Analysis of the Cytomegalovirus Proteome Reveals Unanticipated Complexity

The huge 240,000 base pair HCMV genome was sequenced more than 20 years ago, but the viral proteome (the proteins encode by those genes) has not been studied in a comprehensive fashion.

Investigators at the University of California, San Francisco (USA) have now obtained much of the missing proteomic data. They used state-of-the-art ribosome profiling and transcript analysis linked to mass spectrometry to experimentally define the HCMV translation products (proteins) and follow their temporal expression. In translation, messenger RNA (mRNA) produced by transcription is decoded by the ribosomes to produce specific polypeptides that will later fold into active proteins.

Ribosome profiling is a technique that uses mRNA to determine what proteins are being translated. It produces a “global snapshot” of all the ribosomes active in the cell at a particular moment. Consequently, this enables researchers to identify the location of translation start sites, their distribution, and the speed of the translating ribosomes. Ribosome profiling derived from the old discovery that the mRNA within a ribosome can be isolated through the use of nucleases that degrade unprotected mRNA regions. This technique analyzes the ratio of multiple specific mRNAs to proteins being synthesized, to provide insight into global gene expression.

Results published in the November 23, 2012, issue of the journal Science revealed hundreds of previously unidentified open reading frames (sites of protein translation), a fraction of which were confirmed by means of mass spectrometry. Many of these open reading frames were found to encode for exceptionally short protein sequences (fewer than 100 amino acids), and some of the newly identified open reading frames were sequestered inside other open reading frames.

These results unveiled an unanticipated complexity to the HCMV coding capacity and illustrated the role of the regulated use of alternative transcript start sites in enabling tight temporal control of HCMV protein expression and allowing multiple distinct polypeptides to be generated from a single genomic locus.

"The genome of a virus is just a starting point," said senior author Dr. Jonathan Weissman, professor of cellular and molecular pharmacology and of biochemistry and biophysics at the University of California, San Francisco. "Understanding what proteins are encoded by that genome allows us to start thinking about what the virus does and how we can interfere with it… Each of the proteins we have identified has the potential to tell us how this virus is manipulating its host cell."

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