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Journal of Virology, October 2004, p. 11187-11197, Vol. 78, No. 20
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.20.11187-11197.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of Proteins Associated with Murine Cytomegalovirus Virions

Lisa M. Kattenhorn,1 Ryan Mills,2 Markus Wagner,1 Alexandre Lomsadze,3 Vsevolod Makeev,4 Mark Borodovsky,2,3* Hidde L. Ploegh,1 and Benedikt M. Kessler1,5*

Pathology Functional Proteomics Center,5 Department of Pathology, Harvard Medical School, Boston, Massachusetts,1 State Scientific Center GosNII Genetika, Moscow, Russia,4 School of Biology,3 Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia2

Received 26 March 2004/ Accepted 1 June 2004

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 ORFc225441-226898 (m166.5) and ORF105932-106072. Immunoblot experiments confirmed expression of m166.5 during viral infection.

* Corresponding author. Mailing address for Benedikt Kessler: Pathology Functional Proteomics Center, Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115. Phone: (617) 432-6660. Fax: (617) 432-6562. bkessler@hms.harvard.edu. Mailing address for Mark Borodovsky: School of Biology, Georgia Institute of Technology, 310 Ferst Dr., Atlanta, GA 30332. Phone: (404) 894-8432. Fax: (404) 894-0519. E-mail: mark.borodovsky{at}biology.gatech.edu.

Journal of Virology, October 2004, p. 11187-11197, Vol. 78, No. 20
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.20.11187-11197.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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