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

Effect of bafilomycin on cultured cells before and after VZV infection. BAF can cause toxicity to cultured cells. Many different concentrations of BAF have been used by virologists in the past 25 years (Table 1) . When we reviewed each of the papers listed in Table 1 , we found that concentrations equal to or less than 30 nM were commonly selected for experiments longer than a few hours' duration, whereas higher BAF concentrations for shorter intervals were selected for studies aimed solely at inhibiting viral entry. Because we were not studying VZV entry, we tested the toxic effect of 10 nM BAF on cultured MRC monolayers. BAF was added when the monolayers were approx-imately 90% confluent. We purposely chose monolayers that were less than confluent because VZV infections are typically carried out in such monolayers (18) . The compound was left in the medium for either 24 h or 48 h. Then, the monolayers were visualized by microscopy and photographed. When they were compared with an untreated control monolayer, it was apparent that addition of BAF to the medium inhibited the MRC5 monolayer from reaching confluence ( Fig. 1A to C). We also enumerated the cell populations by trypan blue exclusion. Addition of BAF for 24 h decreased the cell count by 35% compared with a control monolayer; after removal of BAF, the cell count remained the same 24 h later. Addition of BAF for 48 h decreased the cell count by 53% compared with a control monolayer; after removal of BAF, the cell count decreased another 4% over the next 24 h.