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. 2004 Oct;78(20):11187-97.
doi: 10.1128/JVI.78.20.11187-11197.2004.

Identification of proteins associated with murine cytomegalovirus virions

Lisa M Kattenhorn  1 , Ryan Mills, Markus Wagner, Alexandre Lomsadze, Vsevolod Makeev, Mark Borodovsky, Hidde L Ploegh, Benedikt M Kessler
Affiliations

Identification of proteins associated with murine cytomegalovirus virions

Lisa M Kattenhorn et al. J Virol. 2004 Oct.

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.

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Figures

FIG. 1.
FIG. 1.
Characterization of isolated MCMV virions. (A) Electron microscopy picture of purified MCMV particles. (B) Analysis of virus particles isolated from supernatant of infected cells. Isolated protein was separated by SDS-10% PAGE, and individual polypeptides were excised from the gel, digested with trypsin, and analyzed by MS/MS. MCMV-encoded polypeptides and host cell proteins are indicated. Contaminating serum proteins (e.g., serum albumin) and unidentified polypeptides are marked with an asterisk and a caret, respectively.
FIG. 2.
FIG. 2.
Evidence for a 3′ extension of the MCMV m20 gene. (A) MS/MS spectrum of the 1,449.75 (M+H)3+ precursor ion of the tryptic peptide fragment CGETGEATSPSDSWLERPAYTEVSSPSTGFAATPASSQWSR as interpreted by MASCOT with a score of 45. (B) The matching b and y ions of the peptide found by MS/MS analysis. (C) Putative coding region at position 20579 to 21313 overlapping the 5′ region of the m20 gene. The three reading frames of the complementary sequence are displayed. The location of the tryptic peptide identified from this ORF, corresponding to the MS/MS spectrum in panel A, is indicated as a black box. Thin grey bars indicate genes annotated by Rawlinson et al. (45). Black bars represent ORFs predicted as protein coding by GeneMarkS, and grey areas indicate regions of interest with moderate coding potential generated by GeneMark. (D) DNA sequencing analysis revealed an incorrect insertion (G) at position 20958 of the MCMV genome (Smith strain NCBI 004065). Removal of this G extended the C terminus of m20 from position 20805 to position 20579.
FIG. 3.
FIG. 3.
Identification of a frameshift in the MCMV M31 gene. (A) A region with high coding potential was detected at the C-terminal end of the M31 gene in reading frame 3. The three reading frames of the direct sequence are displayed. (B) DNA sequencing analysis determined a missing G at position 38803. (C) Insertion of G38803 restored full-length homology of M31 to RCMV protein R31. The box indicates the amino acid which corresponds to the corrected codon from the insertion.
FIG. 4.
FIG. 4.
Expression of m166.5 confirmed by MS and Western blot analysis. (A) MS/MS spectrum of precursor ion 715.26 (uppercase 2+) was interpreted as peptide GSGTISASAPAAGVQR by MASCOT, with a score of 44. Detected b and y ions are indicated. (B) Sequence of m166.5. Tryptic peptides detected by MS are underlined. (C) GeneMark probability plot of the m166 MCMV gene region. The three reading frames of the complementary sequence are displayed. m166.5 partially overlaps with m167, and similar probabilities of coding potential are seen for both reading frames. Peptides found by MS/MS analysis are indicated as black boxes. Thin grey bars represent genes annotated by Rawlinson et al. (45), black bars are genes predicted by GeneMarkS, and grey areas indicate regions of interest with moderate coding potential. (D) Western blot analysis of NIH 3T3 endothelial cells infected with MCMV containing an HA-tagged m166.5 gene. Infected cells were harvested after the indicated times, lysed, and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody.
FIG. 5.
FIG. 5.
Evidence for expression of the predicted ORF105932-10672. (A) The MS/MS spectrum of the precursor ion 673.21 (uppercase 2+) corresponds to the tryptic fragment AIVGFQDDIVLR with a score of 48 by MASCOT. Matching b and y ions are indicated. This virus-derived peptide was detected in the virion preparation analyzed by SDS-PAGE and isolated from the 20- to 29-kDa range. The tryptic peptide fragment detected within the sequence of ORF105932-106072 by MS is underlined. (B) Gene predicted by GeneMarkS, with high coding potential. The three reading frames of the direct sequence are displayed. A black box indicates the tryptic fragment identified by MS/MS. The predicted gene and regions with moderate coding potential are indicated as black and gray bars, respectively.
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References

    1. Atalay, R., A. Zimmermann, M. Wagner, E. Borst, C. Benz, M. Messerle, and H. Hengel. 2002. Identification and expression of human cytomegalovirus transcription units coding for two distinct Fcγ receptor homologs. J. Virol. 76:8596-8608. - PMC - PubMed
    1. Bahr, U., and G. Darai. 2004. Re-evaluation and in silico annotation of the Tupaia herpesvirus proteins. Virus Genes 28:99-120. - PubMed
    1. Baldick, C. J., Jr., and T. Shenk. 1996. Proteins associated with purified human cytomegalovirus particles. J. Virol. 70:6097-6105. - PMC - PubMed
    1. Bechtel, J. T., and T. Shenk. 2002. Human cytomegalovirus UL47 tegument protein functions after entry and before immediate-early gene expression. J. Virol. 76:1043-1050. - PMC - PubMed
    1. Besemer, J., A. Lomsadze, and M. Borodovsky. 2001. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res. 29:2607-2618. - PMC - PubMed

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