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Identification and characterization of a 50K DNA-binding protein of guinea-pig cytomegalovirus
1Institute for Developmental Research, Aichi Prefectural Colony, Japan.
Abstract:
In a previous study we showed that cells infected with guinea-pig cytomegalovirus (GPCMV) contain large amounts of a non-structural 50K nuclear protein that is detectable by immunoelectron microscopy using monoclonal antibodies. The present study shows that this 50K protein is a DNA-binding protein, as determined by single-stranded DNA affinity chromatography, and a phosphorylated protein as demonstrated by immunoprecipitation using [32P]orthophosphate-labelled cells. This protein binds both viral and host cellular double-stranded and single-stranded DNA, assayed by a simple method using DNA linked to a nylon membrane. Induction of the 50K protein in GPCMV-infected cells was highly dependent on viral DNA synthesis, which was detected by dot hybridization using a cloned GPCMV DNA probe. Synthesis of the 50K protein was significantly impaired when phosphonoacetic acid was added to the culture medium. Induction of the 50K protein was detected about 6 h before the appearance of the 76K viral matrix protein.
Insights
Researchers found that a 50K nuclear protein in guinea-pig cytomegalovirus (GPCMV)-infected cells binds DNA and is phosphorylated. Its production depends on viral DNA synthesis, appearing before viral matrix protein.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- A previous study identified a non-structural 50K nuclear protein in guinea-pig cytomegalovirus (GPCMV)-infected cells.
- This protein was detectable via immunoelectron microscopy using monoclonal antibodies.
Purpose of the Study:
- To characterize the biochemical properties of the 50K nuclear protein.
- To investigate the relationship between 50K protein induction and viral DNA synthesis in GPCMV infection.
Main Methods:
- Single-stranded DNA affinity chromatography was used to assess DNA-binding properties.
- Immunoprecipitation with [32P]orthophosphate-labelled cells identified phosphorylation.
- DNA-binding assays utilized DNA linked to a nylon membrane.
- Dot hybridization with a GPCMV DNA probe monitored viral DNA synthesis.
- The effect of phosphonoacetic acid on 50K protein synthesis was evaluated.
Main Results:
- The 50K protein was confirmed to be a DNA-binding protein.
- It was also identified as a phosphorylated protein.
- The protein binds both viral and host cellular DNA (single- and double-stranded).
- 50K protein induction was highly dependent on viral DNA synthesis.
- Synthesis was impaired by phosphonoacetic acid.
- 50K protein induction preceded the appearance of the 76K viral matrix protein by approximately 6 hours.
Conclusions:
- The 50K nuclear protein is a DNA-binding, phosphorylated viral protein.
- Its synthesis is tightly linked to viral DNA replication.
- This protein may play a role in early stages of GPCMV replication.