Subcellular fractionation method to study endosomal trafficking of Kaposis sarcoma-associated herpesvirus

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)
Lia R.,Akula,Shaw M.,Hussein,Hosni A. M. Walker (Creator)
East Carolina University (ECU )
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Abstract: Background: Virus entry involves multiple steps and is a highly orchestrated process on which successful infectioncollectively depends. Entry processes are commonly analyzed by monitoring internalized virus particles via Westernblotting, polymerase chain reaction, and imaging techniques that allow scientist to track the intracellular location ofthe pathogen. Such studies have provided abundant direct evidence on how viruses interact with receptor molecules on the cell surface, induce cell signaling at the point of initial contact with the cell to facilitate internalization,and exploit existing endocytic mechanisms of the cell for their ultimate infectious agenda. However, there is dearthof knowledge in regards to trafficking of a virus via endosomes. Herein, we describe an optimized laboratory procedure to isolate individual organelles during different stages of endocytosis by performing subcellular fractionation.This methodology is established using Kaposi"s sarcoma-associated herpesvirus (KSHV) infection of human foreskinfibroblast (HFF) cells as a model. With KSHV and other herpesviruses alike, envelope glycoproteins have been widelyreported to physically engage target cell surface receptors, such as integrins, in interactions leading to entry andsubsequent infection.Results: Subcellular fractionation was used to isolate early and late endosomes (EEs and LEs) by performing a seriesof centrifugations steps. Specifically, a centrifugation step post-homogenization was utilized to obtain the postnuclear supernatant containing intact intracellular organelles in suspension. Successive fractionation via sucrosedensity gradient centrifugation was performed to isolate specific organelles including EEs and LEs. Intracellular KSHVtrafficking was directly traced in the isolated endosomal fractions. Additionally, the subcellular fractionation approachdemonstrates a key role for integrins in the endosomal trafficking of KSHV. The results obtained from fractionationstudies corroborated those obtained by traditional imaging studies.Conclusions: This study is the first of its kind to employ a sucrose flotation gradient assay to map intracellular KSHVtrafficking in HFF cells. We are confident that such an approach will serve as a powerful tool to directly study intracellular trafficking of a virus, signaling events occurring on endosomal membranes, and dynamics of molecular eventswithin endosomes that are crucial for uncoating and virus escape into the cytosol.

Additional Information

Language: English
Date: 2016
Fractionation, Ultracentrifugation, Sucrose density gradient, Endosomes, Virus entry, Endocytosis

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