Varicella-zoster virus (VZV) is well known for its propensity to cause polykaryons (syncytia) in the vesicles within infected skin. Similarly in cultured cells, VZV induces extensive syncytial formation by virus-mediated cell-to-cell fusion. Statistical analyses of fusion parameters demonstrated three-way interactive effects among all three tested variables (incubation temperature, cell type and virus strain). For example, fusion was greatly enhanced at 33 degrees C vs 37 degrees C; also fusion was pronounced in epidermal cells but negligible in fibroblast cells. As with all herpesviruses, VZV gH was a major fusogen. VZV cell fusion was inhibited by antibody to gH, but surprisingly was enhanced by antibody to gE. Other evidence implicating a role for VZV gE in the fusion process was provided by two mutant viruses, in which gE cell surface expression was enhanced. Under transfection conditions, VZV fusion formation occurred after expression of the gH/gL complex; in contrast, pseudorabies virus requires expression of gH, gL and gB, while the herpes simplex virus (HSV) types 1 and 2 require the quartet of gH, gL, gB and gD. VZV has no gD gene and no apparent gD functional homologue. On the other hand, VZV gE exerts a greater effect than HSV gE on membrane fusion. Taken together, the data in this review suggest that VZV has evolved viral glycoprotein machinery more geared toward cell-to-cell fusion (fusion-from-within) than toward virus-to-cell fusion (entry/fusion-from-without), as a means for syncytium formation within the human epidermis.
Varicella-zoster virus is considered to have one of the most stable genomes of all human herpesviruses. In 1998, we reported the unanticipated discovery of a wild-type virus that had lost an immunodominant B-cell epitope on the gE ectodomain (VZV-MSP); the gE escape mutant virus exhibited an unusual pattern of egress. Further studies have now documented a markedly enhanced cell-to-cell spread by the mutant virus in cell culture. This property was investigated by laser scanning confocal microscopy combined with a software program that allows the measurement of pixel intensity of the fluorescent signal. For this new application of imaging technology, the VZV immediate early protein 62 (IE 62) was selected as the fluoresceinated marker. By 48 h postinfection, the number of IE 62-positive pixels in the VZV-MSP-infected culture was nearly fourfold greater than the number of pixels in a culture infected with a low-passage laboratory strain. Titrations by infectious center assays supported the above image analysis data. Confirmatory studies in the SCID-hu mouse documented that VZV-MSP spread more rapidly than other VZV strains in human fetal skin implants. Generally, the cytopathology and vesicle formation produced by other strains at 21 days postinfection were demonstrable with VZV-MSP at 14 days. To assess whether additional genes were contributing to the unusual VZV-MSP phenotype, approximately 20 kb of the VZV-MSP genome was sequenced, including ORFs 31 (gB), 37 (gH), 47, 60 (gL), 61, 62 (IE 62), 66, 67 (gI), and 68 (gE). Except for a few polymorphisms, as well as the previously discovered mutation within gE, the nucleotide sequences within most open reading frames were identical to the prototype VZV-Dumas strain. In short, VZV-MSP represents a novel variant virus with a distinguishable phenotype demonstrable in both infected cell cultures and SCID-hu mice.
Common methods for the preparation of cultured cells for concurrent light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) are not completely satisfactory. This article describes how we grow mammalian cells on plastic disks made from Aclar film. Aclar is a transparent fluorinated-chlorinated thermoplastic that contains no volatile components and is, for all practical purposes, chemically inert. Cells adhere to it readily and remain attached after fixation, dehydration, and critical-point drying or embedding. The film also accepts heavy metal coating by ionic bombardment and is extremely stable in the vacuum of the SEM. LM observations are unhindered by Aclar, since the film is as transparent as glass. Fluorescence microscopy is possible with this film, since it exhibits no detectable autofluorescence. During SEM observation, the film has great dimensional stability, and the cells and heavy metal coating remain attached to the Aclar even under high-resolution operating conditions. TEM processing of specimens grown on Aclar is simplified by the fact that Aclar does not stick to the epoxy resins used in EM. Furthermore, Aclar is easily sectioned and does not damage knives used in ultramicrotomy. The use of Aclar film considerably simplifies the preparation of cultured cells for all types of microscopy. This method is particularly useful in correlating surface features between SEM and TEM observations.
SUMMARYWe investigated the ultrastructural development and maturation of cytomegalovirus (CMV) nuclear inclusions (NIs) in human embryo thyroid cells at 1 to 144 h post-infection. At 5 h, most cells had rounded from an initial fibroblastic appearance and contained early NIs. At 24 h, early NIs were larger and better defined. At 48 h, although early NIs were still present, most cells had larger and presumably more mature NIs. These latter NIs consisted of several subunits, each made up of a fibrillar network enclosing an electron-lucent area which contained coarse and delicate granules. Also, at 48 h, virus particles were first seen in the nucleoplasm. At 72 h, in cells with more developed NIs, virus particles were closely associated with the fibrillar network. Between 96 and 144 h, the NIs reached maximum size and were made up of numerous subunits. The results indicate that two types of NIs coexist during CMV infection. The appearance of the early and late N!s coincides with the reported peaks of CMV DNA synthesis and thus may explain the biphasic pattern of DNA synthesis in CMV infection. Morphogenetic features of the NIs conform with the hypothesis that synthesis of CMV DNA may occur in the centre of each NI subunit and that the fibrillar network represents condensing capsid proteins.
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