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

Date Published: April 10, 2014

Publisher: Public Library of Science

Author(s): Scott M. Robinson, Ginger Tsueng, Jon Sin, Vrushali Mangale, Shahad Rahawi, Laura L. McIntyre, Wesley Williams, Nelson Kha, Casey Cruz, Bryan M. Hancock, David P. Nguyen, M. Richard Sayen, Brett J. Hilton, Kelly S. Doran, Anca M. Segall, Roland Wolkowicz, Christopher T. Cornell, J. Lindsay Whitton, Roberta A. Gottlieb, Ralph Feuer, Ted C. Pierson.


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 with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured “fluorescent timer” protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.

Partial Text

Enteroviruses (EV) are among the most common and medically-important human pathogens, and a frequent cause of central nervous system (CNS) disease [1]. Worldwide distribution of EV infection is revealed by the detection of EV-specific antibodies in the sera of approximately 75% of individuals within developed countries. For example, in 1996, approximately 10–15 million diagnosed cases of EV infection occurred in the US alone [2].

Tracking viral infection may be critical in understanding viral dissemination and pathogenesis. For example, vaccinia virus has been shown to undergo repulsion of superinfection by the early expression of A33 and A36 protein which allows the virus to increase viral spread and maximize the replication rate substantially [46]. Also, human T-lymphotropic virus-1 (HTLV-1) utilizes the virological synapse, a specialized area of cell-cell contact promoting transmission of virus [47]. Much remains to be determined with regards to cell-to-cell spread of CVB3 and virus dissemination within the host [27]. The pancreas is a primary target organ for CVB3 infection in mice, and early viral replication here may seed other organs such as the heart and CNS. For example, transgenic mice expressing the antiviral molecule interferon-γ within the pancreas showed reduced viral titers and substantially less myocarditis [22]. Mice are considered an informative model to evaluate the mechanisms of coxsackievirus pathogenesis, and many of diseases caused by coxsackieviruses have been recapitulated in a mouse model [48]. Importantly, understanding factors contributing to early pancreatic infection may help to reduce viral replication and virus dissemination within the host. Therefore, we engineered the “fluorescent timer” protein into our infectious clone of CVB3 in order to monitor virus dissemination both in culture and in vivo. “Fluorescent timer” protein containing a nuclear localization signal has been previously utilized to track the cytoplasmic accumulation of nuclear proteins within infected cells [49]. However, this unique molecular marker has not been employed to follow the progression of virus infection directly.




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