Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells

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

This table lists all the proteins identified as having a visual enrichment or depletion in the infected cell nucleoli compared with the uninfected cell nucleoli. Each protein is listed with the spot number as identified on the 2D gels in Fig. 2 , the accession number, the number of peptides identified, the percent coverage, and a brief description of its function. IRES, internal ribosome entry site. Fig. 1C (top panel) also reinforces the distinct effects of adenovirus infection compared with ActD treatment. Looking at rRNA transcription, upon ActD treatment, RNA polymerase I subunits and accessory transcription factors were similarly depleted from the nucleolus. However, in virally infected cells, nucleolar levels of the RNA polymerase subunits and transcription factor UBF were affected differently (Fig. 1C, bottom panel) . This is consistent with previous observations during adenovirus infection where UBF was depleted from the nucleolus whereas RNA polymerase I remained nucleolar as evidenced by immunofluorescence using antibodies specific for RNA polymerase I, in situ visualization of ongoing RNA synthesis, and S1 nuclease protection assay of rRNA synthesis initiation (11) . This suggests that adenovi- The second row of images shows the same indicated endogenous or expressed tagged protein in cells infected with adenovirus for 18 h. Viral infection was confirmed by anti-DBP serum. In B, a third row is shown in which cells were infected with adenovirus and the location of endogenous UBF was compared directly with that of endogenous Nopp140. A duplicate slide (not shown) confirmed that Ͼ99% of cells were infected in this experiment. In some cases, where compatible serum was available, a control nucleolar antigen, B23.1 or UBF, is also shown alongside the uninfected cell images. VSV, vesicular stomatitis virus; EGFP, enhanced GFP. rus induces a specific removal of UBF from the nucleolus, independently of other components of the rDNA transcription complex. This gives us confidence that our data reflect genuine changes in the nucleolar proteome because we know of no other way of inducing this pattern of changes on the rDNA transcription complex.