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Expression analysis of recombinant herpes simplex virus type 1 DNase
1Forschungsschwerpunkt Genomforschung und Bioinformatik, Deutsches Krebsforschungszentrum, Heidelberg, FRG.
Virus Genes
|December 19, 1998
Summary
Researchers successfully expressed functional herpes simplex virus type 1 (HSV-1) deoxyribonuclease (DNase) in mammalian cells using vaccinia virus and Semliki Forest virus (SFV) vectors. The study identified an 85-kDa protein and analyzed its localization and expression dynamics.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Herpes simplex virus type 1 (HSV-1) deoxyribonuclease (DNase) is a viral enzyme involved in DNA processing.
- Understanding its expression and function is crucial for viral pathogenesis research.
- Mammalian cell expression systems offer powerful tools for studying viral proteins.
Purpose of the Study:
- To analyze the expression of recombinant HSV-1 DNase in BHK-21 cells using different mammalian expression systems.
- To characterize the expressed protein and its enzymatic activity.
- To investigate the intracellular localization of HSV-1 DNase.
Main Methods:
- Utilized vaccinia virus and Semliki Forest virus (SFV) based expression systems in BHK-21 cells.
- Employed rabbit antiserum and mouse monoclonal antibody for protein detection and identification.
- Performed immunofluorescence microscopy to study intracellular localization.
Main Results:
- Efficient expression of soluble and functional HSV-1 DNase achieved using vaccinia virus/T7 RNA polymerase hybrid and recombinant SFV systems.
- Identified a major 85-kDa protein, consistent in size with native HSV-1 DNase, in both expression systems.
- Recombinant SFV system showed optimal enzyme activity between 16-36 hours post-infection, with activity diminishing later.
- Vaccinia/T7 system facilitated small-scale expression analysis and demonstrated colocalization of HSV-1 DNase with cellular chromatin.
Conclusions:
- Recombinant SFV is suitable for biochemical studies of HSV-1 DNase, provided specific infection parameters are met (packaged virus, <24h infection).
- The vaccinia/T7 hybrid system is effective for expression analysis and studying intracellular interactions.
- HSV-1 DNase localizes to cellular chromatin, independent of its C-terminal residues, suggesting no requirement for other viral factors for this association.