due to its lipid modification, control the conformation of the ectodomain of the transmembrane protein and thereby govern membrane fusion.

Stability of AML1 (core) site enhancer mutations in T lymphomas induced by attenuated SL3-3 murine leukemia virus mutants.

1997 ◽  
Vol 71 (7) ◽  
pp. 5080-5087 ◽  
Author(s):  
H W Amtoft ◽  
A B Sørensen ◽  
C Bareil ◽  
J Schmidt ◽  
A Luz ◽  
...  
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Core Site ◽  
T Lymphomas ◽  
Murine Leukemia

scholarly journals Enhancer mutations of Akv murine leukemia virus inhibit the induction of mature B-cell lymphomas and shift disease specificity towards the more differentiated plasma cell stage

Virology ◽  
2007 ◽  
Vol 362 (1) ◽  
pp. 179-191 ◽  
Author(s):  
Karina Dalsgaard Sørensen ◽  
Sandra Kunder ◽  
Leticia Quintanilla-Martinez ◽  
Jonna Sørensen ◽  
Jörg Schmidt ◽  
...  
Keyword(s):  
B Cell ◽  
Plasma Cell ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
B Cell Lymphomas ◽  
Cell Stage ◽  
Disease Specificity ◽  
Murine Leukemia
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