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Subcellular fractionation method to study endosomal trafficking of Kaposi’s sarcoma-associated herpesvirus

dc.contributor.authorWalker, Lia R.
dc.contributor.authorHussein, Hosni Ahmed Mohamed
dc.contributor.authorAkula, Shaw M.
dc.date.accessioned2016-06-23T14:41:45Z
dc.date.available2016-06-23T14:41:45Z
dc.date.issued2016-01
dc.description.abstractBackground Virus entry involves multiple steps and is a highly orchestrated process on which successful infection collectively depends. Entry processes are commonly analyzed by monitoring internalized virus particles via Western blotting, polymerase chain reaction, and imaging techniques that allow scientist to track the intracellular location of the 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 dearth of 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 foreskin fibroblast (HFF) cells as a model. With KSHV and other herpesviruses alike, envelope glycoproteins have been widely reported to physically engage target cell surface receptors, such as integrins, in interactions leading to entry and subsequent infection. Results Subcellular fractionation was used to isolate early and late endosomes (EEs and LEs) by performing a series of centrifugations steps. Specifically, a centrifugation step post-homogenization was utilized to obtain the post-nuclear supernatant containing intact intracellular organelles in suspension. Successive fractionation via sucrose density gradient centrifugation was performed to isolate specific organelles including EEs and LEs. Intracellular KSHV trafficking was directly traced in the isolated endosomal fractions. Additionally, the subcellular fractionation approach demonstrates a key role for integrins in the endosomal trafficking of KSHV. The results obtained from fractionation studies 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 KSHV trafficking 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 events within endosomes that are crucial for uncoating and virus escape into the cytosol.en_US
dc.identifier.citationCell & Bioscience; 6: p. 1-10en_US
dc.identifier.doi10.1186/s13578-015-0066-2
dc.identifier.issn2045-3701
dc.identifier.pmidpmc4714431en_US
dc.identifier.urihttp://hdl.handle.net/10342/5697
dc.relation.urihttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4714431/en_US
dc.subjectFractionationen_US
dc.subjectUltracentrifugationen_US
dc.subjectSucrose density gradient,en_US
dc.subjectEndosomesen_US
dc.subjectVirus entryen_US
dc.subjectEndocytosisen_US
dc.titleSubcellular fractionation method to study endosomal trafficking of Kaposi’s sarcoma-associated herpesvirusen_US
dc.typeArticleen_US
ecu.journal.nameCell & Bioscienceen_US
ecu.journal.pages1-10en_US
ecu.journal.volume6en_US

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