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

Next, we assessed the effects of both VZV infection and BAF treatment on the monolayers (Fig. 1D) . BAF was added at either 24 h postinfection (hpi) or 48 hpi; the cultures were photographed at 72 hpi. There was not a notable difference between the appearances of the infected monolayers treated with BAF and the uninfected monolayers treated with BAF; in other words, the cytopathology was primarily due to the BAF treatment rather than the infection. As expected, the cytopathology in both experiments was more extensive after a 48-h incubation with BAF (Fig. 1D ). Of note, the degree of BAF-induced cytopathology was more than anticipated after reading the papers listed in Table 1 . None of the papers in the table mentioned any observable effects on a monolayer treated with a lower concentration of BAF, such as 10 nM. Effects of bafilomycin on LC3-II and VZV gE production. Because of the known property of BAF to inhibit autophagic flux, we predicted that the production of LC3-II would be increased in BAF-treated cultures. This prediction was confirmed ( Fig. 2A) . We also predicted that the biosynthesis of the VZV glycoproteins would be decreased after BAF treatment of infected cultures. To carry out this experiment, we infected 6 75-cm 2 monolayers with equal numbers of infected cells and then treated 2 of the 6 mono- layers with BAF between 48 and 72 hpi and 2 monolayers with BAF between 24 and 72 hpi. Virus was purified by two sequential sedimentations as described in Materials and Methods, after which the three virus bands were analyzed for the predominant viral glycoprotein VZV gE (Fig. 2B ). As predicted, compared with the untreated control infected culture, the relative amount of gE declined in monolayers treated with BAF, especially for 48 h. As a subsequent confirmatory experiment, we examined similarly treated monolayers by confocal microscopy (Fig. 2C to E). As expected, BAF treatment increased the intensity of autophagosomes within a cell while decreasing the intensity of VZV glycoprotein expression within the cell. We have previously shown that levels of VZV gE expression increase for the first 72 h after infection in the absence of BAF (19) . Because we saw less viral glycoprotein expression in the presence of BAF, we also probed for the Golgi apparatus in another experiment. When we examined the Golgi apparatus with a specific antibody to GM130, the Golgi cisternae in BAF-treated infected cells appeared to be more disassembled than the Golgi cisternae in untreated infected cells ( Fig. 2F and G). However, when we constructed a three-dimensional (3D) image of the Golgi apparatus from a z-stack with Imaris software, the Golgi cisternae were disorganized in both treated and untreated infected cells ( Fig. 2F and G). We also produced images of the Golgi apparatus in uninfected cells; the Golgi cisternae labeled with the anti-GM130 antibody were more linear in these nonstressed cells (see Fig. S1 in the supplemental material).