Varicella zoster virus (VZV) is the causative agent of varicella (chickenpox) and zoster (shingles). Investigating VZV pathogenesis is challenging as VZV is a human-specific virus and infection does not occur, or is highly restricted, in other species. However, the use of human tissue xenografts in mice with severe combined immunodeficiency (SCID) enables the analysis of VZV infection in differentiated human cells in their typical tissue microenvironment. Xenografts of human skin, dorsal root ganglia or foetal thymus that contains T cells can be infected with mutant viruses or in the presence of inhibitors of viral or cellular functions to assess the molecular mechanisms of VZV–host interactions. In this Review, we discuss how these models have improved our understanding of VZV pathogenesis.

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Herpesvirus entry functions of the conserved glycoproteins gB and gH-gL have been delineated, but their role in regulating cell-cell fusion is poorly understood. Varicella-zoster virus (VZV) infection provides a valuable model for investigating cell-cell fusion because of the importance of this process for pathogenesis in human skin and sensory ganglia. The present study identifies a canonical immunoreceptor tyrosine-based inhibition motif (ITIM) in the gB cytoplasmic domain (gBcyt) and demonstrates that the gBcyt is a tyrosine kinase substrate. Orbitrap mass spectrometry confirmed that Y881, central to the ITIM, is phosphorylated. To determine whether the gBcyt ITIM regulates gB/gH-gL-induced cell-cell fusion in vitro, tyrosine residues Y881 and Y920 in the gBcyt were substituted with phenylalanine separately or together. Recombinant viruses with these substitutions were generated to establish their effects on syncytia formation in replication in vitro and in the human skin xenograft model of VZV pathogenesis. The Y881F substitution caused significantly increased cell-cell fusion despite reduced cell-surface gB. Importantly, the Y881F or Y881/920F substitutions in VZV caused aggressive syncytia formation, reducing cell-cell spread. These in vitro effects of aggressive syncytia formation translated to severely impaired skin infection in vivo. In contrast, the Y920F substitution did not affect virus replication in vitro or in vivo. These observations suggest that gB modulates cell-cell fusion via an ITIM-mediated Y881 phosphorylation-dependent mechanism, supporting a unique concept that intracellular signaling through this gBcyt motif regulates VZV syncytia formation and is essential for skin pathogenesis.fusogenicity | mutagenesis | polykaryocyte | virulence T he alphaherpesvirus varicella-zoster virus (VZV) is a human pathogen that spreads from mucosal epithelial sites of initial infection to skin via a T cell-associated viremia (1), causing varicella (chicken pox). Viremia and cutaneous infection enable transfer of VZV to sensory nerve ganglia and establishment of latency in neurons (2). Zoster (shingles) is caused by VZV reactivation from latently infected neurons and can lead to the debilitating condition of postherpetic neuralgia. Live attenuated VZV vaccines are effective against varicella and zoster but are not recommended for immunocompromised patients.Enveloped viruses from several families, including the Herpesviridae, require fusion with cellular membranes for virion entry, and in some cases induce syncytia through cell-cell fusion (2-5). Little is known about the functional role of syncytia during pathogenesis. VZV is a valuable model pathogen for investigating this process because natural infection of the human host involves formation of multinucleated polykaryocytes in skin and fusion of neurons and satellite cells in sensory ganglia (2, 6). In addition, VZV produces syncytia during replication in vitro and triggers fusion between differentiated cells in human skin and dorsal root ganglion xenograf...

Enveloped viruses require membrane fusion for cell entry and replication. For herpesviruses, this event is governed by the multiprotein core complex of conserved glycoproteins (g)B and gH/gL. The recent crystal structures of gH/gL from herpes simplex virus 2, pseudorabies virus, and Epstein-Barr virus revealed distinct domains that, surprisingly, do not resemble known viral fusogens. Varicella-zoster virus (VZV) causes chicken pox and shingles. VZV is an α-herpesvirus closely related to herpes simplex virus 2, enabling prediction of the VZV gH structure by homology modeling. We have defined specific roles for each gH domain in VZV replication and pathogenesis using structure-based site-directed mutagenesis of gH. The distal tip of domain (D)I was important for skin tropism, entry, and fusion. DII helices and a conserved disulfide bond were essential for gH structure and VZV replication. An essential 724 CXXC 727 motif was critical for DIII structural stability and membrane fusion. This assignment of domain-dependent mechanisms to VZV gH links elements of the glycoprotein structure to function in herpesvirus replication and virulence.H erpesviruses are ubiquitous and important pathogens in both immunocompetent and immunocompromised individuals. Three of the eight human herpesviruses, varicella-zoster virus (VZV) and herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), are α-herpesviruses (1). These three viruses initiate infection at mucosal sites. The pathogenesis of primary VZV infection, varicella (chicken pox), then depends on T-cell infection, which transfers VZV to skin and results in the establishment of latency in the sensory nerve ganglia (2). VZV skin lesions consist of polykaryocytes that are generated by cell fusion and release large quantities of virus. In contrast, VZV infection does not induce Tcell fusion, allowing these cells to transport virus to skin and neurons. VZV reactivation from latency causes zoster (shingles) and the debilitating condition postherpetic neuralgia.After host cell attachment, entry by enveloped viruses, including herpesviruses, requires fusion. Enveloped viruses typically use a single fusogenic glycoprotein, but evidence from several herpesviruses indicates that entry fusion is induced by a multiprotein core complex consisting of glycoprotein (g)B and the heterodimer gH/gL (3). gH requires the scaffolding protein gL for maturation and transport, although gL might also promote fusion (4-6). The gH homologs of HSV-1, Epstein-Barr virus (EBV) and pseudorabies virus (PRV), are required for virus entry (7-9). VZV gH and gL are essential for virus replication, and antibodies to gH inhibit both VZV entry and cell fusion (10, 11). Despite extensive evidence that herpesvirus gH proteins are an essential component of the fusion complex, how gH/gL proteins function in fusion has yet to be fully elucidated.gH was originally proposed to act directly as a fusogen, but the newly resolved crystal structures of HSV-2 and EBV gH/gL and PRV gH do not resemble known viral fusion proteins (12-14)...

Glycoprotein B (gB), the most conserved protein in the family Herpesviridae, is essential for the fusion of viral and cellular membranes. Information about varicella-zoster virus (VZV) gB is limited, but homology modeling showed that the structure of VZV gB was similar to that of herpes simplex virus (HSV) gB, including the putative fusion loops. In contrast to HSV gB, VZV gB had a furin recognition motif ([R]-X-[KR]-R-ͦ-X, where ͦ indicates the position at which the polypeptide is cleaved) at residues 491 to 494, thought to be required for gB cleavage into two polypeptides. To investigate their contribution, the putative primary fusion loop or the furin recognition motif was mutated in expression constructs and in the context of the VZV genome. Varicella-zoster virus (VZV), an alphaherpesvirus, causes chicken pox (varicella) as a primary infection and shingles (zoster) upon reactivation from infected ganglia in humans (reviewed in reference 16).Although not yet investigated in VZV, herpesvirus entry requires fusion of the virus envelope with cell membranes governed by viral glycoprotein B (gB) and gH/gL, which are conserved across the family Herpesviridae (12,27,57). gB is the most conserved glycoprotein, with its function as a fusion protein well documented for several of the herpesviruses (10,19,38,48,51,52).Open reading frame 31 (ORF31) codes for the 931 amino acids of VZV gB (18, 37). The successive N-and O-linked glycosylation plus the sialation and sulfation of VZV gB yields a mature protein with a molecular mass of approximately 140 kDa (45). Upon maturation, gB is cleaved, presumably by cellular proteases, into two polypeptides of 66 and 68 kDa. Intracellular trafficking of gB was shown to be dependent upon amino acid motifs in the cytoplasmic domain (24-26). In transfection studies, gB was transported to the cellular surface where it was endocytosed and localized to the trans-Golgi, where envelopment of viral particles is thought to occur.The structures of gB in the two human alphaherpesviruses, VZV and herpes simplex virus type 1 (HSV-1), are likely to be very similar as they have 49% amino acid identity (reviewed in reference 16). The ectodomain of HSV-1 gB was shown to form a spike that consisted of trimers with the structural homology to gG of vesicular stomatitis virus (28). Heldwein et al. (28) proposed that HSV-1 gB is a class II fusion protein based on homology to VSV G. The herpesvirus gB monomer was divided into five domains, I to V. Domain I consisted of a continuous amino acid sequence that folded into a pleckstrin homology-like domain, while domain II was comprised of two discontinuous segments, which also had a pleckstrin homologylike domain. A loop region exposed to the exterior of gB connected domain II with domain III. Domain III was comprised of three discontinuous segments and connected to the external loop by a long ␣ helix that ended in a central coiled coil. Domain IV crowned gB and was connected to domain V, which stretched from the top to the bottom of the gB monomer, forming ...