| title: | Molecular studies of replicase complexes of alphaviruses and alphavirus-like viruses and use of alphaviruses as advanced gene expression vectors. |
|---|---|
| reg no: | ETF5055 |
| project type: | Estonian Science Foundation research grant |
| subject: |
1.8. Molecular Biology 2.7. Biotechnology, Food and Drink Technology |
| status: | completed |
| institution: | Estonian Biocentre |
| head of project: | Andres Merits |
| duration: | 01.01.2002 - 31.12.2005 |
| description: | Alphaviruses are a relatively well studied group of plus-stranded RNA viruss which are evolutionary related to a large group of animal, insect and plant infecting viruses, called the alphavirus-like superfamily. This makes alphaviruses extremely useful as models, since basic research of alphavirus molecular biology reveals virus-specific molecular mechanisms and gives also information about possible gene functions of alphavirus-like viruses. With this project we plan to participate in basic research of molecular biology of alphaviruses, using the Semliki Forest virus (SFV, an alphavirus) and HEV (Hepatitis E virus) as models. First, we plan to study the formation of the SFV replication complex and to characterize structures, functions and properties of replicase proteins of SFV and HEV. Using the expression and purification of recombinant proteins from SFV and HEV replicase complexes their enzymatic properties will be determined. Site-specific mutagenesis will be used to map functionally active domains and sequences of these proteins. Subcellular localization of replicase proteins and their mutant variants will be studied using transfected and infected cells with the aim of determining what functions of these proteins fulfil during normal infection. Based on the results of the basic studies test-systems for evaluating the potential of virus inhibitors can be developed. Second, we plan to improve existing vectors, based on the SFV genome and replicons. To make the gene expression from the SFV 26S RNA promoter controlable, the temperature sensitive mutations, affecting subgenomic RNA synthesis will be identified and introduced into SFV replicon vector. Unlike existing vectors, these improved vectors would allow us to clone and express genes with toxic products. The results, obtained on the research of the functions of mutant SFV replicase proteins will also find their use in the improved SFV vectors. Mutations, which reduce cytotoxicity of SFV, prevent apoptotic cell death of infected cells and minimize virus-induced damages of cytoskeleton and/or membranous structures will be introduced into SFV based vector. This new generation of SFV based vectors will be safe and easy to use for multiple research and biotechnological applications. |
| project group | ||||
|---|---|---|---|---|
| no | name | institution | position | |
| 1. | Kaja Kiiver | TU | Ph.D. student | |
| 2. | Andres Merits | Estonian Biocentre | Senior scientist | |
| 3. | Eva Žusinaite | EBC | Ph.D. student | |