Murine helix-loop-helix transcriptional activator proteins binding to the E-box motif of the Akv murine leukemia virus enhancer identified by cDNA cloning

1992 ◽  
Vol 12 (8) ◽  
pp. 3449-3459
Author(s):  
A L Nielsen ◽  
N Pallisgaard ◽  
F S Pedersen ◽  
P Jørgensen
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Transcriptional Activator ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
E Box ◽  
Nih 3T3 ◽  
Murine Leukemia

The enhancer region of Akv murine leukemia virus contains the sequence motif ACAGATGG. This sequence is homologous to the E-box motif originally defined as a regulatory element in the enhancers of immunoglobulin mu and kappa genes. We have used double-stranded oligonucleotide probes, corresponding to the E box of the murine leukemia virus Akv, to screen a randomly primed lambda gt11 cDNA expression library made from mouse NIH 3T3 fibroblast RNA. We have identified seven lambda clones expressing DNA-binding proteins representing two different genes termed ALF1 and ALF2. The results of sequencing ALF2 cDNA suggests that we have recovered the gene for the basic-helix-loop-helix transcription factor A1, the murine analog of the human transcription factor E47. The cDNA sequence of ALF1 codes for a new member of the basic-helix-loop-helix protein family. Two splice variants of ALF1 cDNA have been found, differing by a 72-bp insertion, coding for putative proteins of 682 and 706 amino acids. The two ALF1 mRNAs are expressed at various levels in mouse tissues. In vitro DNA binding assays, using prokaryotically expressed ALF1 proteins, demonstrated specific binding of the ALF1 proteins to the Akv murine leukemia virus E-box motif ACAGATGG. Expression in NIH 3T3 fibroblasts of GAL4-ALF1 chimeric protein stimulated expression from a minimal promoter linked to a GAL4 binding site, indicating the existence of a transcriptional activator domain in ALF1.

scholarly journals Murine helix-loop-helix transcriptional activator proteins binding to the E-box motif of the Akv murine leukemia virus enhancer identified by cDNA cloning.

1992 ◽  
Vol 12 (8) ◽  
pp. 3449-3459 ◽  
Author(s):  
A L Nielsen ◽  
N Pallisgaard ◽  
F S Pedersen ◽  
P Jørgensen
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Transcriptional Activator ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
E Box ◽  
Nih 3T3 ◽  
Murine Leukemia

The enhancer region of Akv murine leukemia virus contains the sequence motif ACAGATGG. This sequence is homologous to the E-box motif originally defined as a regulatory element in the enhancers of immunoglobulin mu and kappa genes. We have used double-stranded oligonucleotide probes, corresponding to the E box of the murine leukemia virus Akv, to screen a randomly primed lambda gt11 cDNA expression library made from mouse NIH 3T3 fibroblast RNA. We have identified seven lambda clones expressing DNA-binding proteins representing two different genes termed ALF1 and ALF2. The results of sequencing ALF2 cDNA suggests that we have recovered the gene for the basic-helix-loop-helix transcription factor A1, the murine analog of the human transcription factor E47. The cDNA sequence of ALF1 codes for a new member of the basic-helix-loop-helix protein family. Two splice variants of ALF1 cDNA have been found, differing by a 72-bp insertion, coding for putative proteins of 682 and 706 amino acids. The two ALF1 mRNAs are expressed at various levels in mouse tissues. In vitro DNA binding assays, using prokaryotically expressed ALF1 proteins, demonstrated specific binding of the ALF1 proteins to the Akv murine leukemia virus E-box motif ACAGATGG. Expression in NIH 3T3 fibroblasts of GAL4-ALF1 chimeric protein stimulated expression from a minimal promoter linked to a GAL4 binding site, indicating the existence of a transcriptional activator domain in ALF1.

scholarly journals Efg1, a Morphogenetic Regulator in Candida albicans, Is a Sequence-Specific DNA Binding Protein

2001 ◽  
Vol 183 (13) ◽  
pp. 4090-4093 ◽  
Author(s):  
Ping Leng ◽  
Philip R. Lee ◽  
Hong Wu ◽  
Alistair J. P. Brown
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Human Pathogen ◽  
Hyphal Development ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
E Box

ABSTRACT Efg1 is essential for hyphal development in the human pathogenCandida albicans under most conditions. Efg1 is related to basic helix-loop-helix regulators, and therefore most workers presume that Efg1 is a transcription factor. Here we confirm that Efg1 is a DNA binding protein that can interact specifically with the E box.

Identification of transactivation and repression functions of the dioxin receptor and its basic helix-loop-helix/PAS partner factor Arnt: inducible versus constitutive modes of regulation

1994 ◽  
Vol 14 (12) ◽  
pp. 8343-8355
Author(s):  
M L Whitelaw ◽  
J A Gustafsson ◽  
L Poellinger
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Ligand Binding ◽  
Transactivation Domain ◽  
Response Element ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
C Terminus ◽  
Heterodimeric Complex ◽  
Dioxin Receptor

Gene regulation by dioxins is mediated via the dioxin receptor, a ligand-dependent basic helix-loop-helix (bHLH)/PAS transcription factor. The latent dioxin receptor responds to dioxin signalling by forming an activated heterodimeric complex with a specific bHLH partner, Arnt, an essential process for target DNA recognition. We have analyzed the transactivating potential within this heterodimeric complex by dissecting it into individual subunits, replacing the dimerization and DNA-binding bHLH motifs with heterologous zinc finger DNA-binding domains. The uncoupled Arnt chimera, maintaining 84% of Arnt residues, forms a potent and constitutive transcription factor. Chimeric proteins show that the dioxin receptor also harbors a strong transactivation domain in the C terminus, although this activity was silenced by inclusion of 82 amino acids from the central ligand-binding portion of the dioxin receptor. This central repression region conferred binding of the molecular chaperone hsp90 upon otherwise constitutive chimeras in vitro, indicating that hsp90 has the ability to mediate a cis-repressive function on distant transactivation domains. Importantly, when the ligand-binding domain of the dioxin receptor remained intact, the ability of this hsp90-binding activity to confer repression became conditional rather than irreversible. Our data are consistent with a model in which crucial activities of the dioxin receptor, such as dimerization with Arnt and transactivation, are conditionally repressed by the central ligand- and-hsp90-binding region of the receptor. In contrast, the Arnt protein appears to be free from any repressive activity. Moreover, within the context of the dioxin response element (xenobiotic response element), the C terminus of Arnt conferred a potent, dominating transactivation function onto the native bHLH heterodimeric complex. Finally, the relative transactivation potencies of the individual dioxin receptor and Arnt chimeras varied with cell type and promoter architecture, indicating that the mechanisms for transcriptional activation may differ between these two subunits and that in the native complex the transactivation pathway may be dependent upon cell-specific and promoter contexts.

scholarly journals Caveola-Dependent Endocytic Entry of Amphotropic Murine Leukemia Virus

2005 ◽  
Vol 79 (16) ◽  
pp. 10776-10787 ◽  
Author(s):  
Christiane Beer ◽  
Ditte S. Andersen ◽  
Aleksandra Rojek ◽  
Lene Pedersen
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
3T3 Cells ◽  
Coated Pits ◽  
Caveolin 1 ◽  
Amphotropic Murine Leukemia Virus ◽  
Triton X ◽  
Nih 3T3 ◽  
Murine Leukemia ◽  
Nih 3T3 Cells

ABSTRACT Early results suggested that the amphotropic murine leukemia virus (A-MLV) does not enter cells via endocytosis through clathrin-coated pits and this gammaretrovirus has therefore been anticipated to fuse directly with the plasma membrane. However, here we present data implicating a caveola-mediated endocytic entry route for A-MLV via its receptor Pit2. Caveolae belong to the cholesterol-rich microdomains characterized by resistance to nonionic detergents such as Triton X-100. Extraction of murine fibroblastic NIH 3T3 cells in cold Triton X-100 showed the presence of the A-MLV receptor Pit2 in detergent-insoluble microdomains. Using coimmunoprecipitation of cell extracts, we were able to demonstrate direct association of Pit2 with caveolin-1, the structural protein of caveolae. Other investigations revealed that A-MLV infection in contrast to vesicular stomatitis virus infection is a slow process (t ≈5 h), which is dependent on plasma membrane cholesterol but independent of NH4Cl treatment of cells; NH4Cl impairs entry via clathrin-coated pits. Furthermore, expression of dominant-negative caveolin-1 decreased the susceptibility to infection via Pit2 by approximately 70%. These results show that A-MLV can enter cells via a caveola-dependent entry route. Moreover, increase in A-MLV infection by treatment with okadaic acid as well as entry of fusion-defective fluorescent A-MLV virions in NIH 3T3 cells further confirmed our findings and show that A-MLV can enter mouse fibroblasts via an endocytic entry route involving caveolae. Finally, we also found colocalization of fusion-defective fluorescent A-MLV virions with caveolin-1 in NIH 3T3 cells. This is the first time substantial evidence has been presented implicating the existence of a caveola-dependent endocytic entry pathway for a retrovirus.

scholarly journals Macropinocytosis Is the Entry Mechanism of Amphotropic Murine Leukemia Virus

2014 ◽  
Vol 89 (3) ◽  
pp. 1851-1866 ◽  
Author(s):  
Izabela Rasmussen ◽  
Frederik Vilhardt
Keyword(s):  
Cell Surface ◽  
Actin Cytoskeleton ◽  
Murine Leukemia Virus ◽  
Mammalian Cells ◽  
Leukemia Virus ◽  
Amphotropic Murine Leukemia Virus ◽  
Conditional Expression ◽  
Nih 3T3 ◽  
Entry Mechanism ◽  
Murine Leukemia

ABSTRACTThe entry mechanism of murine amphotropic retrovirus (A-MLV) has not been unambiguously determined. We show here that A-MLV is internalized not by caveolae or other pinocytic mechanisms but by macropinocytosis. Thus, A-MLV infection of mouse embryonic fibroblasts deficient for caveolin or dynamin, and NIH 3T3 cells knocked down for caveolin expression, was unaffected. Conversely, A-MLV infection of NIH 3T3 and HeLa cells was sensitive to amiloride analogues and actin-depolymerizing drugs that interfere with macropinocytosis. Further manipulation of the actin cytoskeleton through conditional expression of dominant positive or negative mutants of Rac1, PAK1, and RhoG, to increase or decrease macropinocytosis, similarly correlated with an augmented or inhibited infection with A-MLV, respectively. The same experimental perturbations affected the infection of viruses that use clathrin-coated-pit endocytosis or other pathways for entry only mildly or not at all. These data agree with immunofluorescence studies and cryo-immunogold labeling for electron microscopy, which demonstrate the presence of A-MLV in protrusion-rich areas of the cell surface and in cortical fluid phase (dextran)-filled macropinosomes, which also account for up to a half of the cellular uptake of the cell surface-binding lectin concanavalin A. We conclude that A-MLV use macropinocytosis as the predominant entry portal into cells.IMPORTANCEBinding and entry of virus particles into mammalian cells are the first steps of infection. Understanding how pathogens and toxins exploit or divert endocytosis pathways has advanced our understanding of membrane trafficking pathways, which benefits development of new therapeutic schemes and methods of drug delivery. We show here that amphotropic murine leukemia virus (A-MLV) pseudotyped with the amphotropic envelope protein (which expands the host range to many mammalian cells) gains entry into host cells by macropinocytosis. Macropinosomes form as large, fluid-filled vacuoles (up to 10 μm) following the collapse of cell surface protrusions and membrane scission. We used drugs or the introduction of mutant proteins that affect the actin cytoskeleton and cell surface dynamics to show that macropinocytosis and A-MLV infection are correlated, and we provide both light- and electron-microscopic evidence to show the localization of A-MLV in macropinosomes. Finally, we specifically exclude some other potential entry portals, including caveolae, previously suggested to internalize A-MLV.

scholarly journals Increased Induction of Osteopetrosis, but Unaltered Lymphomagenicity, by Murine Leukemia Virus SL3-3 after Mutation of a Nuclear Factor 1 Site in the Enhancer

1999 ◽  
Vol 73 (12) ◽  
pp. 10406-10415 ◽  
Author(s):  
Steen Ethelberg ◽  
Barbara D. Tzschaschel ◽  
Arne Luz ◽  
Salvador J. Diaz-Cano ◽  
Finn Skou Pedersen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Transcription Factor Binding Sites ◽  
Nuclear Factor ◽  
Wild Type ◽  
Factor Binding ◽  
Nuclear Factor 1 ◽  
Murine Leukemia

ABSTRACT SL3-3 is a murine leukemia virus which is only weakly bone pathogenic but highly T-cell lymphomagenic. A major pathogenic determinant is the transcriptional enhancer comprising several transcription factor binding sites, among which are three identical sites for nuclear factor 1 (NF1). We have investigated the pathogenic properties of NF1 site enhancer mutants of SL3-3. Two different mutants carrying a 3-bp mutation either in all three NF1 sites or in the central site alone were constructed and assayed in inbred NMRI mice. The wild type and both mutants induced lymphomas in all mice, with a mean latency period of 9 weeks. However, there was a considerable difference in osteopetrosis induction. Wild-type SL3-3 induced osteopetrosis in 11% of the mice (2 of 19), and the triple NF1 site mutant induced osteopetrosis in none of the mice (0 of 19), whereas the single NF1 site mutant induced osteopetrosis in 56% (10 of 18) of the mice, as determined by X-ray analysis. A detailed histological examination of the femurs of the mice was carried out and found to support this diagnosis. Thus, the NF1 sites of SL3-3 are major determinants of osteopetrosis induction, without determining lymphomagenesis. This conclusion was further supported by evaluation of the bone pathogenicity of other SL3-3 enhancer variants, the lymphomagenicity of which had been examined previously. This evaluation furthermore strongly indicated that the core sites, a second group of transcription factor binding sites in the viral enhancer, are necessary for the osteopetrosis induction potential of SL3-3.

Immunologic Localization of Murine Leukemia Virus Antigens in Thin Sections

1981 ◽  
Vol 39 ◽  
pp. 402-403
Author(s):  
Ray A. Weigand ◽  
Carol I. Paquette ◽  
Clifford Longley ◽  
Philip Furmanski
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Intracellular Localization ◽  
Protein A ◽  
Thin Sections ◽  
Virus Particles ◽  
Tumor Virus ◽  
Labelling Procedure ◽  
Nih 3T3 ◽  
Murine Leukemia

In order to examine the temporal sequence of intracellular events which culminates in the release of infectious virus particles from the cell surface, we have developed immunoferritin techniques to localize viral antigens in thin sections. We previously described the intracellular localization of murine mammary tumor virus p28 protein in thin sections using an unlabelled antibody procedure with ferritin-antiferritin. We now describe the labelling of murine leukemia virus (MuLV) in thin sections using anti-MuLV and Protein A-ferritin.Cultures of F-MuLV in NIH/3T3 cells were grown to 90% confluence, fixed, dehydrated, and embedded in Epon as before. Thin sections were picked up on nickel grids, incubated in 20% H2O2 for 20 min, rinsed in PBS, and subjected to the labelling procedure. The labelling protocol included: preincubation in 5% BSA in PBS, rinse in 1% BSA in PBS, 3 hour incubation in rabbit anti-MuLV, rinse in 1% BSA in PBS, incubation in apoferritin, incubation in Protein A-ferritin (Zymed Labs., Burlingame, CA), rinses in 1% BSA in PBS and PBS, and incubation in 2% glutaraldehyde in PBS.

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