Abstract
Many pathogens, including viruses, bacteria, as well as bacterial toxins, enter their target cells by endocytosis leading to accumulation of pathogenic and cellular proteins in endosomes. Here, we present detailed experimental instructions on isolation of endosomes after virus infection and their subsequent biomolecular characterization. The isolation of endosomes is based on discontinuous sucrose gradient centrifugation, where different endosomal compartments accumulate at a specific sucrose interface. This enables the enrichment and separation of the virus-interacting and co-internalized cell-surface receptors and membrane-associated proteins. The endosomal fractions can be further analyzed by Western blot or quantitative real-time PCR, which reveals changes in the viral protein or DNA content during the processes of endocytosis and endosomal escape. In addition, comparative quantitative mass spectrometry enables the identification of unknown host-cell factors required for infection.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Mercer J, Schelhaas M, Helenius A (2010) Virus entry by endocytosis. Annu Rev Biochem 79:803–833
Spoden GA, Sapp M, Florin L (2012) Host cell factors involved in papillomavirus entry. Med Microbiol Immunol 201(4):437–448
Khan MN, Savoie S, Bergeron JJ et al (1986) Characterization of rat liver endosomal fractions: in vitro activation of insulin-stimuable kinase in these structures. J Biol Chem 261(18):8462–8472
Aniento F, Gruenberg J (2004) Subcellular fractionation of tissue culture cells. Curr Protoc Protein Sci Chapter 4, Unit 4 3.
Gorvel JP, Chavrier P, Zerial M et al (1991) rab5 controls early endosome fusion in vitro. Cell 64:915–925
de Araujo ME, Huber LA, Stasyk T (2008) Isolation of endocitic organelles by density gradient centrifugation. Methods Mol Biol 424:317–331
Lee YH, Tan HT, Chung MC (2010) Subcellular fractionation methods and strategies for proteomics. Proteomics 10(22):3935–3956
Popa-Wagner R, Porwal M, Kann M et al (2012) Impact of VP1-specific protein sequence motifs on AAV2 intracellular trafficking and nuclear entry. J Virol 86(17):9163–9174. doi: 10.1128/JVI.00282-12
Spoden GA, Freitag K, Husmann M et al (2008) Clathrin- and caveolin-independent entry of human papillomavirus type 16-involvement of tetraspanin-enriched microdomains (TEMs). PLoS One 3(10):e331
Scheffer KD, Gawlitza A, Spoden GA et al (2013) Tetraspanin CD151 mediates papillomavirus type 16 endocytosis. J Virol 87(6):3435–3446. doi:10.1128/JVI.02906-12
Lambert C, Doring T, Prange R (2007) Hepatitis B virus maturation is sensitive to functional inhibition of ESCRT-III, Vps4, and gamma 2-adaptin. J Virol 81:9050–9060
Cordwell SJ, Thingholm TE (2010) Technologies for plasma membrane proteomics. Proteomics 10(4):611–627
Veldwijk MR, Topaly J, Laufs S et al (2002) Development and optimization of a real-time PCR-based method for titration of AAV-2 vector stocks. Mol Ther 6(2):272–278
Spoden GA, Besold K, Krauter S et al (2012) Polyethylenimine is a strong inhibitor of human papillomavirus and cytomegalovirus infection. Antimicrob Agents Chemother 56(1):75–82
Acknowledgments
This work was supported by grants to L.F. from the German Research Foundation (DFG, SFB490) and the Johannes Gutenberg University Mainz (intern-funding program). Thanks to Stefan Tenzer for mass spectrometry information.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Scheffer, K.D., Popa-Wagner, R., Florin, L. (2013). Isolation and Characterization of Pathogen-Bearing Endosomes Enable Analysis of Endosomal Escape and Identification of New Cellular Cofactors of Infection. In: Bailer, S., Lieber, D. (eds) Virus-Host Interactions. Methods in Molecular Biology, vol 1064. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-601-6_7
Download citation
DOI: https://doi.org/10.1007/978-1-62703-601-6_7
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-600-9
Online ISBN: 978-1-62703-601-6
eBook Packages: Springer Protocols