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

Abstract

Varicella-zoster virus (VZV) is an alphaherpesvirus that lacks the herpesviral neurovirulence protein ICP34.5. The underlying hypothesis of this project was that inhibitors of autophagy reduce VZV infectivity. We selected the vacuolar proton ATPase inhibitor bafilomycin A1 for analysis because of its well-known antiautophagy property of impeding acidification during the late stage of autophagic flux. We documented that bafilomycin treatment from 48 to 72 h postinfection lowered VZV titers substantially (P ≤ 0.008). Because we were unable to define the site of the block in the infectious cycle by confocal microscopy, we turned to electron microscopy. Capsids were observed in the nucleus, in the perinuclear space, and in the cytoplasm adjacent to Golgi apparatus vesicles. Many of the capsids had an aberrant appearance, as has been observed previously in infections not treated with bafilomycin. In contrast to prior untreated infections, however, secondary envelopment of capsids was not seen in the trans-Golgi network, nor were prototypical enveloped particles with capsids (virions) seen in cytoplasmic vesicles after bafilomycin treatment. Instead, multiple particles with varying diameters without capsids (light particles) were seen in large virus assembly compartments near the disorganized Golgi apparatus. Bafilomycin treatment also led to increased numbers of multivesicular bodies in the cytoplasm, some of which contained remnants of the Golgi apparatus. In summary, we have defined a previously unrecognized property of bafilomycin whereby it disrupted the site of secondary envelopment of VZV capsids by altering the pH of the trans-Golgi network and thereby preventing the correct formation of virus assembly compartments. IMPORTANCE This study of VZV assembly in the presence of bafilomycin A1 emphasizes the importance of the Golgi apparatus/trans-Golgi network as a platform in the alphaherpesvirus life cycle. We have previously shown that VZV induces levels of autophagy far above the basal levels of autophagy in human skin, a major site of VZV assembly. The current study documented that bafilomycin treatment led to impaired assembly of VZV capsids after primary envelopment/de-envelopment but before secondary reenvelopment. This VZV study also complemented prior herpes simplex virus 1 and pseudorabies virus studies investigating two other inhibitors of endoplasmic reticulum (ER)/Golgi apparatus function: brefeldin A and monensin. Studies with porcine herpesvirus demonstrated that primary enveloped particles accumulated in the perinuclear space in the presence of brefeldin A, while studies with herpes simplex virus 1 documented an impaired secondary assembly of enveloped viral particles in the presence of monensin.

Exocytosis of Progeny Infectious Varicella-Zoster Virus Particles via a Mannose-6-Phosphate Receptor Pathway without Xenophagy following Secondary Envelopment

“…At this point, we reexamined our large archive of electron micrographs from past experiments and confirmed that even one or two naked capsids within a single micrograph were very difficult to find in the cytoplasm of VZV-infected MRC-5 or human melanoma cells (29). Further, we noted in our prior paper that naked capsids were sometimes in the cytoplasm of BAF-treated MRC-5 cells, often surrounding but not within vacuolar compartments (8). However, the number was fewer than that seen in VZV-infected Pompe cells.…”

“…In one set of experiments, we examined VZV-infected cells by electron microscopy at early time points of 15 min, 8 h, 20 h, and 30 h after infection to document the minimal presence of inoculum virus. Therefore, we have an archive of over 1,000 electron micrographs, some on photographic paper and, more recently, in digital format (8). For this new research project, we observed an additional 483 digital electron micrographs of VZV-infected cells.…”

“…The inoculum was layered over an existent Pompe cell monolayer; uninfected Pompe cells were not cocultivated with infected MRC-5 cells. Some cultures were incubated in the presence of the autophagy inhibitor BAF (Sigma; B1793), as described previously (8). Altogether, over 30 experiments have been carried out in Pompe cells.…”

“…Many investigators have selected various inhibitors of autophagy to gauge whether decreased autophagy has an effect on virus assembly (6). For example, we recently reported that the inhibitor of autophagic flux bafilomycin A1 (BAF) inhibited VZV secondary envelopment, confirming and expanding an earlier herpes simplex virus 1 (HSV1) observation (7,8). Although widely used, inhibitors may exert unintended or unknown effects on cellular pathways that alter virus assembly (9)(10)(11)(12).…”

“…Recently, investigators have discovered a deficiency in autophagic flux in Pompe disease (15,16). For our virology studies, we selected the alphaherpesvirus VZV as the test pathogen because of related prior publications (8,17). When we infected cells obtained from a child with Pompe disease, we observed during the first experiments that VZV infection and assembly were markedly decreased in Pompe cells compared with VZV infection and assembly in common cell substrates.…”