Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells

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

doi:10.1074/mcp.m900338-mcp200

Yun W Lam, Vanessa C Evans + Show 3 more

Open Access

https://doi.org/10.1074/mcp.m900338-mcp200

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Abstract

Adenoviruses replicate primarily in the host cell nucleus, and it is well established that adenovirus infection affects the structure and function of host cell nucleoli in addition to coding for a number of nucleolar targeted viral proteins. Here we used unbiased proteomics methods, including high throughput mass spectrometry coupled with stable isotope labeling by amino acids in cell culture (SILAC) and traditional two-dimensional gel electrophoresis, to identify quantitative changes in the protein composition of the nucleolus during adenovirus infection. Two-dimensional gel analysis revealed changes in six proteins. By contrast, SILAC-based approaches identified 351 proteins with 24 proteins showing at least a 2-fold change after infection. Of those, four were previously reported to have aberrant localization and/or functional relevance during adenovirus infection. In total, 15 proteins identified as changing in amount by proteomics methods were examined in infected cells using confocal microscopy. Eleven of these proteins showed altered patterns of localization in adenovirus-infected cells. Comparing our data with the effects of actinomycin D on the nucleolar proteome revealed that adenovirus infection apparently specifically targets a relatively small subset of nucleolar antigens at the time point examined.

Highlights

Human adenoviruses comprise a non-enveloped icosahedral particle of 90 nm in diameter containing a linear doublestranded DNA genome of ϳ36 kbp
We have previously shown that nucleolar antigens UBF and B23 are visibly depleted from the nucleolus during adenovirus infection and play functional roles in viral DNA replication [11,12,13]
Confocal Microscopy of Candidate Proteins Identified by 2D Gel Analysis—As with the SILAC data, we examined using immunofluorescence a number of candidate proteins identified by 2D gel analysis that were altered in abundance following infection

Summary

Introduction

Human adenoviruses comprise a non-enveloped icosahedral particle of 90 nm in diameter containing a linear doublestranded DNA genome of ϳ36 kbp. After virus attachment and entry to the host cell, the genome is delivered to the host cell nucleus, initiating a cascade of viral gene expression that results in viral DNA replication and accumulation of viral proteins that eventually form new infectious particles. The use of modern proteomics approaches to investigate viral infections is still relatively new, but recently several notable studies have been reported that illustrate the power and utility of these techniques [22,23,24,25,26]. Most of these have used 2D gel electrophoresis-based approaches with some success. A key study examined the changes in composition of the nucleolus after inhibition of rRNA synthesis with actinomycin D (ActD), quantitating the effects on almost 500 proteins in the nucleolus [29]

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Discussion

Conclusion