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

Abstract

BackgroundVirus 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.ResultsSubcellular 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.ConclusionsThis 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.

Hsp90 Mediates Membrane Deformation and Exosome Release

“…in vitro MVB fusion assay MVBs were isolated from adult flies expressing evi-GFP under control of the general Actin-Gal4 driver using a protocol based on (Walker et al, 2016). About 10 mL of flies were crushed with a sterile pestle (VWR) in 4 mL of ice-cold homogenization buffer (250 mM sucrose (MP Biomedicals), 1 mM EDTA (Sigma Aldrich) containing a Complete Protease Inhibitors Cocktail (Roche)).…”

Myristoylated methionine sulfoxide reductase A is a late endosomal protein

“…To prepare a fraction enriched in late endosomes, subcellular fractionation was performed using a modification of a published procedure (56). 100 mg of liver from wild-type or msrA knockout mice was washed with cold phosphate buffered saline.…”

“…Following electrophoresis and blotting, the membranes were probed with various antibodies. With this method, late endosomes will be enriched in fractions 1 and 2 (56).…”

Rapid and facile quantitation of polyplex endocytic trafficking

“…The location of particles within different organelles can also be determined after the organelles have been separated by subcellular fractionation in density-gradient centrifugation [20][21][22]. This method is experimentally challenging and requires careful optimization [23,24].…”