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Phosphorylation and nucleic acid binding properties of m1 Moloney murine sarcoma virus-specific pP60gag
Abstract:
The pP60gag polyprotein of the feline leukemia virus pseudotype of m1 Moloney murine sarcoma virus [m1MSV(FeLV)] was previously shown to be MSV specific and to contain murine p30 and smaller structural polypeptides. This protein was detected in m1MSV-transformed cells, and in pulse-chase studies it was found to be stable. In this study virion P60 was shown to contain murine pp12, to be phosphorylated, and to bind to nucleic acids. 32P-labeled m1MSV[FeLV) was fractionated by guanidine agarose chromatography and analyzed by gel electrophoresis. Both P60 and pp12 were found to be the major phosphoproteins, phosphorylated in both serine and threonine residues. Virion P60 bound preferentially to single-stranded DNA and RNA in a competition filter binding assay, using 125I-labeled single-stranded calf thymus DNA and various unlabeled nucleic acids. Similar phosphorylation and DNA binding properties were demonstrated for cellular P60. Thus, immunoprecipitation of cellular extracts showed that P60 was phosphorylated in both producer and nonproducer transformed cells, indicating that phosphorylation occurs independently of virus assembly. Moreover, P60 from cytoplasmic extracts was retained on single-stranded DNA-Sepharose columns, demonstrating that cellular P60 binds to DNA.
Insights
The feline leukemia virus protein P60 is phosphorylated and binds to nucleic acids, including DNA and RNA. This phosphorylation occurs independently of virus assembly in transformed cells.
Area of Science:
- Virology
- Molecular Biology
- Oncogenesis
Background:
- The feline leukemia virus (FeLV) pseudotype of m1 Moloney murine sarcoma virus [m1MSV(FeLV)] encodes a pP60gag polyprotein.
- This protein is specific to m1MSV and contains murine p30 and smaller structural polypeptides.
- Previous studies detected pP60gag in m1MSV-transformed cells and found it to be stable.
Purpose of the Study:
- To investigate the biochemical properties of virion P60, specifically its phosphorylation status and nucleic acid binding capabilities.
- To determine if cellular P60 shares similar phosphorylation and DNA binding characteristics with virion P60.
- To ascertain whether P60 phosphorylation is dependent on virus assembly.
Main Methods:
- Guanidine agarose chromatography and gel electrophoresis were used to analyze 32P-labeled m1MSV(FeLV).
- Competition filter binding assays with 125I-labeled single-stranded DNA and various unlabeled nucleic acids were employed.
- Immunoprecipitation of cellular extracts and single-stranded DNA-Sepharose column chromatography were utilized.
Main Results:
- Virion P60 was identified as a major phosphoprotein containing murine pp12, with phosphorylation occurring on serine and threonine residues.
- Virion P60 exhibited preferential binding to single-stranded DNA and RNA.
- Cellular P60 displayed similar phosphorylation patterns and demonstrated binding to DNA, independent of virus assembly.
Conclusions:
- The pP60gag protein of m1MSV(FeLV) is a phosphorylated molecule that binds to nucleic acids.
- Cellular P60 shares these biochemical properties with virion P60.
- Phosphorylation of P60 occurs in transformed cells irrespective of the virus assembly process.