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Generation of mammalian cells expressing stably measles virus proteins via bicistronic RNA
1Department of Microbiology, University of Washington School of Medicine, Seattle 98195.
Abstract:
The proteins of measles virus are believed to be cytotoxic, and have never been expressed stably from the cloned genes in cultured cells. We found that measles viral proteins can be expressed via a bicistronic RNA. The dominantly selectable DHFR* protein-coding region encoding a mutant dihydrofolate reductase was inserted into the 3'-untranslated regions of the measles viral genes encoding nucleoprotein (N), matrix (M) protein, and hemagglutinin (H). The tandemly arranged cistrons were placed under control by the inducible promoter of human metallothionein IIA gene, or the noninducible early promoter of simian virus 40. Upon transfecting into mammalian cells, these gene constructs synthesized bicistronic RNAs. The downstream DHFR* gene conferred resistance to methotrexate (MTX). Cells that survived MTX selection expressed stably the N, M, or H protein of measles virus. Expression of N protein was further inducible by cadmium chloride treatment. This system will be useful for studying the protein functions of measles virus, and could be applied to express other potentially toxic gene products.
Insights
Researchers developed a novel bicistronic RNA system for stable measles virus protein expression in cultured cells. This breakthrough enables the study of cytotoxic viral proteins and the expression of other toxic gene products.
Area of Science:
- Virology
- Molecular Biology
- Gene Expression
Background:
- Measles virus proteins are potentially cytotoxic and difficult to express stably in cultured cells.
- Previous attempts to express cloned measles viral genes in cultured cells have been unsuccessful.
- Understanding measles virus protein function is crucial for developing effective antiviral strategies.
Purpose of the Study:
- To develop a novel system for the stable expression of measles virus proteins in cultured mammalian cells.
- To overcome challenges associated with expressing potentially cytotoxic viral proteins.
- To facilitate research into measles virus protein functions and applications for other toxic gene products.
Main Methods:
- Constructed bicistronic RNA vectors containing measles virus genes (N, M, H) linked to a selectable marker (DHFR*).
- Utilized inducible (metallothionein IIA) and noninducible (SV40 early) promoters for gene expression.
- Introduced constructs into mammalian cells and selected for stable expression using methotrexate (MTX).
- Investigated inducibility of N protein expression with cadmium chloride.
Main Results:
- Successfully synthesized bicistronic RNAs and achieved stable expression of measles virus nucleoprotein (N), matrix (M), and hemagglutinin (H) proteins.
- DHFR* gene conferred methotrexate resistance, enabling selection of cells expressing viral proteins.
- N protein expression was further inducible by cadmium chloride, demonstrating controlled expression.
- The developed system allows for the stable expression of previously difficult-to-express measles virus proteins.
Conclusions:
- A novel bicistronic RNA expression system enables stable expression of potentially cytotoxic measles virus proteins in cultured cells.
- This system overcomes previous limitations in expressing measles viral genes, facilitating functional studies.
- The methodology is applicable for expressing other toxic gene products, broadening its utility in biological research.