VZV-32 is a low passage laboratory strain; its genome has been completely sequenced (GenBank DQ479961.1) (Peters et al., 2006). rVZV/34.5 is a recombinant virus, in which herpes simplex virus 1 (HSV1) ICP34.5 gene (F strain) previously cloned into a pSG5 expression vector (Stratagene) was cut out along with its SV40 early promoter and poly A signal and inserted into rVZV-Oka (Cohen and Siedel, 1993). MRC-5 human fibroblast cells were grown on coverslips in six well tissue culture plates in Minimum Essential Medium (MEM; Gibco, Life Technologies) supplemented with 7% fetal bovine serum (FBS), L-glutamine, non-essential amino acids, and penicillin/streptomycin. When monolayers were nearly confluent, they were inoculated with trypsin-dispersed VZV-infected cells at a ratio of one infected cell to eight uninfected cells (Grose and Brunel, 1978).

De-identified foreskins from circumcision procedures were collected at the University of Iowa Hospital under an exempt Institutional Review Board protocol. As described (Taylor and Moffat, 2005; Jarosinski et al., 2018), we prepared the foreskin for culture by sterilizing the outside of the skin with 1 min incubation in 10% providone-iodine solution followed by rinsing in 70% ethanol and MEM. The skin was then divided into 6 mm round pieces with a biopsy punch and placed onto the mesh bottom of tissue culture inserts in a 12 well plate (Corning) containing 1.4 ml of complete MEM supplemented with nystatin and ciprofloxin. Thereafter, the plate was incubated at 32^oC in a humidified incubator with 5% CO₂. We have carried out 9 independent explant procedures.

After 24–48 h in culture, pieces of explanted human skin were inoculated with cell associated VZV. A VZV infected monolayer (25 sq. cm.) at 70–80% cytopathology was trypsin-dispersed, sedimented by low speed centrifugation and resuspended into 1 ml of MEM. Each skin piece was pierced to the depth of 1 mm in multiple sites with a hypodermic needle and then an aliquot (500,000 cells in 200 μl) of VZV-infected cells was injected onto the surface of the epidermis and allowed to penetrate into the holes created by piercing the skin surface. Infected skin explants were then incubated for as long as 28 days. Mock infections were handled similarly except for inoculation with uninfected cells. Preparation of the skin samples for cryosectioning and subsequent immunohistochemistry (IHC) protocols have been described in detail (Jarosinski et al., 2018).

Slides containing the skin samples were prepared for confocal microscopy by published methods (Carpenter et al., 2008). The primary antibody (1:1000) was added for 2 h at ambient temperature and overnight at 4^oC; the secondary antibody (1:1250) and Hoechst 33342 DNA stain (1:1000) were added for 2 h. All samples were viewed on a Zeiss 710 confocal fluorescent microscope. Murine monoclonal antibody (MAb) 3B3 against VZV gE, MAb 233 against gC and MAb 251D9 against the capsid protein were produced in this laboratory (Grose et al., 1983; Friedrichs and Grose, 1986; Storlie et al., 2008); rabbit antibody against microtubule-associated protein light chain 3 (LC3) was obtained from Santa Cruz (sc-28266) and mouse MAb against STAT3 was obtained from ThermoFisher Scientific (13-7000). Rabbit monospecific antibody against HSV1 ICP34.5 was given to us by Dr. Ian Mohr (New York University).

Quantitation of autophagosomes by visual inspection of 2D confocal micrographs is highly specific when combined with appropriate controls (Klionsky et al., 2007; Carpenter et al., 2011). Quantitation has been further improved by the Imaris software program (BitPlane), which converts z-stacks of confocal images into 3D animations; the Spot function within Imaris software locates and enumerates autophagosomes within each 3D animation based on size and intensity threshold (Jackson et al., 2013; Lamb et al., 2017). We examined at least 3 separate images from each experimental group in order to determine the number of autophagosomes per cell in each image. The standard error of the mean (SEM) across each set of images was used to generate error bars. The P values were determined by unpaired, two-tailed Student’s t tests, using GraphPad Prism software.

A major difference between VZV infection of cultured cells and human skin biopsies during herpes zoster is that mature viral particles are produced in human skin in abundance whereas most viral particles produced in cell culture are aberrant (Tournier et al., 1957; Storlie et al., 2008; Carpenter et al., 2009). A representative example of a VZV-infected SOC is shown in Figure 1. In this figure, the cells were prepared for IHC and labeled with an anti-capsid antibody because the nuclei at 14 dpi are filled with capsids. Cell-to-cell virus spread within the epidermis is designated by arrows (Figure 1A); higher magnifications of the capsid-labeled infected cells within the SOC are shown in panels B and C.

A marker of the late phase of the alpha herpesvirus infectious cycle is the true late protein gC (Honess and Roizman, 1974). Expression of VZV gC is sparse in cultured cells but plentiful in human skin during herpes zoster (Storlie et al., 2008). Therefore, we investigated the appearance of the gC protein in the VZV infected SOC by confocal microscopy. As seen in the 3D images of the infected SOC, gC expression was easily detected along the linear piercing sites in the skin explant created with the syringe needle (Figure 2). As demonstrated by 6 different orientations of the Imaris animation (panels A–F), VZV infection had spread cell-to cell via a piercing site into the epidermis by 14 dpi and then outward from this site. This outward spread into the epidermis was not uniform, rather the spread was meandering.

We previously detected elevated levels of the IL-6 protein in media overlying infected SOC (Jarosinski et al., 2018). A recent report has provided evidence that IL-6 can act as a pro-autophagy cytokine within the IL-6-STAT3 pathway (Xue et al., 2016). Therefore, we looked for evidence of STAT3 expression in the VZV infected SOC. We observed STAT3 immunoreactivity in the VZV-infected skin, in particular, STAT3 expression was found in bystander cells in the near vicinity of an infectious focus as well as within numerous cells of an infectious focus (Figures 3A–H). In uninfected skin, only a few randomly distributed STAT3-immunoreactive cells were seen, while no foci of reactivity were observed with the anti-VZV gE antibody (Figures 3I,J). The lower panel K represents a landscape image of another infectious focus at a higher magnification (Figure 3K).

We also carried out a series of confocal microscopy experiments on the VZV infected SOC, in order to investigate the degree of autophagy (Figure 4). We first examined a mock-infected SOC during each of 6 experiments and observed a minimal number of autophagosomes (Figure 4A). After examination of numerous confocal images of VZV infected SOC, we concluded that we had visualized a very robust autophagy response. Measurement of changes in autophagy by enumeration of LC3-positive puncta is one of the most specific assays, especially when combined with analysis by the Imaris software program, which creates a 3D rendering of each z-stack of confocal micrographs as described in Methods. It was apparent that the maximal autophagy response in each cell within VZV-infected human skin was statistically greater than that seen in mock-infected skin (p = 0.0003) (Figure 4B). As expected, the number of autophagosomes counted within a 3D image of an infected cell was also greater than the number counted in a single 2D image (Figures 4C,D). Finally, we selected two images created by the Imaris animation. There were 3,740 autophagosomes detectable in the cytoplasm above the infectious focus when we set the estimated XY diameter of an autophagosome at 500 nm (Figure 4), much higher when performed on 3D renderings of cells rather than on 2D images seen in a confocal micrograph.

After our analyses in SOC described previously as well as the above new data (Jarosinski et al., 2018), we carried out an experiment with a recombinant VZV genome containing the HSV ICP34.5 neurovirulence gene. In our earlier publication, we showed that infection of SOC with wild type VZV closely followed the SCID/human skin model: namely, VZV infection progressed and achieved near maximal distribution by 14 dpi. Although ICP34.5 contributes to HSV neurovirulence in the brain by attaching to Beclin-1 and suppressing autophagy (Orvedahl et al., 2007), the role of ICP34.5 in HSV1-infected non-neuronal tissues has been a subject of debate in the HSV1 literature (Yordy and Iwasaki, 2013). We propose that repeating similar experiments in a VZV/SOC system is an appropriate control in which to explore the effects of ICP34.5 on autophagy outside of the neuronal system. To this end, the ICP 34.5 gene was cloned into the varicella vaccine genome. We first documented that the ICP34.5 protein was expressed by immunolabeling the protein after rVZV/34.5 infection (Figure 5A). As in the SCID model of VZV infection, spread of varicella vaccine virus occurred more slowly in SOC than wild-type VZV (Moffat et al., 1998); at 14 dpi, there were only small foci of infection with the rVZV/34.5 virus (Figure 5C).

Thereafter, we examined the infected SOC culture at 28 dpi (Figures 5D–H). Time points beyond 21 dpi were not examined often in the SCID model. But at 28 dpi in SOC, we observed that the foci of infection with the rVZV/34.5 virus had enlarged and were similar in size to foci with wild type virus at 14 dpi (compare Figures 4, 5). Since wild type virus infection does not progress greatly between 14 and 28 dpi, the foci in a control experiment with wild type virus were similar in size to that of rVZV/34.5 at 28 dpi. Then, we obtained images from 6 individual infectious foci in order to quantitate puncta by confocal microscopy combined with Imaris software. Since the samples were labeled with antibody to VZV gE, the predominant VZV glycoprotein, we documented the extensive production of VZV gE throughout the cytoplasm of the infected SOC (Figures 5E–H). When we enumerated the puncta within individual infected cells by the Imaris Spot program, we found no significant difference in puncta per infected cell when wild-type and rVZV/34.5 infection were compared (Figure 5D; p = 0.3). We have previously shown that autophagy and LC3 production were not diminished in cells infected with vaccine virus (Buckingham et al., 2016). This further experiment documented that ICP34.5 when expressed within a VZV genome did not limit autophagosome production in human skin tissue.