Related Experiment Videos
Characterization of cell lines that inducibly express the adeno-associated virus Rep proteins
1Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo 43699-0008.
Abstract:
The replication (rep) gene of adeno-associated virus (AAV) is involved in AAV DNA replication, gene regulation, and inhibition of cellular transformation induced by various oncogenes. To study the rep gene's antiproliferative effects, we have developed cell lines which express the replication proteins under the control of an inducible mouse metallothionein transcription promoter. The Rep78 protein produced in these cell lines binds to the AAV terminal repeat sequences in vitro and supports AAV DNA replication and trans activation of the AAV p40 transcription promoter in vivo. These cell lines are capable of assembling infectious viruses containing a mutant rep gene or a vector bearing a heterologous gene. Growth rate and colony formation efficiency assays indicated that rep gene expression substantially altered cellular proliferation. Long-term induction of the cell lines followed by removal of the inducing agent suggested that constitutive expression of the Rep proteins does not necessarily result in cell death and that the cells can recover from the cytostatic effects. Flow cytometry analysis indicated that the presence of the Rep proteins increased the population of cells in the S phase of the cell cycle. Thus the rep gene's antiproliferative effects may be realized by interference with cellular DNA replication.
Insights
The adeno-associated virus (AAV) replication (rep) gene inhibits cell proliferation by interfering with cellular DNA replication. Inducible cell lines show rep gene expression alters cell growth and cycle progression.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- The adeno-associated virus (AAV) replication (rep) gene plays a crucial role in viral DNA replication, gene regulation, and suppressing oncogene-induced cellular transformation.
- Understanding the antiproliferative mechanisms of the AAV rep gene is essential for its therapeutic applications.
Purpose of the Study:
- To investigate the antiproliferative effects of the AAV rep gene.
- To develop and characterize cell lines expressing AAV rep proteins under inducible control.
Main Methods:
- Development of inducible cell lines expressing AAV replication proteins via a mouse metallothionein promoter.
- In vitro binding assays of Rep78 to AAV terminal repeats.
- In vivo studies of AAV DNA replication and p40 promoter transactivation.
- Cell proliferation assays (growth rate, colony formation efficiency).
- Flow cytometry to analyze cell cycle distribution.
Main Results:
- Rep78 protein binds AAV terminal repeats and supports viral DNA replication and p40 promoter activity.
- Rep gene expression significantly altered cellular proliferation and colony formation.
- Cells recovered from cytostatic effects after removal of the inducing agent, indicating Rep proteins do not necessarily cause cell death.
- Flow cytometry revealed an accumulation of cells in the S phase of the cell cycle upon Rep protein expression.
Conclusions:
- The AAV rep gene exhibits significant antiproliferative effects on host cells.
- These effects are likely mediated by interference with cellular DNA replication processes.
- The rep gene's impact on cell cycle progression, particularly S phase, is a key mechanism underlying its antiproliferative activity.