Egress of Light Particles among Filopodia on the Surface of Varicella-Zoster Virus-Infected Cells

Abstract

Varicella-zoster virus (VZV) is renowned for its very low titer when grown in cultured cells. There remains no single explanation for the low infectivity. In this study, viral particles on the surfaces of infected cells were examined by several imaging technologies. Few surface particles were detected at 48 h postinfection (hpi), but numerous particles were observed at 72 and 96 hpi. At 72 hpi, 75% of the particles resembled light (L) particles, i.e., envelopes without capsids. By 96 hpi, 85% of all particles resembled L particles. Subsequently, the envelopes of complete virions and L particles were investigated to determine their glycoprotein constituents. Glycoproteins gE, gI, and gB were detected in the envelopes of both types of particles in similar numbers; i.e., there appeared to be no difference in the glycoprotein content of the L particles. The viral particles emerged onto the cell surface amid actin-based filopodia, which were present in abundance within viral highways. Viral particles were easily detected at the base of and along the exterior surfaces of the filopodia. VZV particles were not detected within filopodia. In short, these results demonstrate that VZV infection of cultured cells produces a larger proportion of aberrant coreless particles than has been seen with any other previously examined alphaherpesvirus. Further, these results suggested a major disassociation between capsid formation and envelopment as an explanation for the invariably low VZV titer in cultured cells.Varicella-zoster virus (VZV) is renowned for its extremely low titer when grown in cultured cells. Explanations for this low titer have been pursued for decades. In this investigation, we hypothesized that one of the earliest explanations, an aberrant structure, was at least partly responsible for the markedly reduced infectivity in cell culture.In 1968, Cook and Stevens reported that extranuclear VZ virions were distinguishable from herpes simplex virions by the presence of pleomorphism: VZV had incomplete coats and loss of central dense cores (2). Those authors suggested that aberrant assembly was a potential reason for the lack of cellfree virus. In their work, Cook and Stevens did not describe in detail the characteristics of extracellular VZV. The cell-associated nature of VZV has limited the repertoire of techniques available to those wishing to study VZV outside the cellular environment. Earlier studies (41) using scanning electron microscopy (SEM) illustrated the presence of viral particles arranged in clusters on the surface of infected human melanoma cells (MeWo). In 1995, Harson and Grose (18) reported a very distinguishing characteristic of VZV-infected cells. Thousands of extracellular viral particles were seen on the surfaces of fused cell monolayers. Instead of being randomly distributed over the cell surface, egressed viral particles were arranged in an elongated pattern termed "viral highways." This viral egress phenotype was seen with all VZV strains examined in MeWo cells, including the vaccine strain VZV...

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

“…The samples were extracted from the tissue culture dish. Sections made with a diamond knife were stained with uranyl acetate and lead citrate and observed under a JEOL 1230 electron microscope, as described previously (4).…”

“…A recent paper from Storlie et al (42), reporting a delayed accumulation of the late VZV protein glycoprotein C (gC), suggested that an impairment in the expression of specific viral genes during infection of cultured cells could contribute to such low titers (4). An alternative and not mutually exclusive explanation for the low yields of VZV comes from a separate set of reports, which showed that egressing VZV particles acquire the final envelope at the level of the trans-Golgi network (13) and are then detectable both on the cell surface and within large cytoplasmic vacuoles.…”

“…Although infectious VZV particles are very abundant in skin vesicles during natural infection, in cultured cells the virus grows to very low titers and remains extremely cell associated, with noninfectious light particles (L particles) constituting the majority of particles on the surface of infected cells (4). A recent paper from Storlie et al (42), reporting a delayed accumulation of the late VZV protein glycoprotein C (gC), suggested that an impairment in the expression of specific viral genes during infection of cultured cells could contribute to such low titers (4).…”

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

“…Samples of infected and uninfected cells were prepared for confocal microscopy by methods described previously (7). Briefly, the samples were fixed with paraformaldehyde and permeabilized with 0.05% Triton X-100 in phosphate-buffered saline (T-PBS) and then blocked in 5% nonfat milk with 2.5% normal goat serum for 2 h at room temperature (RT).…”

“…Samples were processed for scanning electron microscopy by methods described previously (7). Briefly, following initial fixation with paraformaldehyde and antibody labeling steps, the sample was further fixed with 4% glutaraldehyde in PBS and then stained with 1% OsO 4 in double-distilled water (ddH 2 O), followed by dehydration in a graded series of ethanol-water mixtures from 25% ethanol to 100% ethanol and then 100% hexamethylene disilane (HMDS).…”