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

摘要: Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, pancreatitis in humans. We genetically engineered unique molecular marker, “fluorescent timer” protein, within our infectious CVB3 clone isolated high-titer recombinant stock (Timer-CVB3) following transfection HeLa cells. “Fluorescent protein undergoes slow conversion fluorescence from green to red over time, Timer-CVB3 can be utilized track virus infection dissemination real time. Upon with Timer-CVB3, cells, neural progenitor stem cells (NPSCs), C2C12 myoblast slowly changed 72 hours as determined by microscopy or flow cytometric analysis. The infected could interrupted fixation, suggesting that fluorophore was stabilized formaldehyde cross-linking reactions. Induction type I interferon response ribavirin treatment reduced progression cell-to-cell spread NPSCs Timer-CVB3. Time lapse photography partially differentiated revealed substantial intracellular membrane remodeling assembly discrete replication organelles which color an asynchronous fashion cell. colocalized closely 3A organelles. Intriguingly, induced release abundant extracellular microvesicles (EMVs) containing matured representing novel route dissemination. virions were readily observed purified EMVs transmission electron microscopy, identified low-density isopycnic iodixanol gradient fractions consistent association. preferential detection lipidated form LC3 (LC3 II) released harboring suggests autophagy pathway plays crucial role microvesicle shedding release, similar process previously described autophagosome-mediated exit without lysis (AWOL) during poliovirus replication. Through use this provides more dynamic information static fluorescent images, we hope gain better understanding tropism, reorganization, virus-associated host.