Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells

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

Confocal Microscopy of Candidate Proteins Identified by SILAC-Because adenovirus induces significant changes in nuclear structure, it is possible that the nucleolar isolation protocol no longer reliably enriches intact nucleoli. Moreover, although many of the proteins identified could conceivably play a role in adenovirus infection, we wanted to independently verify any potential changes in the nucleolar proteome. This would have a further key benefit of prioritizing future work relating these changes to function. We therefore used a range of antisera and tagged expression constructs to examine the fate of a number of proteins identified as being enriched or depleted in the nucleolus. We concentrated on those proteins for which reagents and published data on subcellular location were already available so we could be confident of their locations in uninfected cells. We compared the location of target antigens relative to adenovirus DBP, a key component of the adenovirus DNA replication machinery that is expressed to high levels in the nucleoplasm but not the nucleolus in virus-infected cells. DBP has a distinct pattern of fluorescence and well defined localization relative to viral mRNA transcription and de novo viral DNA synthesis (50). Fig. 3 shows the effects of infection on those proteins apparently depleted from the nucleolus during viral infection. Two nucleolar antigens, hPOP1 and Nopp140, were clearly depleted from the nucleolus (Fig. 3, A and B) . hPOP1 was sequestered from the nucleolus into a mottled pattern within the nucleoplasm and was excluded from the DBP-rich centers (Fig. 3A) .