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

Observations on bafilomycin-treated uninfected cells. In an extensive search of the literature, we found very few transmission electron micrographs of BAF-treated BAF was added at either 48 hpi or 24 hpi, and the cultures were titrated at 72 hpi. Statistical significance was tested by paired t tests and P values. Since no plaques were observed when BAF was added at 24 hpi, there is a green bar immediately above the zero bar. (B) One representative well (1:10 dilution) from the plaque assay to titrate VZV from untreated cultures. Plaques are outlined by white circles. (C) One representative well (1:10 dilution) from the plaque assay to titrate VZV from a monolayer treated with BAF for 48 h; no plaques were seen. monolayers that were not further subjected to infection or other stress factors. Therefore, we performed a detailed comparative analysis of BAF-treated uninfected MRC5 monolayers (Fig. 6 ). BAF treatment led to disorganization and shortening of the Golgi cisternae, often with no discernible cis or trans face (Fig. 6A to D) . Large clusters of vesicles often surrounded the remnants of the cisternae. We observed a markedly increased number of distinctive vacuoles in BAF-treated monolayers, namely, multivesicular bodies (MVBs), which were not readily detectable in the untreated monolayers. MVBs closely resembled in morphology the late endosome/MVB fraction described in an earlier paper using BAF (23) . Some MVBs contained over 50 vesicles in a twodimensional (2D) slice (Fig. 6C) . Some investigators have also used the term "amphisome" to describe a larger MVB filled with cargo (24, 25) . We also included examples of Golgi apparatuses in uninfected and untreated cells (Fig. 6E and F) . Tubular structures typical of the TGN were seen in one micrograph (Fig. 6F) . Numerous vesicles were visible near the trans-Golgi network, but few MVBs were seen.