Autophagy Quantification and STAT3 Expression in a Human Skin Organ Culture Model for Innate Immunity to Herpes Zoster

Abstract

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.

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

“…We have reproduced these results both in infected cell cultures and in infected human skin xenografts within the severe combined immunodeficient (SCID) mouse model for varicella (8, 9). More recently, these results have been duplicated in a human skin organ culture model for herpes zoster infection (10). In another set of experiments, we documented autophagic flux in VZV-infected cells (9).…”

“…Cells exhibit basal levels of autophagy even during herpesvirus infection (13). However, the levels of autophagy induced by VZV infection in both (i) human skin during the VZV disease called herpes zoster (shingles) and (ii) human skin explants in either the skin organ culture model or the severe combined immunodeficient mouse model of VZV infection are far above basal levels (10). Further, we found that inhibition of autophagy diminishes VZV cell-to-cell spread and infectivity (8).…”

The Role of Autophagy in Varicella Zoster Virus Infection

“…Furthermore, HeLa cells transfected with VZV glycoproteins presented with an enlarged ER and increased LC3-punctae formation [45]. Importantly, these results, obtained in cell cultures, have been confirmed and extended to human skin biopsies, human skin organ cultures and the severe combined immunodeficiency mouse model transplanted with human skin [45,48,51]. Observations in human skin further add to the biological relevance of the findings, illustrating autophagy to be a factor involved in disease pathogenesis, as confirmed by studies in human skin organ cultures, in which a significant increase in autophagic vesicles upon VZV infection was demonstrated [51].…”

Inhibition of ULK1 and Beclin1 by an α-herpesvirus Akt-like Ser/Thr kinase limits autophagy to stimulate virus replication

“…Us3-like kinases may allow viruses lacking ICP34.5 to limit autophagy. Not all α-herpesviruses,however, limit autophagy, as autophagy is induced in cells infected with varicella-zoster virus (55,56). The overall extent to which the substrate specificity of other α-herpesvirus Us3 homologs overlaps with HSV-1 Us3, however, remains to be defined and could be a defining determinant that limits autophagy.…”