Coxsackievirus B exits the host cell in shed microvesicles displaying autophagosomal markers.

Scott M Robinson,Christopher T Cornell,J Lindsay Whitton,M Richard Sayen,Vrushali Mangale,Shahad Rahawi,Ginger Tsueng,Casey Cruz,Ralph Feuer,Jon Sin,Kelly S Doran,Anca M Segall,Roberta A Gottlieb,Roland Wolkowicz,Brett J Hilton,David P Nguyen,Laura L Mcintyre,Nelson Kha,Bryan Hancock ,William V Williams

doi:10.1371/journal.ppat.1004045

Scott M Robinson, Christopher T Cornell + Show 18 more

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https://doi.org/10.1371/journal.ppat.1004045

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Abstract

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.

Highlights

Enteroviruses (EV) are among the most common and medicallyimportant human pathogens, and a frequent cause of central nervous system (CNS) disease [1]
Lasting consequences have be observed in the CNS following Coxsackievirus B3 (CVB3) infection [18], and neural progenitor and stem cells (NPSCs) may represent a site of virus persistence in surviving mice infected shortly after birth [19] [20]
Following the infection of HeLa RW cells with Timer-CVB3 virus stock, ‘‘fluorescent timer’’ protein expression was observed by fluorescence microscopy

Summary

Introduction

Enteroviruses (EV) are among the most common and medicallyimportant human pathogens, and a frequent cause of central nervous system (CNS) disease [1]. Coxsackieviruses (CV), members of the enterovirus genus, are significant human pathogens, and the neonatal central nervous system (CNS) and heart are major targets for infection. Infants infected with CV have been shown to be extremely susceptible to myocarditis, meningitis and encephalitis with a subsequent mortality rate as high as 10%. Severe demyelinating diseases may occur following infection, including acute disseminated encephalomyelitis [5] and acute transverse myelitis [6]. We have shown that CVB3 preferentially targets neural progenitor and stem cells (NPSCs) in the CNS [12] [13] [14] [15] [16] [17]. CVB3 can infect the bone marrow and reduce hematopoietic progenitor cell populations [21]

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