Coxsackievirus B exits the host cell in shed microvesicles displaying autophagosomal markers.
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.
The Integrated Regenerative Research Institute (IRRI) at San Diego State University, Cell & Molecular Biology Joint Doctoral Program, Department of Biology, San Diego State University, San Diego, California, United States of America.
Scott M
SM
Robinson
The Integrated Regenerative Research Institute (IRRI) at San Diego State University, Cell & Molecular Biology Joint Doctoral Program, Department of Biology, San Diego State University, San Diego, California, United States of America.
Ginger
G
Tsueng
Donald P. Shiley BioScience Center, San Diego State University, San Diego, California, United States of America.
Jon
J
Sin
The Integrated Regenerative Research Institute (IRRI) at San Diego State University, Cell & Molecular Biology Joint Doctoral Program, Department of Biology, San Diego State University, San Diego, California, United States of America.
Vrushali
V
Mangale
The Integrated Regenerative Research Institute (IRRI) at San Diego State University, Cell & Molecular Biology Joint Doctoral Program, Department of Biology, San Diego State University, San Diego, California, United States of America.
Shahad
S
Rahawi
The Integrated Regenerative Research Institute (IRRI) at San Diego State University, Cell & Molecular Biology Joint Doctoral Program, Department of Biology, San Diego State University, San Diego, California, United States of America.
Laura L
LL
McIntyre
The Integrated Regenerative Research Institute (IRRI) at San Diego State University, Cell & Molecular Biology Joint Doctoral Program, Department of Biology, San Diego State University, San Diego, California, United States of America.
Wesley
W
Williams
The Integrated Regenerative Research Institute (IRRI) at San Diego State University, Cell & Molecular Biology Joint Doctoral Program, Department of Biology, San Diego State University, San Diego, California, United States of America.
Nelson
N
Kha
Department of Biology and Center for Microbial Sciences, San Diego State University, San Diego, California, United States of America.
Casey
C
Cruz
Department of Biology and Center for Microbial Sciences, San Diego State University, San Diego, California, United States of America.
Bryan M
BM
Hancock
The Integrated Regenerative Research Institute (IRRI) at San Diego State University, Cell & Molecular Biology Joint Doctoral Program, Department of Biology, San Diego State University, San Diego, California, United States of America.
David P
DP
Nguyen
Donald P. Shiley BioScience Center, San Diego State University, San Diego, California, United States of America.
M Richard
MR
Sayen
The Integrated Regenerative Research Institute (IRRI) at San Diego State University, Cell & Molecular Biology Joint Doctoral Program, Department of Biology, San Diego State University, San Diego, California, United States of America.
Brett J
BJ
Hilton
Department of Biology and Center for Microbial Sciences, San Diego State University, San Diego, California, United States of America.
Kelly S
KS
Doran
Department of Biology and Center for Microbial Sciences, San Diego State University, San Diego, California, United States of America.
Anca M
AM
Segall
The Integrated Regenerative Research Institute (IRRI) at San Diego State University, Cell & Molecular Biology Joint Doctoral Program, Department of Biology, San Diego State University, San Diego, California, United States of America.
Roland
R
Wolkowicz
Department of Immunology and Microbial Science, SP30-2110, The Scripps Research Institute, La Jolla, California, United States of America.
Christopher T
CT
Cornell
Department of Immunology and Microbial Science, SP30-2110, The Scripps Research Institute, La Jolla, California, United States of America.
J Lindsay
JL
Whitton
Donald P. Shiley BioScience Center, San Diego State University, San Diego, California, United States of America.
Roberta A
RA
Gottlieb
The Integrated Regenerative Research Institute (IRRI) at San Diego State University, Cell & Molecular Biology Joint Doctoral Program, Department of Biology, San Diego State University, San Diego, California, United States of America.
Ralph
R
Feuer
D000818:Animals; D055252:Cell-Derived Microparticles / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D029822:Enterovirus B, Human / Q000502:physiology*; D004769:Enterovirus Infections / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D010588:Phagosomes / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D017201:Virus Shedding / Q000502:physiology*
C500427:3A protein, coxsackievirus B; C477012:MAP1LC3A protein, human; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; D014764:Viral Proteins