Coxsackievirus B Exits the Host Cell in Shed Microvesicles Displaying Autophagosomal Markers

作者：Scott M. Robinson, Ginger Tsueng, Jon Sin, Vrushali Mangale, Shahad Rahawi, Laura Lynn McIntyre, Wesley Williams, Nelson Kha, Casey Cruz, Bryan Hancock, David P. Nguyen, M. Richard Sayen, Brett J. Hilton, Kelly S. Doran, Anca Mara Segall, Roland Wolkowicz, Christopher T. Cornell, J. Lindsay Whitton, Roberta A. Gottlieb, Ralph Feuer · 发表于：PLoS Pathogens · 年份：2014 · DOI：10.1371/journal.ppat.1004045 · 被引用次数：334 · 研究领域：Viral Infections and Immunology Research、Cytomegalovirus and herpesvirus research、RNA regulation and disease

Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, "fluorescent timer" protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. "Fluorescent timer" protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of "fluorescent timer" protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. "Fluorescent timer" protein colocalized closely wit...