Exocytosis of Progeny Infectious Varicella-Zoster Virus Particles via a Mannose-6-Phosphate Receptor Pathway without Xenophagy following Secondary Envelopment

Abstract

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 Role of Autophagy in Varicella Zoster Virus Infection

“…Comparisons of healthy and autophagy-defective cells also indicated the existence of two distinct egress pathways for VZV. Transmission EM analysis showed that in healthy cells, viral capsids seem to accumulate in large vacuoles containing a high number of particles, whereas in the autophagy-defective cells, only small vacuoles, containing a single viral particle each, appear [52]. Importantly, no signs of xenophagy were observed in either the healthy or the deficient cells.…”

“…However, in view of the strong cell-type dependency that has been observed in the context of autophagy, further studies in various different cell or organ cultures are required to better understand this process in greater detail. To date, upregulated autophagy following VZV infection has been described in fibroblasts, mainly MRC-5, but also in primary cells, MeWo cells and keratinocytes [44,52,55]. Furthermore, HeLa cells transfected with VZV glycoproteins presented with an enlarged ER and increased LC3-punctae formation [45].…”

“…Given the apparent absence of autophagy evasion/inhibition of VZV, the question arises as to how the virus escapes the fate of degradation, instead making its way to the plasma membrane to exit the cell. In 2020, Girsch et al [52] were able to demonstrate significantly reduced viral titers in cells defective in autophagy, indicating the role of autophagy in promoting exocytosis of VZV. Further co-localization and co-purification studies demonstrated that the virus can indeed be found in autophagic vesicles.…”

Modulation of Endosome Function, Vesicle Trafficking and Autophagy by Human Herpesviruses

“…On the other hand, VZV utilizes an amalgam of endosomal and autophagosomal pathways to perform exocytosis of complete viral particles, as observed by electron microscopy, which shows virions in single-membraned vesicles carrying both Rab11 and LC3, which resembled amphisomes [ 209 ]. Alternatively, VZV has been shown to have the ability to bind to the mannose-6-phosphate receptor in late endosomes as a mechanism to egress from infected melanoma cells or fibroblasts, regardless of the autophagosome pathway [ 210 ]. A recent report indicates that the VZV glycoprotein M (gM) contains two tyrosine-XX-bulk hydrophobic amino acid domains and one dileucine trafficking-domain associated with trans-Golgi sorting vesicles, which implies that gM is targeted to endosomal-lysosomal compartments to help viral maturation [ 211 ].…”

Viral Interactions with Adaptor-Protein Complexes: A Ubiquitous Trait among Viral Species

“…The γ1AP-1/μ1B also binds, like γ1AP-1/μ1A, to the TGN and it even can substitute γ1AP-1/μ1A in MPR sorting [ 32 ]. Due to these ubiquitous house-keeping functions of MPR300-mediated protein sorting, it is not surprising that several viruses, like HIV-1, VZV, and rotaviruses, bind MPR300 for cell entry via CME, as well as for cell egress [ 33 , 34 , 35 , 36 , 37 ]. Another excellent example of exploiting the specificity of AP-1 are HCV and herpesviruses that skillfully select adaptins to avoid apical surfaces and avoid host immune response.…”