Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways

Abstract

Varicella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (∼100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome.IMPORTANCE VZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an intersection of viral exocytosis and autophagy pathways. Specifically, both LC3-II and Rab11 proteins copurified with some infectious VZV particles. The results suggested that a subpopulation of VZV particles were carried to the cell surface in single-walled vesicles with attributes of an amphisome, an organelle formed from the fusion of an endosome and an autophagosome. Our results also addressed the interpretation of autophagy/xenophagy results with mutated herpes simplex virus lacking its ICP34.5 neurovirulence gene (HSVΔ34.5). The VZV genome lacks an ICP34.5 ortholog, yet we found no evidence of VZV particles housed in a double-membraned autophagosome. In other words, xenophagy, a degradative process documented after infection with HSVΔ34.5, was not observed in VZV-infected cells.

Localization of VZV in saliva of zoster patients

“…Even though it suggests that this subject may be showing some type of persistent, asymptomatic infection, with the positive staining seen by confocal microscopy one would assume that this would be detectable by PCR. Although there is strong evidence to the specificity of the antibody used for VZV with no cross‐reactivity to other viruses, this does not eliminate the possibility of cross‐reactivity with other viruses which may provide an explanation of the results obtained. Some studies have even suggested non‐specific staining of VZV by confocal microscopy in dying cells (Chuck Grose, personal communication).…”

Infectious Bursal Disease Virus Subverts Autophagic Vacuoles To Promote Viral Maturation and Release

“…Taken together, the results show that the autophagic endosomal pathway is one mechanism that facilitates IBDV maturation, release, and reinternalization. A recent study claims that a subpopulation varicella-zoster virus particles are sorted into a vesicle resembling an amphisome for their final trafficking to the outer cell membrane (33). Overall, these researches suggest that highly divergent DNA and RNA viruses may share similar exit pathways.…”

“…However, the mechanism of viral maturation and release needs further investigation. A recent study indicated that exocytosis of varicella-zoster virus virions involves a convergence of endosomal and autophagy pathways (33). The investigators demonstrated that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B).…”

ORF7 of Varicella-Zoster Virus Is Required for Viral Cytoplasmic Envelopment in Differentiated Neuronal Cells

“…A typical VZV replication cycle consists of the following steps: virus entry and uncoating, viral gene expression and genome replication, nucleocapsid assembly and egress from nucleus to cytoplasm, virion assembly in the Golgi apparatus, and virion release and transcellular transmission from host cell to neighboring cells (8)(9)(10). The VZV genome encodes at least 20 tegument proteins with regulatory function or tegumentation (11).…”