Truncation of the gHcyt does not affect VZV particle formation or egress.

<p>Electronmicrographs of melanoma cells infected with (A–F) pOka or (G–L) pOka-gH[Δ834-841] at 48 hours post infection. (A) (G) Images of viral particles on the cell surface, with micrographs D and J represent magnified images of the boxed area of micrographs A and G, respectively. Representative images of (B) (H) viral particle, (E) (K) crystalline arrays of nucleocapsids, (C) (I) nuclei, and (F) (L) vesicles containing viral particles. The C and N denote cytoplasmic and nuclear space, respectively, with the white arrows indicating representative capsids. The scale bar represents 200 nm.</p

Truncation of the gHcyt causes enhanced <i>in vitro</i> cell-cell fusion.

<p>Cell-cell fusion induced by gB and gH mutants were quantified by the Cre reporter fusion assay, with fusion events represented as a percentage relative to fusion events of gB/gH[TL]-gL. gB[WT], gB[Y881F], and gB[Y881/920F] were coexpressed with either gH[TL] or gH[WT] and gL. Vector only represents transfection with empty vectors. gB[WT] only represents expression of gB[WT] with empty vectors. gH[TL]-gL only and gH[WT]-gL only represent expression of gH[TL] and gH[WT] with gL, respectively, with an empty vector. The plus and minus symbols indicate the presence or absence of gL expressing vector, respectively. Data shown represents the mean of two independent experiments with the whiskers indicating the standard error of the mean (SEM). All values were evaluated by ANOVA analysis and found to be significantly different from gB/gH[TL]-gL (P<0.001).</p

Proposed model for limited skin pathogenesis of VZV caused by exaggerated syncytia formation.

<p>Schematic of human skin with the three main layers denoted: G = Granular, S = Spinous, and B = Basal. (A) Exaggerated syncytia formation by pOka-gH[Δ834-841] and pOka-gH[TL] limits the virus from being able to spread effectively and penetrate the skin, causing the lesion to remain near the surface. (B) In contrast, normal syncytia formation by pOka allows the virus to easily spread and penetrate the basal layer allowing for expansion of the lesion.</p

The Cytoplasmic Domain of Varicella-Zoster Virus Glycoprotein H Regulates Syncytia Formation and Skin Pathogenesis

<div><p>The conserved herpesvirus fusion complex consists of glycoproteins gB, gH, and gL which is critical for virion envelope fusion with the cell membrane during entry. For Varicella Zoster Virus (VZV), the complex is necessary for cell-cell fusion and presumed to mediate entry. VZV causes syncytia formation via cell-cell fusion in skin and in sensory ganglia during VZV reactivation, leading to neuronal damage, a potential contributory factor for the debilitating condition of postherpetic neuralgia. The gH cytoplasmic domain (gHcyt) is linked to the regulation of gB/gH-gL-mediated cell fusion as demonstrated by increased cell fusion <i>in vitro</i> by an eight amino acid (aa834-841) truncation of the gHcyt. The gHcyt regulation was identified to be dependent on the physical presence of the domain, and not of specific motifs or biochemical properties as substitution of aa834-841 with V5, cMyc, and hydrophobic or hydrophilic sequences did not affect fusion. The importance of the gHcyt length was corroborated by stepwise deletions of aa834-841 causing incremental increases in cell fusion, independent of gH surface expression and endocytosis. Consistent with the fusion assay, truncating the gHcyt in the viral genome caused exaggerated syncytia formation and significant reduction in viral titers. Importantly, infection of human skin xenografts in SCID mice was severely impaired by the truncation while maintaining the gHcyt length with the V5 substitution preserved typical replication <i>in vitro</i> and in skin. A role for the gHcyt in modulating the functions of the gB cytoplasmic domain (gBcyt) is proposed as the gHcyt truncation substantially enhanced cell fusion in the presence of the gB[Y881F] mutation. The significant reduction in skin infection caused by hyperfusogenic mutations in either the gHcyt or gBcyt demonstrates that both domains are critical for regulating syncytia formation and failure to control cell fusion, rather than enhancing viral spread, is severely detrimental to VZV pathogenesis.</p></div

Amino acids 834-841 of the gHcyt are dispensable for intracellular trafficking and endocytosis of gH <i>in vitro</i>.

<p>Confocal microscopy of melanoma cells transiently expressing gH[WT] or gH mutants with gL at 24 hours post transfection. Cells were stained for gH (red), early endosome antigen (EEA1; green), trans-Golgi network (TGN46; green), and nuclei (Hoechst 33342; blue). White arrows indicate colocalization of EEA1 and gH, which highlight representative endocytic vesicles containing gH. The scale bars represent 20 µm.</p

Viral particles are not observed in melanoma cells transfected with dual mutant gB[Y881F]/gH[Δ834-841] BAC.

<p>Electronmicrographs of FACS-enriched melanoma cells transfected with pOka-TK-GFP, pOka- TK-GFP-gH[Δ834-841], pOka-TK-GFP-gB[Y881F], and pOka-TK-GFP-gB[Y881F]/gH[Δ834-841] BACs at 72 hours post transfection. Images in the left and right column focus on the nuclei and cytoplasm of syncytium, respectively. The images in the bottom left corner represent magnified images of the areas boxed in white. The C and N denote cytoplasmic and nuclear space, respectively. The scale bars represents 1 µm.</p

Truncation of the gHcyt causes exaggerated syncytia formation in VZV-infected melanoma cells.

<p>Merged phase contrast and fluorescence microscopy images of melanoma cells infected with pOka, pOka-gH[TL], and pOka-gH[Δ834-841] viruses at 24, 36, and 48 hours post infection. Nuclei were stained with Hoechst 33342 (blue). Visually detectable syncytia are outlined in red with white arrows indicating extended cytoplasm of VZV-induced syncytia. The scale bars represent 200 µm.</p

Amino acids 834-841 of the gHcyt are dispensable for intracellular trafficking and endocytosis of gH during infection of melanoma cells.

<p>Confocal microscopy of melanoma cells at 24(Uninfected) or infection with pOka-gH[Δ834-841], pOka-gH[TL], pOka-gH[Y835A], pOka-gH[Y835F], pOka-gH[V5], pOka-gH[834StopV5], or pOka viruses. Cells were stained for gH (red), early endosome antigen (EEA1; green), trans-Golgi network (TGN46; green) and nuclei (Hoechst 33342; blue). White arrows indicate colocalization of EEA1 and gH, which highlight representative endocytic vesicles containing gH. The scale bars represent 20 µm.</p

Amino acids 834-841 of the gHcyt are dispensable for gH maturation during infection of melanoma cells.

<p>Western blot of gH immunoprecipitated (IP) from lysates of melanoma cells either mock-infected (uninfected) or infected with pOka-gH[Δ834-841], pOka-gH[TL], pOka-gH[Y835A], pOka-gH[Y835F], pOka-gH[V5], pOka-gH[834StopV5], or pOka for 48 hours. To determine the level of expression of essential viral proteins, the lysates from the same infected cells were also probed for IE63 and glycoprotein E (gE). Alpha tubulin was probed as a loading control. Arrows indicate molecular weights of gH, gE, IE63, and α-tubulin. Molecular weight standard is indicated on the left. All molecular weights are in kilodaltons (kDa).</p

Truncation of the gHcyt reduces viral titers in melanoma cells.

<p>(A) Replication kinetics of pOka-gH[834StopV5], pOka-gH[Δ834-841], pOka-gH[TL], and pOka in melanoma cells over six days. Individual points represent the mean of harvested infected cells titrated in triplicate, including the titrated inoculum at Day 0. Titers were measured in log₁₀ of plaque forming units per milliliter (PFU/mL). The error bars represent the SEM. Statistical differences between pOka-gH[TL] and the other viruses were evaluated by two-way ANOVA (*P<0.05, **P<0.01, ***P<0.001). (B) Replication kinetics of pOka-gH[Y835A], pOka-gH[Y835F], pOka-gH[V5], and pOka in melanoma cells over six days with no statistical differences were observed between pOka and the mutant viruses. (C and D) Box and whisker plots of plaque sizes measured (n = 30) in melanoma cells infected with (C) pOka-gH[834StopV5], pOka-gH[Δ834-841], pOka-gH[TL], and pOka or (D) pOka-gH[Y835A], pOka-gH[Y835F], pOka-gH[V5], and pOka at four days post infection. Area of plaques was measured in millimeter squared (mm²). The boxes represent the 10–90% percentile with the median indicated by the band and the outliers (<10% or >90% percentile) represented as individual black dots.</p