Fig 1.
hTert immortalized human choroid plexus epithelial cells support JCPyV infection.
(A) Choroid plexus epithelial cells immortalized by serial infection with hTert lentivirus are morphologically similar to primary choroid plexus epithelial cells and express transthyretin (green; DAPI in blue). (B) Quantification of virus infection in primary and immortalized CPE cells compared to infection of the human glial cell line, SVG-A based on expression of the late viral protein VP1. VP1 expression was normalized relative to the infection in SVG-A control cells and set to 1. (C) Indirect immunofluorescent analysis of VP1 positive cells in infected cultures at 5 days post infection (VP1, green; DAPI, blue).
Fig 2.
Extracellular vesicles concentrated from CPEP and CPEL cells display similar characteristics and express extracellular vesicle markers.
(A) Nanoparticle tracking analysis of extracellular vesicles (EV) produced by primary and immortalized choroid plexus epithelial cells. (B) EV size and distribution is similar between CPEP and CPEL cells. (C) Extracellular vesicles concentrated from CPEP and CPEL cells by differential centrifugation express the characteristic EV markers Annexin V, TSG101, Flotillin-1, and CD9. Markers of contaminating cellular organelles (cytochrome c, calnexin, GM130) were detected in whole cell lysates but not in EV fractions.
Fig 3.
JCPyV associates with vesicles from infected CPEP and CPEL cells.
(A) Nanoparticle tracking analysis of extracellular vesicles produced by JCPyV infected primary and immortalized CPE cells show similar vesicle size and distribution, averaging 150-200nm (B) EV yield from infected CPE cultures as quantified by NTA. (C) EV from infected CPEP and CPEL cells express characteristic EV markers and the major viral capsid protein VP1 is found in the EV fraction. (D) Transmission electron microscopy of EV from infected CPE cells shows viral particles bound to the outside of EV and enclosed within EV.
Fig 4.
Extracellular vesicles from JCPyV infected CPEP and CPEL cells readily transmit the infection to SVG-A cells and to primary human astrocytes but not to HEK293T cells.
(A) CPEP EVJC+, CPEL EVJC+, and purified JCPyV were used to infect SVG-A, primary human astrocyte, and HEK293T cells. VP1 was quantified by indirect immunofluorescent analysis at 4 days post infection. Infection is normalized relative to the infection in SVG-A control cells which was set to 1. (B) Quantification of protected viral genomes in CPE origin EVJC+. Infectious EV preparations were analyzed by qPCR for total protected viral genome content.
Fig 5.
EV-mediated infection of glial cells is not sensitive to treatment with neuraminidase and is not blocked by neutralizing antibodies.
(A) Neuraminidase treatment (+NA) of SVG-A cells reduces infection by purified virus (CsCl JCPyV) but has no effect on EV mediated infection (EVJC+). (B) Uptake of PKH67 labeled vesicles is not significantly reduced on neuraminidase (+NA) treated SVG-A cells compared to untreated cells (UT). (C) Titration of NA treatment on SVGA cells showed a dose dependent decrease in purified JCPyV-488 binding as NA concentration is increased. (D) Pretreatment with anti-JC blocking antibody inhibits CsCl JCPyV infection but has no effect on EVJC+ mediated infection in SVG-A cells. (E) Pretreatment with anti-JC blocking antibody does not inhibit PKH67 labeled EV internalization. (F) Titration of the anti-JCPyV antisera used to inhibit virus infection in panels D and E. * = p < 0.05.
Fig 6.
EV mediated infection of SVG-A cells with externally associated virus can be blocked with anti-JCPyV antisera.
(A) EVJC+ from infected CPEL cells were concentrated by differential ultracentrifugation and purified over a qEV column. Western blot analysis of each fraction showed that the viral protein VP1 and the EV marker CD9 co-purifed in fraction 7. (B) EVs from uninfected CPEL cells were concentrated by differential ultracentrifugation and then spiked with 10e9 copies of purified JCPyV particles. This mixture was then subjected to fractionation over the qEV column and analyzed by western blot for CD9 and VP1. VP1 and CD9 co-purifed in fractions 7 and 8. (C) Fraction 7 from each was used to infect neuraminidase treated SVG-A cells. Neuraminidase only inhibited infection by purified JCPyV and had no effect on the transmission of virus by EV isolated from infected cells or from EV isolated from uninfected cells and spiked with purified virus. (D) Anti-JCPyV antisera (Anti-JC) but not preimmune sera (PI) inhibited infection by purified JCPyV and by EV isolated from uninfected cells and subsequently spiked with purified virus. Anti-JC had no effect on the transmission of virus from EV isolated from infected CPEL cells (EVJC+). * = p < 0.01.
Fig 7.
Infectious entry of EV isolated from infected CPE cells occurs by both clathrin dependent and independent mechanisms.
(A) Pretreatment of SVG-A cells with the macropinocytosis inhibitor, EIPA. EIPA targets sodium/proton exchangers and effectively inhibits the uptake of labeled dextran (control) and labeled extracellular vesicles isolated from virus infected CPEP (CPEP EVJC+) and CPEL (CPEL EVJC+) cells into SVG-A cells. The inhibition of uptake is partially reversible by removing the drug and allowing the cells time to recover (Rescue) (B) Pretreatment of SVG-A cells with the clathrin mediated endocytosis inhibitor Pitstop2 effectively inhibits uptake of both purified JCPyV virus (control) and labeled extracellular vesicles isolated from virus infected CPEP (CPEP EVJC+) and CPEL (CPEL EVJC+) cells into SVG-A cells. The inhibition of uptake is partially reversible by removing the drug and allowing the cells time to recover (Rescue) (C) EIPA treatment of SVG-A cells reduces infection by purified virus and by infectious EV isolated from CPEP cells but not from CPEL cells. (D) Pitstop treatment of SVG-A cells reduces infection by purified virus and by infectious EV isolated from CPEP and CPEL cells. * p < 0.05; ** p < 0.01.
Fig 8.
Model of JCPyV trafficking in EV to the CNS via the choroid plexus.
Left panel: Hematoxylin and Eosin [H&E] stain of normal human choroid plexus and brain parenchyma; magnification: 200x. Right panel: Proposed model of JCPyV trafficking to the brain via the choroid plexus. JCPyV (either free virions or enclosed in vesicles) enters the stroma from the bloodstream through fenestrated capillaries to infect the choroid plexus epithelium. Infected CPE packages JCPyV into vesicles and secretes these EVs into the CSF, which subsequently enter the brain parenchyma and infect target glial cells.