Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells

Lam, Yun W.; Evans, Vanessa C.; Heesom, Kate J.; Lamond, Angus I.; Matthews, David A.

We next investigated the adenovirus-induced changes on nucleolar composition. Of the proteins shown to be depleted, UBF was the most affected in accordance with previous reports (11) . Protein B23.1 has also been shown to be sequestered into viral replication centers (12) , and this depletion was also detected by our high throughput proteomics analysis as an almost 2-fold depletion compared with uninfected cells. Accordingly, we concentrated on proteins that, like B23.1 and UBF, showed a 2-fold depletion or enrichment in the nucleoli of infected cells (Table I) . 2D Gel Analysis of Virally Infected Nucleoli-We repeated the isolation of nucleoli from uninfected and infected cells a further three times at 18 h postinfection. Western blotting confirmed the purity of the nucleolar fractions (data not shown). The nucleoli from these experiments were examined using 2D gel analysis, which showed that only a small number of proteins had clearly altered amounts at this time point. Fig. 2 shows a typical pair of gels, and seven proteins showing a change in quantity are marked. These spots were picked and sequenced as described under "Experimental Procedures." Their identities are listed in Table II .