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A stem cell-specific silencer in the primer-binding site of a retrovirus
R Petersen1, G Kempler, E Barklis
1Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
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.
Insights
Researchers identified a silencer element (RBS) in Moloney murine leukemia virus (M-MuLV) that blocks retrovirus expression in embryonal carcinoma (EC) cells. This RBS is recognized by a stem cell repressor, offering insights into viral gene regulation.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Retrovirus expression is often repressed in embryonal carcinoma (EC) cells due to inefficient viral promoter function.
- Specific mutations in Moloney murine leukemia virus (M-MuLV) within the tRNA primer-binding site (PBS) can overcome this EC cell-specific repression.
Purpose of the Study:
- To map the repressor-binding site (RBS) responsible for EC cell-specific retrovirus silencing.
- To elucidate the mechanism of action and identify factors involved in this repression.
Main Methods:
- Construction of recombinant retroviruses with altered PBS regions.
- Mapping of the RBS element using a recombinant virus backbone.
- Analysis of RBS function in different orientations, positions, and in introns.
- In vitro DNA-binding assays to identify specific binding factors.
Main Results:
- A glutamine PBS, replacing the proline PBS of M-MuLV, enabled retrovirus expression in EC cells.
- The EC cell-specific repressor-binding site (RBS) was mapped to M-MuLV nucleotides 147-174.
- The RBS functions as a DNA-level silencer, independent of precise positioning or orientation, and can repress heterologous promoters.
- A stem cell repressor (binding factor A) and a hemimethylated RBS-specific factor (binding factor Hp) were identified.
Conclusions:
- The identified RBS acts as a transcriptional silencer, likely mediated by trans-acting factors like binding factor A.
- The findings suggest a mechanism for stem cell-specific retroviral silencing involving DNA-level regulation and potentially methylation.
- This research provides a molecular basis for understanding retroviral gene regulation in EC cells.