Progeny Varicella-Zoster Virus Capsids Exit the Nucleus but Never Undergo Secondary Envelopment during Autophagic Flux Inhibition by Bafilomycin A1

Girsch, James H.; Walters, Katherine; Jackson, Wallen; Grose, Charles

Confocal microscopy. Cells were plated on glass coverslips in 6-well tissue culture plates and incubated at 37°C until they were 90% confluent. The cells were inoculated with VZV-infected cells at a ratio of 1 infected cell to 8 uninfected cells. The infected cells were treated with 10 nM bafilomycin at 24 or 48 hpi. At 72 hpi, the cells were fixed with 2% paraformaldehyde with 0.02% Triton X-100 at room temperature for 1 h. The fixed cells were washed 5 times for 5 min each time in PBS and blocked for 30 min in PBS with 5% nonfat dry milk. The coverslips were incubated in primary antibodies diluted 1:2,000 in PBS-1% milk for 1 h at room temperature and washed 5 times for 5 min each time in PBS. The coverslips were then incubated in goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 546, washed 5 times for 5 min each time, and mounted on glass slides. Images were collected on a Zeiss 710 laser scanning confocal microscope (6) . Z-stacks of 2D images were converted into 3D images with Imaris software (Oxford Instruments).