Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells

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

SILAC Analysis of Virus-infected Cell Nucleoli-A total of 351 proteins were quantified by SILAC analysis comparing virally infected host cell nucleoli to uninfected nucleoli that were ranked according to whether there appeared to be depletion or enrichment in the infected cell nucleolus ( Fig. 1 and supplemental Tables 1 and 2). Functional classification (Fig. 1A) revealed that the distribution of the functional roles of the detected proteins was similar to that of the previously reported human nucleolar proteome (29) , indicating that the nucleoli isolated in this study were of purity comparable to the nucleolar preparations used in our previous proteomics studies. Interestingly, no viral proteins were detected in the nucleoli of the virally infected cells using adenoviral genome data in MASCOT searches, potentially reflecting a lack of abundance at that time point. Fig. 1B shows the changes in abundance of the detected proteins in isolated nucleoli after viral infection. Remarkably, a majority of the nucleolar proteins remained relatively unaffected by viral infection; very few proteins exhibited significant changes in levels (more than Ϯ1-fold). A closer comparison of the nucleolar responses to viral infection and with previously reported data (29) on the effects of transcription inhibition by ActD is shown in Fig. 1C . The SILAC -fold changes of the nucleolar levels of selected proteins after viral inhibition and ActD treatment were very different, indicating that the human nucleolus reacts to these two perturbations in highly distinct manners. In particular, the effects of viral infection on components of the rRNA transcription machinery were distinct from the effects of ActD treatment. The SILAC ratios of ribosomal proteins showed a mild decrease after viral infection (Fig. 1D ), whereas ActD treatment had a significant impact on a number of ribosomal proteins.