A stem cell-specific silencer in the primer-binding site of a retrovirus

Richard Petersen, Geraldine Kempler, Eric Barklis

Research output: Contribution to journalArticle

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Abstract

Retrovirus expression in embryonal carcinoma (EC) cells is blocked at a postintegration stage of the viral life cycle, in part because of the inadequate function of the viral long terminal repeat promoter in this cell type. However, selection for retrovirus expression in EC cells has identified mutations in Moloney murine leukemia virus (M-MuLV) located in the tRNA primer-binding site (PBS) region which relieve the EC cell-specific repression. We have found that exchanging the M-MuLV proline PBS for a glutamine one in a recombinant virus permits expression in EC cells. By using the recombinant virus as a backbone, the EC cell-specific repressor-binding site (RBS) element has been mapped to M-MuLV nucleotides 147 to 174. The RBS does not require precise positioning downstream of the M-MuLV promoter and can function in either orientation and in an intron, indicating that the regulatory effect is probably at the DNA, rather than RNA, level. We also show that the RBS element can repress heterologous promoters from an upstream position. Our results indicate that the RBS acts as a silencer that its inhibitory effect is mediated by a trans-acting factor, and that the mechanism of action is probably at the level of transcription. Through in vitro binding assays we have identified a binding factor which specifically recognizes the wild-type RBS sequence (binding factor A). The binding characteristics of factor A suggest that it is a stem cell repressor which acts at the M-MuLV RBS. Our DNA-binding assays also have identified a unique binding factor (binding factor Hp) which specifically recognizes a hemimethylated form of the wild-type RBS. This factor may play a role in methylation mediated control of retrovirus expression in EC cells.

Original languageEnglish (US)
Pages (from-to)1214-1221
Number of pages8
JournalMolecular and Cellular Biology
Volume11
Issue number3
StatePublished - Mar 1991

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Retroviridae
Embryonal Carcinoma Stem Cells
Stem Cells
Moloney murine leukemia virus
Binding Sites
Viruses
Virus Attachment
Trans-Activators
Terminal Repeat Sequences
DNA
Transfer RNA
Life Cycle Stages
Glutamine
Proline
Introns
Methylation
Nucleotides
RNA
Mutation

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

A stem cell-specific silencer in the primer-binding site of a retrovirus. / Petersen, Richard; Kempler, Geraldine; Barklis, Eric.

In: Molecular and Cellular Biology, Vol. 11, No. 3, 03.1991, p. 1214-1221.

Research output: Contribution to journalArticle

Petersen, Richard ; Kempler, Geraldine ; Barklis, Eric. / A stem cell-specific silencer in the primer-binding site of a retrovirus. In: Molecular and Cellular Biology. 1991 ; Vol. 11, No. 3. pp. 1214-1221.
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