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

Geraldine Kempler, Breton Freitag, Brent Berwin, Oliver Nanassy, Eric Barklis

Research output: Contribution to journalArticle

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Abstract

The Moloney murine leukemia virus (M-MuLV) repressor binding site (RBS) mediates cell-type-specific repression in embryonal carcinoma (EC) cells of expression from several different promoters, including the M-MuLV long terminal repeat promoter. Silencing has been shown to depend on an element normally located in the proviral 5′ noncoding region and occurs at the DNA level in the absence of retroviral proteins. Using fragments of the RBS region, we now show that the minimal size of the silencer corresponds to M-MuLV nt 147-163 and overlaps with the retroviral primer binding site region by 17 of its 18 bp. A panel of point mutations within the RBS has been examined to yield a consensus RBS sequence which is consistent with the notion that a previously identified nuclear factor (binding factor A) mediates RBS repression. Viral vectors using neomycin, β-galactosidase, and luciferase reporters have been employed to show that RBS-mediated repression occurs in EC and embryonal stem, but not in other tested cell types. Repression was observed to occur within 48 hr of infection, prior to when global methylation of proviruses has been reported to occur. Repression also occurred after azacytidine treatment of EC cells, supporting the notion that the RBS functions independently of provirus methylation. However, levels of provirus methylation in selected cells were increased in the presence of a wild-type RBS, and methylation correlated with a secondary stage of virus repression. Thus, the M-MuLV RBS acts directly to control expression in EC cells and also appears to trigger a secondary level of repression which is coincident with provirus methylation.

Original languageEnglish (US)
Pages (from-to)690-699
Number of pages10
JournalVirology
Volume193
Issue number2
DOIs
StatePublished - Apr 1993

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Moloney murine leukemia virus
Stem Cells
Binding Sites
Proviruses
Methylation
Embryonal Carcinoma Stem Cells
Virus Attachment
Galactosidases
Embryonal Carcinoma
Azacitidine
Neomycin
Terminal Repeat Sequences
Luciferases
Point Mutation
Viruses

ASJC Scopus subject areas

  • Infectious Diseases
  • Virology

Cite this

Characterization of the Moloney Murine Leukemia Virus Stem Cell-Specific Repressor Binding Site. / Kempler, Geraldine; Freitag, Breton; Berwin, Brent; Nanassy, Oliver; Barklis, Eric.

In: Virology, Vol. 193, No. 2, 04.1993, p. 690-699.

Research output: Contribution to journalArticle

Kempler, Geraldine ; Freitag, Breton ; Berwin, Brent ; Nanassy, Oliver ; Barklis, Eric. / Characterization of the Moloney Murine Leukemia Virus Stem Cell-Specific Repressor Binding Site. In: Virology. 1993 ; Vol. 193, No. 2. pp. 690-699.
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