Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells

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

In this study, we isolated nucleoli from infected and mock infected HeLa cells and compared the differences in the isolated proteins by quantitative proteomics. To test the suitability of this approach, we infected a HeLa cell line stably ex-pressing a GFP-tagged ribosomal protein RPL27 (34) with Ad5. RPL27-GFP was used as a nucleolar marker in live cells and in isolated nucleoli. At 18 h postinfection, the cells were harvested, and nucleoli were isolated using a standard protocol (29, 35) . As shown in supplemental Fig. 1 , live cell imaging revealed that the RPL27-GFP signal in the nucleolus of infected cells (supplemental Fig. 1C ) was similar to that in untreated cells (supplemental Fig. 1A ). The GFP signals in nucleoli isolated from untreated (supplemental Fig. 1B ) and infected (supplemental Fig. 1D ) cells were also similar, although the nucleoli from infected cells appeared slightly larger. Interestingly, adenovirus infection induced the appearance of RPL27-GFP in threadlike structures in the nucleoplasm. The significance of the accumulation of this ribosomal protein in these structures or the apparent slight enlargement of nucleoli is unknown. The threadlike GFP-containing structures were not observed in the isolated nucleolar preparation, suggesting that the preparation was not contaminated with nucleoplasmic materials and was suitable for future proteomics analysis.