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. 2020 Mar 31;94(8):e01957-19. doi: 10.1128/JVI.01957-19

FIG 2.

FIG 2

H129 viral particles are transported mainly in the form of a capsid in cortical neuron axon by live-imaging analysis. (A) (a1) Schematic of recombinant H129 genome and viral particles; VP26 fused with mCherry and gD fused with green fluorescent protein (GFP), H129-G/R. (a2) EM images for H129-G/R, dually labeled H129 virions. Cell-free viruses in the supernatant were collected at 24 hpi and then processed for immunoEM analysis. Ultrathin sections were probed with antibodies against mCherry or GFP, followed by colloidal gold-conjugated secondary antibodies. Shown are representative images for VP26-mCherry (10 nm gold, left) or gD-GFP (5 nm gold, right) stained on complete virions. Scale bar: 100 nm. (a3) Colocalization of VP26-mCherry, gD-GFP, and gB. Neurons cultured for 14 days were infected with H129-G/R for 24 h and then fixed with 4% paraformaldehyde and immunostained with mouse-anti-gB. Shown are progeny viral particles in neurites. Arrows indicate VP26-mCherry (red) colocalized with both gD-GFP (green) and gB (blue). (B) (b1) The schematic of the microfluidic chamber and culture and infection of cortical neurons in the microfluidic chamber. (b2) Neurons were cultured for 14 days and then infected by adding H129-G/R into the soma (blue) side at an MOI of 1. Movement of viral particles in infected neuron axons was observed by live-cell imaging from 12 hpi to 24 hpi. (b3) Frames of capsid (white arrow) and enveloped capsid (purple arrow) were captured by continuous 1.5-s sequential exposures. Example time-lapse series of a single progeny capsid moving toward axon terminal are shown. Scale bar: 5 μm. (b4) Analysis of the moving viral particles in axons. The numbers of moving unenveloped and enveloped capsids were counted. The proportions of unenveloped and enveloped capsids of the total number of running capsids are shown as mean ± SEM; statistical significance was analyzed by Student’s t test. A total of 591 unenveloped capsids and 38 enveloped capsids were collected from 3 independent experiments. ***, P < 0.001.