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Viral DNA synthesis in vitro with the inclusions isolated from adenovirus 12-infected cells
Abstract:
A fraction defined as the inclusions was isolated by banding in CsC1 gradients from nuclei of adenovirus 12-infected KB cells. When examined by electron microscopy, the isolated inclusions were relatively homogeneous, finely granular materials of moderate electron density, possibly representing the disintegrated type II or IV inclusions. The conditions of endogenous DNA synthesis in vitro with the inclusions were determined. The product of DNA synthesis in vitro with the inclusions was mainly viral and scarcely cellular, as revealed by DNA-DNA hybridization and methylated albumin kieselgur column chromatography. However, viral DNA synthesized in vitro was smaller (18S, 22S) than viral DNA in virions (31 S, 34 S) in neutral and alkaline sucrose gradients. Effects of various treatment of the inclusions on the DNA-synthesizing activity showed that phospholipase C inhibited the activity efficiently. The in vitro DNA synthesis was stimulated by addition of the cytoplasmic extract from adenovirus 12-infected cells and not that from unifected cells. The analysis of the composition of the inclusions showed that the inclusions contained DNA, protein, phospholipid and a small amount of RNA and carbohydrate.
Insights
Researchers isolated adenovirus 12 inclusions from infected cells to study DNA synthesis. In vitro synthesis primarily produced viral DNA, though smaller than virion DNA, and was inhibited by phospholipase C.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Adenovirus 12 infection of KB cells leads to nuclear inclusions.
- These inclusions are potential sites for viral DNA replication.
Purpose of the Study:
- To characterize the isolated inclusions and their DNA-synthesizing activity in vitro.
- To determine the nature of the synthesized DNA and factors affecting synthesis.
Main Methods:
- Isolation of inclusions using cesium chloride gradients.
- Electron microscopy for structural analysis.
- In vitro DNA synthesis assays.
- DNA-DNA hybridization and chromatography for product analysis.
- Sucrose gradient centrifugation for DNA size determination.
- Treatment with phospholipase C and cytoplasmic extracts.
Main Results:
- Isolated inclusions were homogeneous, granular material, likely disintegrated inclusion bodies.
- In vitro DNA synthesis predominantly produced viral DNA, smaller than mature viral DNA.
- Phospholipase C significantly inhibited DNA synthesis.
- Cytoplasmic extracts from infected cells stimulated in vitro DNA synthesis.
Conclusions:
- The isolated inclusions possess endogenous DNA-synthesizing activity, primarily generating viral DNA fragments.
- Phospholipase C and cytoplasmic factors play roles in regulating this viral DNA synthesis.