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Use of free‐flow electrophoresis for the analysis of cellular uptake of picornaviruses
Author(s) -
Kronenberger Peter,
Schober Daniela,
Prchla Elisabeth,
Blaas Dieter,
Fuchs Renate
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150181407
Subject(s) - endosome , poliovirus , intracellular , free flow electrophoresis , centrifugation , cytoplasm , biology , virus , vesicle , differential centrifugation , rna , hela , microbiology and biotechnology , membrane , chemistry , virology , biochemistry , cell , polyacrylamide gel electrophoresis , gene , gel electrophoresis of proteins , enzyme
Abstract Free‐flow electrophoresis is a powerful tool to separate subcellular vesicles such as early and late endosomes from plasma membranes. Using this technique, the intracellular distribution of poliovirus type 2 Sabin (PV2) and its derived subviral particles was analyzed upon infection of HeLa cells. Comparison of various infection conditions showed that maximally 30% of total cell associated PV2 was found in endosomal compartments with the remainder being associated with plasma membrane fractions; 2% of viral label was recovered from the cytoplasm in form of free virions. Sucrose gradient centrifugation analysis of the viral material recovered from the respective fractions revealed that intracellular virus was exclusively in its native conformation. This is in sharp contrast to human rhinovirus serotype 2 (HRV2), which is rapidly modified to RNA‐free subviral particles upon accumulation in endosomes. The data suggest that productive poliovirus uncoating can occur at the plasma membrane whereas internalized virus is most probably aborted.