Related Experiment Videos
Repression of human cytomegalovirus gene expression associated with a novel immediate early regulatory region binding
S L Shelbourn1, S K Kothari, J G Sissons
1Department of Medicine, University of Cambridge, Addenbrooke's Hospital, UK.
Abstract:
A major determinant controlling reactivation of persistent viruses is likely to be the level of cellular factors which regulate virus transcription. Human cytomegalovirus (the largest human herpesvirus) does not replicate in human teratocarcinoma (T2) cells due to a block in transcription of immediate early (IE) gene expression, but these cells become permissive upon retinoic acid induced differentiation. We have analysed changes in DNA binding factors to the major IE promoter/regulatory region of HCMV that occur during differentiation of T2 cells to a permissive phenotype. We show that undifferentiated T2 cells contain a specific nuclear factor that binds to a far upstream region of the major IE regulatory region. Differentiation of T2 cells is associated with a major decrease in this factor and deletion of its specific binding site from IE expression vectors also results in increased levels of expression in undifferentiated cells. Consequently, this novel factor present in undifferentiated cells is a candidate for a differentiation specific negative regulator of HCMV IE gene expression which binds to an element upstream of the major IE enhancer.
Insights
Undifferentiated cells contain a factor that blocks human cytomegalovirus (HCMV) immediate-early (IE) gene expression. Differentiation reduces this factor, allowing HCMV replication.
Area of Science:
- Virology
- Molecular Biology
- Cellular Biology
Background:
- Viral reactivation is often controlled by cellular transcription factors.
- Human cytomegalovirus (HCMV) replication is blocked in teratocarcinoma (T2) cells due to impaired immediate-early (IE) gene transcription.
- T2 cells become permissive to HCMV upon retinoic acid-induced differentiation.
Purpose of the Study:
- To investigate changes in DNA-binding factors affecting the HCMV major IE promoter during T2 cell differentiation.
- To identify cellular factors regulating HCMV IE gene expression in T2 cells.
Main Methods:
- Analysis of DNA-binding factors to the HCMV major IE promoter/regulatory region.
- Electrophoretic mobility shift assays (EMSAs) to detect specific nuclear factors.
- Use of IE expression vectors with and without specific binding sites.
Main Results:
- Undifferentiated T2 cells possess a specific nuclear factor binding to a far upstream region of the HCMV major IE regulatory region.
- T2 cell differentiation leads to a significant decrease in this nuclear factor.
- Deletion of the binding site for this factor enhances IE expression in undifferentiated T2 cells.
Conclusions:
- A novel, differentiation-specific nuclear factor acts as a negative regulator of HCMV IE gene expression in undifferentiated T2 cells.
- This factor binds to an element upstream of the major IE enhancer, inhibiting transcription.
- Understanding these regulatory mechanisms is crucial for controlling HCMV reactivation.