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.

The literature on egress of different herpesviruses after secondary envelopment is contradictory. In this report, we investigated varicella-zoster virus (VZV) egress in a cell line from a child with Pompe Disease, a glycogen storage disease caused by a defect in the enzyme required for glycogen digestion. In Pompe cells, both the late autophagy pathway and the mannose-6-phosphate receptor (M6PR) pathway are interrupted. We have postulated that intact autophagic flux was required for higher recoveries of VZV infectivity. To test that hypothesis, we infected Pompe cells and then assessed the VZV infectious cycle. We discovered that the infectious cycle in Pompe cells was remarkably different from either fibroblast or melanoma cells. No large late endosomes filled with VZV particles were observed in Pompe cells; only individual viral particles in small vacuoles were seen. The distribution of the M6PR pathway (trans Golgi network to late endosome) was constrained in infected Pompe cells. When analyzed with two different anti-M6PR antibodies, extensive co-localization of the major VZV glycoprotein gE (known to contain M6P residues) and the M6PR was documented in the viral highways at the surface of non-Pompe cells after maximum intensity projection of confocal z-stacks, but neither gE nor M6PR was seen in abundance at the surface of infected Pompe cells. Taken together, our results suggested that (i) Pompe cells lacked a VZV trafficking pathway within M6PR-positive large endosomes and (ii) most infectious VZV in conventional cell substrates was transported via large M6PR-positive vacuoles without degradative xenophagy to the plasma membrane. Importance. The long term goal of this research has been to determine why VZV, when grown in cultured cells, invariably is more cell associated with lower titer as compared with other alpha herpes viruses such as herpes simplex virus 1 (HSV1) or pseudorabies virus (PRV). Data from both HSV1 and PRV laboratories have identified a Rab6 secretory pathway for transport of single enveloped viral particles from the trans Golgi network within small vacuoles to the plasma membrane. In contrast, post-secondary envelopment in fibroblast or melanoma cells, multiple infectious VZV particles accumulated within large M6PR-positive late endosomes that were not degraded en route to the plama membrane. We propose that this M6PR pathway is most utilized in VZV infection, least utilized in HSV1 infection, with PRV being closer to HSV1. Supportive data from other VZV, PRV and HSV1 laboratories about evidence for two egress pathways are included in the Discussion.

The goal of this project was to document the autophagy response in human neonatal skin organ culture (SOC) after infection with varicella-zoster virus (VZV). The VZV-infected SOC model has attributes of herpes zoster, in that an injection of virus into the skin is analogous to exit of virus from the sensory nerve termini into skin during herpes zoster. Cultures were maintained for 28 days and periodically examined for an autophagy response by quantitation of autophagosomes with Imaris software. Expression of the STAT3 protein was plentiful in the VZV-infected SOC. Abundant autophagy was observed in VZV-infected SOC between 14 and 28 days after infection, while autophagy in mock-infected SOC was minimal (p = 0.0003). The autophagic response after infection of SOC with a recombinant VZV genome containing the herpes simplex virus ICP34.5 neurovirulence gene was similar to wild-type VZV (p = 0.3). These results suggested that the VZV-infected SOC system resembled biopsy data from herpes zoster infection of skin. An enhanced autophagy response has now been reported after infection with two additional alpha herpesviruses besides VZV, namely, pseudorabies virus and duck enteritis herpes virus; both lack the ICP34.5 protein.

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