scholarly journals The Moloney Murine Leukemia Virus Repressor Binding Site Represses Expression in Murine and Human Hematopoietic Stem Cells

2003 ◽  
Vol 77 (17) ◽  
pp. 9439-9450 ◽  
Author(s):  
Dennis L. Haas ◽  
Carolyn Lutzko ◽  
Aaron C. Logan ◽  
Gerald J. Cho ◽  
Dianne Skelton ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Binding Site ◽  
Lentiviral Vector ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Mouse Bone Marrow ◽  
Primary Mouse ◽  
Genome Context ◽  
Repressor Binding Site ◽  
Murine Leukemia

ABSTRACT The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34+ CD38− hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.

Characterization of the Moloney Murine Leukemia Virus Stem Cell-Specific Repressor Binding Site

Virology ◽  
1993 ◽  
Vol 193 (2) ◽  
pp. 690-699 ◽  
Author(s):  
Geraldine Kempler ◽  
Breton Freitag ◽  
Brent Berwin ◽  
Oliver Nanassy ◽  
Eric Barklis
Keyword(s):  
Stem Cell ◽  
Binding Site ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Repressor Binding Site ◽  
Murine Leukemia

Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to Moloney murine leukemia virus–based vectors

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3385-3391 ◽  
Author(s):  
Stephane Barrette ◽  
Janet L. Douglas ◽  
Nancy E. Seidel ◽  
David M. Bodine
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Lines ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Lentivirus Vector ◽  
Hematopoietic Stem ◽  
Lentivirus Vectors ◽  
Murine Leukemia

Abstract The low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34+ cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin− bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.

Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to Moloney murine leukemia virus–based vectors

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3385-3391 ◽  
Author(s):  
Stephane Barrette ◽  
Janet L. Douglas ◽  
Nancy E. Seidel ◽  
David M. Bodine
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Lines ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Lentivirus Vector ◽  
Hematopoietic Stem ◽  
Lentivirus Vectors ◽  
Murine Leukemia

The low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34+ cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin− bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.

Hydrophobic binding site(s) on Moloney-murine leukemia virus P30

Virology ◽  
1978 ◽  
Vol 85 (1) ◽  
pp. 211-221 ◽  
Author(s):  
Stephen K. Swanson ◽  
Eugene Sulkowski ◽  
Kenneth F. Manly
Keyword(s):  
Binding Site ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Hydrophobic Binding ◽  
Murine Leukemia

scholarly journals Interaction of Moloney Murine Leukemia Virus Capsid with Ubc9 and PIASy Mediates SUMO-1 Addition Required Early in Infection

2006 ◽  
Vol 80 (1) ◽  
pp. 342-352 ◽  
Author(s):  
Andrew Yueh ◽  
Juliana Leung ◽  
Subarna Bhattacharyya ◽  
Lucy A. Perrone ◽  
Kenia de los Santos ◽  
...  
Keyword(s):  
Binding Site ◽  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Viral Gene ◽  
Alanine Scanning Mutagenesis ◽  
Viral Dna ◽  
Murine Leukemia

ABSTRACT Yeast two-hybrid screens led to the identification of Ubc9 and PIASy, the E2 and E3 small ubiquitin-like modifier (SUMO)-conjugating enzymes, as proteins interacting with the capsid (CA) protein of the Moloney murine leukemia virus. The binding site in CA for Ubc9 was mapped by deletion and alanine-scanning mutagenesis to a consensus motif for SUMOylation at residues 202 to 220, and the binding site for PIASy was mapped to residues 114 to 176, directly centered on the major homology region. Expression of CA and a tagged SUMO-1 protein resulted in covalent transfer of SUMO-1 to CA in vivo. Mutations of lysine residues to arginines near the Ubc9 binding site and mutations at the PIASy binding site reduced or eliminated CA SUMOylation. Introduction of these mutations into the complete viral genome blocked virus replication. The mutants exhibited no defects in the late stages of viral gene expression or virion assembly. Upon infection, the mutant viruses were able to carry out reverse transcription to synthesize normal levels of linear viral DNA but were unable to produce the circular viral DNAs or integrated provirus normally found in the nucleus. The results suggest that the SUMOylation of CA mediated by an interaction with Ubc9 and PIASy is required for early events of infection, after reverse transcription and before nuclear entry and viral DNA integration.

scholarly journals Identification of Directly Infected Cells in the Bone Marrow of Neonatal Moloney Murine Leukemia Virus-Infected Mice by Use of a Moloney Murine Leukemia Virus-Based Vector

1999 ◽  
Vol 73 (2) ◽  
pp. 1617-1623 ◽  
Author(s):  
Michael A. Okimoto ◽  
Hung Fan
Keyword(s):  
Bone Marrow ◽  
Murine Leukemia Virus ◽  
Bone Marrow Cells ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Small Cells ◽  
Nonspecific Esterase ◽  
Hematopoietic Progenitors ◽  
Infected Cells ◽  
Murine Leukemia

ABSTRACT Early bone marrow infection of Moloney murine leukemia virus (M-MuLV)-infected mice was studied. Previous experiments indicated that early bone marrow infection is essential for the efficient development of T lymphoma. In order to identify the cellular pathway of infection in the bone marrow, infection of mice with a helper-free replication-defective M-MuLV-based retroviral vector was carried out. Such a vector will undergo only one round of infection, without spreading to other cells; thus, cells infected by the initially injected virus (directly infected cells) can be identified. For these experiments, the BAG vector that expresses bacterial β-galactosidase was employed. Neonatal NIH/Swiss mice were inoculated intraperitoneally with ca. 106 infectious units of a BAG vector pseudotyped with M-MuLV proteins, and bone marrow cells were recovered 2 to 12 days postinfection. Single-cell suspensions were tested for infection by staining with X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) or by immunofluorescence with an anti-β-galactosidase antibody. Two sizes of infected cells were evident: large multinucleated cells and small nondescript (presumptively hematopoietic) cells. Secondary stains for lineage-specific markers indicated that the large cells were osteoclasts. Some of the small cells expressed nonspecific esterase, which placed them in the myeloid lineage, but they lacked markers for hematopoietic progenitors (mac-1, gr-1, sca-1, and CD34). These results provide evidence for primary M-MuLV infection of osteoclasts or osteoclast progenitors in the bone marrow, and they suggest that known hematopoietic progenitors are not primary targets for infection. However, the subsequent spread of infection to hematopoietic progenitors was indicated, since bone marrow from mice infected in parallel with replication-competent wild-type M-MuLV showed detectable infection in small cells positive for mac-1 or CD34, as well as in osteoclasts.

scholarly journals Diminished Potential for B-Lymphoid Differentiation after Murine Leukemia Virus Infection In Vivo and in EML Hematopoietic Progenitor Cells

2007 ◽  
Vol 81 (13) ◽  
pp. 7274-7279 ◽  
Author(s):  
Samantha L. Finstad ◽  
Naomi Rosenberg ◽  
Laura S. Levy
Keyword(s):  
Bone Marrow ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Selective Advantage ◽  
Moloney Murine Leukemia Virus ◽  
Vitro Model ◽  
B Lymphoid ◽  
Murine Leukemia

ABSTRACT Infection with a recombinant murine-feline gammaretrovirus, MoFe2, or with the parent virus, Moloney murine leukemia virus, caused significant reduction in B-lymphoid differentiation of bone marrow at 2 to 8 weeks postinfection. The suppression was selective, in that myeloid potential was significantly increased by infection. Analysis of cell surface markers and immunoglobulin H gene rearrangements in an in vitro model demonstrated normal B-lymphoid differentiation after infection but significantly reduced viability of differentiating cells. This reduction in viability may confer a selective advantage on undifferentiated lymphoid progenitors in the bone marrow of gammaretrovirus-infected animals and thereby contribute to the establishment of a premalignant state.

Replication of a Moloney Murine Leukemia Virus Mutant Lacking the ELP Binding Site

Virology ◽  
1994 ◽  
Vol 203 (1) ◽  
pp. 162-165
Author(s):  
Jose Coulombe ◽  
Manon Dube ◽  
Douglas A. Gray
Keyword(s):  
Binding Site ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Virus Mutant ◽  
Murine Leukemia
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