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Characterization of novel populations of MVM virions containing covalent DNA-protein complexes
E A Faust1, K Brudzynska, J Morgan
1Cancer Research Laboratory, University of Western Ontario, London, Canada.
Abstract:
Virions of minute virus of mice were purified by sedimentation in sucrose gradients and chromatography on DEAE-cellulose columns and shown to consist of single-stranded viral DNA and the viral capsid polypeptides VP-1 (83 kDa) and VP-2 (64.5 kDa). A 63-kDa polypeptide distinct from the viral capsid polypeptide VP-3 (61.4 kDa) was found in some virion preparations. Virions sedimented at 135 and 110 S. The genomic single strands associated with purified 135 and 110 S virions were covalently bound to a protein as judged by the anomalous electrophoretic mobility of the DNA in agarose gels at pH 12.5. The protein was removed from the DNA by Pronase but remained bound after heating at 98 degrees in the presence of 0.1% sodium dodecyl sulfate. Nuclease digestion of the purified DNA-protein complex released several polypeptides ranging in size from 58 to 65 kDa. Restriction enzyme analysis of the purified DNA protein complex following its conversion to a duplex RF DNA in vitro showed that the protein was attached to the 5' termini of the DNA.
Insights
Minute virus of mice virions contain single-stranded DNA and capsid proteins. A novel protein is covalently bound to the viral DNA
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Minute virus of mice (MVM) virions are composed of single-stranded DNA and capsid proteins.
- Understanding the MVM virion structure and DNA-protein interactions is crucial for viral replication studies.
Purpose of the Study:
- To characterize the protein components of minute virus of mice virions.
- To investigate the nature of the association between viral DNA and associated proteins.
Main Methods:
- Purification of MVM virions using sucrose gradient sedimentation and DEAE-cellulose chromatography.
- Analysis of viral polypeptides by SDS-PAGE.
- Electrophoretic analysis of DNA-protein complexes under various conditions (pH, heat, enzymatic digestion).
- Restriction enzyme analysis of converted duplex DNA.
Main Results:
- Purified MVM virions contained capsid polypeptides VP-1 and VP-2, with VP-3 occasionally present.
- A distinct 63-kDa polypeptide was identified in some preparations.
- Genomic single-stranded DNA in MVM virions was covalently bound to a protein, resistant to heat and SDS but sensitive to Pronase.
- Nuclease digestion released polypeptides (58-65 kDa) from the DNA-protein complex.
- Restriction analysis indicated the protein binds to the 5' termini of the viral DNA.
Conclusions:
- MVM virions possess a complex protein-DNA association.
- A novel protein, distinct from capsid proteins, is covalently linked to the MVM genome's 5' end.