Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells

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

Comparison with Other Established Effects of Adenovirus on Nucleolus-The SILAC data point to depletion of UBF and B23.1 during viral infection, which reflects previously published data by ourselves and others that UBF and B23.1 are functionally sequestered upon adenovirus infection into viral DNA replication centers (11) (12) (13) . Another example is U2AF 65 , a splicing factor known to play a role in splicing of adenovirus transcripts (52, 53) . Finally, exportin 5 is known to export adenovirus VA RNA, competing with cellular microRNA export and potentially modulating cellular protein expression (54) . The identification of these proteins by SILAC/MS analysis further validates our approach because they are all well established functional partners in adenovirus infection. This gives us confidence that some, if not all, of the novel proteins Novel Proteins Depleted from Nucleolus on Infection-Of those proteins examined that show a clear nucleolar localization in uninfected cells, all had a dramatic redistribution in infected cells. For example, hPOP1 is normally involved in rRNA processing (42, 55) , and its mislocalization may reflect data showing that adenovirus eventually inhibits rRNA processing during infection (2) . Nopp140 mislocalization also fits with these observations because it is a chaperone for small nucleolar RNA (56, 57) . However, its co-localization with UBF and DBP implies that it may be an accessory factor in viral DNA replication during viral infection. Notably, in interphase cells, UBF and Nopp140 are co-localized and are known to be recruited together (56) . RBM4 has been reported to be either predominantly nuclear or predominantly nucleolar, depending on experimental conditions (43, 58 -60) . In our hands, the protein was nucleolar in Ͼ80% of cells. RBM4 has been implicated in the modulation of splice site selection, something of clear relevance to adenovirus infection, but it has also FIG. 4 . Distribution of proteins identified by SILAC as being enriched in nucleolus during viral infection. All the images are of a fixed focal plane ϳ0.3 m in depth, the DAPI stain is in blue in all cases, and the bar represents 10 m. A-C, in each case the top row of images is representative of the location of the indicated endogenous protein or expressed tagged fusion protein in Ͼ80% of cells examined. 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, the use of a cyan fluorescent protein (CFP) fusion protein precluded the use of DAPI as a nuclear marker, and so antiserum to U2AF65 was used instead. In A, UBF is also shown alongside the uninfected cell images as a marker for the nucleolus.