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

Abstract

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 in vitro 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 in vitro 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.

The Glycoprotein B Cytoplasmic Domain Lysine Cluster Is Critical for Varicella-Zoster Virus Cell-Cell Fusion Regulation and Infection

“…5B). A 0.5-log 10 decrease in viral titers was also observed for pOka-gH[Δ834-841] at 3 dpi, which correlated with previous observations (31). Thus, the positive charge of the gBcyt lysine cluster contributes to the cell-cell fusion regulation required for effective VZV propagation.…”

“…This provided further evidence that combining the ITIM and lysine cluster mutations further disrupts gB function. The pOka-gH[Δ834-841] virus, which has the last eight amino acids of the gH cytoplasmic domain (gHcyt) deleted, also exhibited the exaggerated syncytium formation phenotype, with a median value of 126 nuclei/ syncytium, consistent with past observations (31). Thus, the positive charge of the gBcyt lysine cluster is critical for the regulation of cell-cell fusion in infected melanoma cells.…”

“…The reduced replication kinetics of mutant VZV with dysregulated cell-cell fusion has been attributed to the limited VZV spread caused by the exaggerated syncytium formation phenotype (31). To ascertain if the gBcyt mutations affected VZV spread in melanoma cells, the plaque sizes of the mutant viruses were compared to pOka at 4 dpi (Fig.…”

“…Truncation of the terminal 8 aa of the 18-aa cytoplasmic domain of gH (gH[Δ834-841]) induced a hyperfusion phenotype that was also observed in an in vitro cell-cell fusion assay (31). The gHcyt fusion regulation was identified to be dependent upon the length rather than the sequence of the domain.…”

“…The length of the HSV-1 gHcyt has also been shown to contribute to gB/gH-gL/gD cell-cell fusion (28). Similar to VZV gB[Y881F], the gH[Δ834-841] virus also caused exaggerated syncytium formation that translated into reduced replication kinetics of VZV in cultured melanoma cells and limited skin pathogenesis (31).…”