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Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins
Published on: August 31, 2017
Identification of proteins associated with murine cytomegalovirus virions
Lisa M Kattenhorn1, Ryan Mills, Markus Wagner
1Pathology Functional Proteomics Center, Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
Abstract:
Proteins associated with the murine cytomegalovirus (MCMV) viral particle were identified by a combined approach of proteomic and genomic methods. Purified MCMV virions were dissociated by complete denaturation and subjected to either separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in-gel digestion or treated directly by in-solution tryptic digestion. Peptides were separated by nanoflow liquid chromatography and analyzed by tandem mass spectrometry (LC-MS/MS). The MS/MS spectra obtained were searched against a database of MCMV open reading frames (ORFs) predicted to be protein coding by an MCMV-specific version of the gene prediction algorithm GeneMarkS. We identified 38 proteins from the capsid, tegument, glycoprotein, replication, and immunomodulatory protein families, as well as 20 genes of unknown function. Observed irregularities in coding potential suggested possible sequence errors in the 3'-proximal ends of m20 and M31. These errors were experimentally confirmed by sequencing analysis. The MS data further indicated the presence of peptides derived from the unannotated ORFs ORF(c225441-226898) (m166.5) and ORF(105932-106072). Immunoblot experiments confirmed expression of m166.5 during viral infection.
Insights
Researchers identified 38 known and 20 unknown proteins in murine cytomegalovirus (MCMV) using advanced proteomic and genomic techniques. This study also corrected sequence errors in MCMV genes m20 and M31.
Area of Science:
- Virology
- Proteomics
- Genomics
Background:
- Murine cytomegalovirus (MCMV) is a significant model organism for studying herpesvirus infections.
- Comprehensive identification of viral proteins is crucial for understanding viral replication and pathogenesis.
Purpose of the Study:
- To identify and characterize proteins associated with the MCMV virion.
- To refine the MCMV genome annotation by identifying novel open reading frames (ORFs) and correcting sequence errors.
Main Methods:
- Purified MCMV virions were analyzed using a combination of proteomics (LC-MS/MS) and genomics.
- Proteins were digested and peptides analyzed via tandem mass spectrometry.
- MS/MS spectra were searched against an MCMV-specific ORF database, followed by sequencing analysis and immunoblotting.
Main Results:
- Identified 38 known MCMV proteins across various functional families (capsid, tegument, glycoprotein, replication, immunomodulatory).
- Discovered 20 MCMV genes with unknown functions.
- Confirmed sequence errors in the 3'-proximal ends of m20 and M31 and identified peptides from unannotated ORFs m166.5 and ORF(105932-106072).
- Experimentally validated the expression of m166.5 during MCMV infection.
Conclusions:
- This study provides a more complete proteomic map of the MCMV virion.
- The findings enhance the accuracy of MCMV genome annotation and contribute to a better understanding of MCMV biology.
