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Analysis of structural proteins of purified murine cytomegalovirus
Abstract:
Murine cytomegalovirus propagated in mouse embryo fibroblasts was purified by the following procedures. (i) Extracellular virus was concentrated by centrifugation at 100,000 x g for 90 min. (ii) The concentrated virus was passed through a Bio-Rad Bio-Gel A-15m column to eliminate contaminating materials smaller than 15 x 10(6) daltons. Most of the virus was recovered in the void volume of the column. (iii) Two consecutive centrifugations through 20 to 50% potassium tartrate gradients were performed. After the second tartrate gradient centrifugation, symmetrical, coinciding peaks of plaque titer, protein, and radioactivity were found at a density between 1.20 g/cm3 and 1.21 g/cm3. To establish purification criteria, virus was purified from two different mixtures: [35S]methionine-labeled extracellular virus, mixed with an equal volume of unlabeled normal culture fluid, and unlabeled extracellular virus mixed with an equal volume of [35S]methionine-labeled normal culture fluid. At the end of the procedure, the extent of purification, as judged by the ratio of cellular to viral radioactivity was at least 70-fold. Virus proteins were analyzed by electrophoresis on a 5 to 20% gradient polyacrylamide gel slab. After gel electrophoresis,, Coomassie brilliant blue staining profiles and autoradiograms of the purified virus preparations were compared. At least 33 virus structural protein bands were present. The molecular weights of these proteins ranged from 11,500 to 255,000. The sum of the molecular weights of the virus structural proteins was 2,462,000. Autoradiograms obtained from electrophoresis of purified [14C]glucosamine-labeled virus showed that at lease 6 of the 33 viral structural proteins were glycoproteins.
Insights
This study details a purification method for murine cytomegalovirus (MCMV) using ultracentrifugation and gel filtration. The optimized protocol yields highly purified MCMV, revealing at least 33 structural proteins, including glycoproteins.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Murine cytomegalovirus (MCMV) is a significant pathogen in research models.
- Efficient purification of infectious MCMV is crucial for studying its structure and function.
- Existing purification methods may not adequately remove all contaminants.
Purpose of the Study:
- To develop and validate a robust purification protocol for infectious murine cytomegalovirus (MCMV).
- To characterize the structural protein composition of purified MCMV.
- To establish purification criteria for high-quality MCMV preparations.
Main Methods:
- Virus concentration via high-speed centrifugation.
- Size exclusion chromatography using Bio-Gel A-15m for contaminant removal.
- Potassium tartrate gradient centrifugation for density-based purification.
- Analysis of viral proteins using SDS-PAGE and autoradiography.
Main Results:
- A multi-step purification protocol involving centrifugation and chromatography was established.
- Purified MCMV exhibited a density of 1.20-1.21 g/cm³.
- Purification achieved at least a 70-fold enrichment, assessed by radioactivity ratios.
- Electrophoretic analysis identified at least 33 distinct viral structural proteins.
- Molecular weights of viral proteins ranged from 11,500 to 255,000 Da.
- At least 6 of the 33 structural proteins were identified as glycoproteins.
Conclusions:
- The developed protocol effectively purifies infectious MCMV from cell culture.
- The structural proteome of MCMV includes at least 33 proteins, with several being glycoproteins.
- This purification strategy provides a reliable method for obtaining high-purity MCMV for further research.