Varicella-Zoster Virus Infection Induces Autophagy in both Cultured Cells and Human Skin Vesicles

Abstract

When grown in cultured cells, varicella-zoster virus (VZV) forms many aberrant light particles and produces low titers. Various studies have explored the reasons for such a phenotype and have pointed to impaired expression of specific late genes and at lysosomal targeting of egressing virions as possible causes. In the studies presented here, we report that the autophagic degradation pathway was induced at late time points after VZV infection of cultured cells, as documented by immunoblot analysis of the cellular proteins LC3B and p62/SQSTM1, along with electron microscopy analysis, which demonstrated the presence of both early autophagosomes and late autophagic compartments. Autophagy was induced in infected cells even in the presence of phosphonoacetic acid, an inhibitor of viral late gene expression, thus suggesting that accumulation of immediate-early and early viral gene products might be the major stimulus for its induction. We also showed that the autophagic response was not dependent on a specific cell substrate, virus strain, or type of inoculum. Finally, using immunofluorescence imaging, we demonstrated autophagosome-specific staining in human zoster vesicles but not in normal skin. Thus, our results document that this innate immune response pathway is a component of the VZV infectious cycle in both cultured cells and the human skin vesicle, the final site of virion formation in the infected human host.

Autophagosome Formation during Varicella-Zoster Virus Infection following Endoplasmic Reticulum Stress and the Unfolded Protein Response

“…Thus, we have greatly extended earlier findings in which we detected autophagosomes in a biopsy sample from one case of human zoster (47). In other words, we have demonstrated that autophagy occurs during both primary VZV infection and VZV reactivation within the skin vesicle, the final site of virion assembly and envelopment.…”

“…Effects of tunicamycin on autophagy in uninfected and VZVinfected cells. We previously described a prominent punctate LC3B staining pattern in VZV-infected MRC-5 cells compared with a diffuse pattern in uninfected cells (47). Here we show that the punctate pattern of LC3B immunoreactivity in VZV-infected MRC-5 cells was virtually identical to the punctate pattern seen in uninfected MRC-5 cells treated with tunicamycin ( Fig.…”

“…VZV lacks an ICP34.5-homologous protein (10). Therefore, it was of interest when we showed that autophagy is a prominent feature in cultured cells infected with VZV, as demonstrated by immunoblotting for the LC3B marker protein as well as the autophagy adaptor p62/SQSTM1 (47). Further, we observed numerous cytoplasmic organelles with distinctive double outer membranes characteristic of autophagosomes within electron microscopic images of the same VZV-infected cells.…”

Autophagic flux without a block differentiates varicella-zoster virus infection from herpes simplex virus infection

“…We had previously investigated VZV autophagic flux by measurement of stress-inducible intracellular p62/SQSTM1 protein levels in infected cells, and showed that VZV-induced autophagy was completed, leading to the degradation of p62/ SQSTM1 (15). To confirm and extend our observations, we measured autophagic flux by a traditional pulse-chase assay using radiolabeled amino acids.…”

“…In early experiments, we documented the presence of autophagosomes in skin vesicles from immunocompetent children with varicella or adults with herpes zoster (15,16). To confirm that the autophagy seen in the VZV-infected skin xenografts in SCID mice, which lack adaptive immune responses, recapitulated the response seen in human skin vesicles from children with varicella, we performed a direct comparison.…”