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

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.

Protein composition of the hepatitis A virus quasi-envelope

“…Recent studies have brought increasing recognition to the fact that multiple members of the Picornaviridae are released from cells enclosed in membranous vesicles in the absence of cell lysis (2)(3)(4)(5)(6). This mode of cellular egress has been suggested to play an important role in disease pathogenesis by masking viral antigens from the immune system (2) or increasing the number of viral genomes delivered to single infected cells (6).…”

“…Poliovirus and other enteroviruses appear to egress nonlytically from infected cells in autophagosome-derived vesicles containing LC3-II (4,5,28). These enteroviral vesicles are much larger than eHAV, and contain a much greater number of viral capsids than eHAV vesicles (2,6).…”

“…Coxsackievirus B, poliovirus, and many other members of the genus Enterovirus typically cause lytic infections in cultured cells. However, viruses in this genus are also released within extracellular vesicles before cell lysis, often with many capsids packaged within a single vesicle (4)(5)(6). The cellular egress of these classically nonenveloped viruses in membrane-bound vesicles has blurred the distinction between enveloped and nonenveloped viruses and has important implications for pathogenesis (2).…”

“…Although the size and density of eHAV vesicles are consistent with an exosome-like origin in multivesicular bodies (MVBs) (2, 3), eHAV biogenesis remains incompletely defined and the protein composition of the membranes surrounding the quasi-enveloped capsid is unknown. In contrast, the extracellular vesicles involved in egress of enteroviruses have been suggested to be derived from autophagosomes, as they contain lipidated LC3-II, a marker of autophagy, and disrupting autophagy blocks their release (4,6). However, the protein composition of these extracellular vesicles also has yet to be comprehensively investigated.…”

The autophagic machinery ensures nonlytic transmission of mycobacteria

“…Even though no molecular mechanism has been proposed, autophagosome interactions with the plasma membrane have been suggested to allow the release of virus particles (32) and the cell-to-cell transmission of Brucella (2). Recently, using a fluorescent timer approach, it was shown that the autophagic pathway contributes to the shedding of microvesicles that harbor infectious coxsackievirusB and contribute to the spreading of the virus from cell to cell (33).…”