Human induced pluripotent stem cell-derived salivary gland organoids model SARS-CoV-2 infection and replication.

Salivary glands act as virus reservoirs in various infectious diseases and have been reported to be targeted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the mechanisms underlying infection and replication in salivary glands are still enigmatic due to the lack of proper in vitro models. Here, we show that human induced salivary glands (hiSGs) generated from human induced pluripotent stem cells can be infected with SARS-CoV-2. The hiSGs exhibit properties similar to those of embryonic salivary glands and are a valuable tool for the functional analysis of genes during development. Orthotopically transplanted hiSGs can be engrafted at a recipient site in mice and show a mature phenotype. In addition, we confirm SARS-CoV-2 infection and replication in hiSGs. SARS-CoV-2 derived from saliva in asymptomatic individuals may participate in the spread of the virus. hiSGs may be a promising model for investigating the role of salivary glands as a virus reservoir.

A Variant Allele in Varicella-Zoster Virus Glycoprotein B Selected during Production of the Varicella Vaccine Contributes to Its Attenuation.

Attenuation of the live varicella Oka vaccine (vOka) has been attributed to mutations in the genome acquired during cell culture passage of pOka (parent strain); however, the precise mechanisms of attenuation remain unknown. Comparative sequence analyses of several vaccine batches showed that over 100 single-nucleotide polymorphisms (SNPs) are conserved across all vaccine batches; 6 SNPs are nearly fixed, suggesting that these SNPs are responsible for attenuation. By contrast, prior analysis of chimeric vOka and pOka recombinants indicates that loci other than these six SNPs contribute to attenuation. Here, we report that pOka consists of a heterogenous population of virus sequences with two nearly equally represented bases, guanine (G) or adenine (A), at nucleotide 2096 of the ORF31 coding sequence, which encodes glycoprotein B (gB) resulting in arginine (R) or glutamine (Q), respectively, at amino acid 699 of gB. By contrast, 2096A/699Q is dominant in vOka (>99.98%). gB699Q/gH/gL showed significantly less fusion activity than gB699R/gH/gL in a cell-based fusion assay. Recombinant pOka with gB669Q (rpOka_gB699Q) had a similar growth phenotype as vOka during lytic infection in cell culture including human primary skin cells; however, rpOka_gB699R showed a growth phenotype similar to pOka. rpOka_gB699R entered neurons from axonal terminals more efficiently than rpOka_gB699Q in the presence of cell membrane-derived vesicles containing gB. Strikingly, when a mixture of pOka with both alleles equally represented was used to infect human neurons from axon terminals, pOka with gB699R was dominant for virus entry. These results identify a variant allele in gB that contributes to attenuation of vOka. IMPORTANCE The live-attenuated varicella vaccine has reduced the burden of chickenpox. Despite its development in 1974, the molecular basis for its attenuation is still not well understood. Since the live-attenuated varicella vaccine is the only licensed human herpesvirus vaccine that prevents primary disease, it is important to understand the mechanism for its attenuation. Here we identify that a variant allele in glycoprotein B (gB) selected during generation of the varicella vaccine contributes to its attenuation. This variant is impaired for fusion, virus entry into neurons from nerve terminals, and replication in human skin cells. Identification of a variant allele in gB, one of the essential herpesvirus core genes, that contributes to its attenuation may provide insights that assist in the development of other herpesvirus vaccines.

Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection.

Primary varicella-zoster virus (VZV) infection leads to varicella and the establishment of lifelong latency in sensory ganglion neurons. Reactivation of latent VZV causes herpes zoster, which is frequently associated with chronic pain. Latent viral gene expression is restricted to the VZV latency-associated transcript (VLT) and VLT-ORF63 (VLT63) fusion transcripts. Since VLT and VLT63 encode proteins that are expressed during lytic infection, we investigated whether pVLT and pVLT-ORF63 are essential for VZV replication by performing VZV genome mutagenesis using CRISPR/Cas9 and BAC technologies. We first established that CRISPR/Cas9 can efficiently mutate VZV genomes in lytically VZV-infected cells through targeting non-essential genes ORF8 and ORF11 and subsequently show recovery of viable mutant viruses. By contrast, the VLT region was markedly resistant to CRISPR/Cas9 editing. Whereas most mutants expressed wild-type or N-terminally altered versions of pVLT and pVLT-ORF63, only a minority of the resulting mutant viruses lacked pVLT and pVLT-ORF63 coding potential. Growth curve analysis showed that pVLT/pVLT-ORF63 negative viruses were viable, but impaired in growth in epithelial cells. We confirmed this phenotype independently using BAC-derived pVLT/pVLT-ORF63 negative and repaired viruses. Collectively, these data demonstrate that pVLT and/or pVLT-ORF63 are dispensable for lytic VZV replication but promote efficient VZV infection in epithelial cells.

Nectin-1 Is an Entry Mediator for Varicella-Zoster Virus Infection of Human Neurons.

Varicella-zoster virus (VZV) maintains lifelong latency in neurons following initial infection and can subsequently be reactivated to result in herpes zoster or severe neurological manifestations such as encephalitis. Mechanisms of VZV neuropathogenesis have been challenging to study due to the strict human tropism of the virus. Although neuronal entry mediators of other herpesviruses, including herpes simplex virus, have been identified, little is known regarding how VZV enters neurons. Here, we utilize a human stem cell-based neuronal model to characterize cellular factors that mediate entry. Through transcriptional profiling of infected cells, we identify the cell adhesion molecule nectin-1 as a candidate mediator of VZV entry. Nectin-1 is highly expressed in the cell bodies and axons of neurons. Either knockdown of endogenous nectin-1 or incubation with soluble forms of nectin-1 produced in mammalian cells results in a marked decrease in infectivity of neurons. Notably, while addition of soluble nectin-1 during viral infection inhibits infectivity, addition after infection has no effect on infectivity. Ectopic expression of human nectin-1 in a cell line resistant to productive VZV infection confers susceptibility to infection. In summary, we have identified nectin-1 as a neuronal entry mediator of VZV. IMPORTANCE Varicella-zoster virus (VZV) causes chickenpox, gains access to neurons during primary infection where it resides lifelong, and can later be reactivated. Reactivation is associated with shingles and postherpetic neuralgia, as well as with severe neurologic complications, including vasculitis and encephalitis. Although the varicella vaccine substantially decreases morbidity and mortality associated with primary infection, the vaccine cannot prevent the development of neuronal latency, and vaccinated populations are still at risk for reactivation. Furthermore, immunocompromised individuals are at higher risk for VZV reactivation and associated complications. Little is known regarding how VZV enters neurons. Here, we identify nectin-1 as an entry mediator of VZV in human neurons. Identification of nectin-1 as a neuronal VZV entry mediator could lead to improved treatments and preventative measures to reduce VZV related morbidity and mortality.

Varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency.

Varicella-zoster virus (VZV) establishes lifelong neuronal latency in most humans world-wide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. How VZV establishes, maintains and reactivates from latency is largely unknown. VZV transcription during latency is restricted to the latency-associated transcript (VLT) and RNA 63 (encoding ORF63) in naturally VZV-infected human trigeminal ganglia (TG). While significantly more abundant, VLT levels positively correlated with RNA 63 suggesting co-regulated transcription during latency. Here, we identify VLT-ORF63 fusion transcripts and confirm VLT-ORF63, but not RNA 63, expression in human TG neurons. During in vitro latency, VLT is transcribed, whereas VLT-ORF63 expression is induced by reactivation stimuli. One isoform of VLT-ORF63, encoding a fusion protein combining VLT and ORF63 proteins, induces broad viral gene transcription. Collectively, our findings show that VZV expresses a unique set of VLT-ORF63 transcripts, potentially involved in the transition from latency to lytic VZV infection.

Decoding the Architecture of the Varicella-Zoster Virus Transcriptome.

Varicella-zoster virus (VZV), a double-stranded DNA virus, causes varicella, establishes lifelong latency in ganglionic neurons, and reactivates later in life to cause herpes zoster, commonly associated with chronic pain. The VZV genome is densely packed and produces multitudes of overlapping transcripts deriving from both strands. While 71 distinct open reading frames (ORFs) have thus far been experimentally defined, the full coding potential of VZV remains unknown. Here, we integrated multiple short-read RNA sequencing approaches with long-read direct RNA sequencing on RNA isolated from VZV-infected cells to provide a comprehensive reannotation of the lytic VZV transcriptome architecture. Through precise mapping of transcription start sites, splice junctions, and polyadenylation sites, we identified 136 distinct polyadenylated VZV RNAs that encode canonical ORFs, noncanonical ORFs, and ORF fusions, as well as putative noncoding RNAs (ncRNAs). Furthermore, we determined the kinetic class of all VZV transcripts and observed, unexpectedly, that transcripts encoding the ORF62 protein, previously designated Immediate-Early, were expressed with Late kinetics. Our work showcases the complexity of the VZV transcriptome and provides a comprehensive resource that will facilitate future functional studies of coding RNAs, ncRNAs, and the biological mechanisms underlying the regulation of viral transcription and translation during lytic VZV infection.IMPORTANCE Transcription from herpesviral genomes, executed by the host RNA polymerase II and regulated by viral proteins, results in coordinated viral gene expression to efficiently produce infectious progeny. However, the complete coding potential and regulation of viral gene expression remain ill-defined for the human alphaherpesvirus varicella-zoster virus (VZV), causative agent of both varicella and herpes zoster. Here, we present a comprehensive overview of the VZV transcriptome and the kinetic class of all identified viral transcripts, using two virus strains and two biologically relevant cell types. Additionally, our data provide an overview of how VZV diversifies its transcription from one of the smallest herpesviral genomes. Unexpectedly, the transcript encoding the major viral transactivator protein (pORF62) was expressed with Late kinetics, whereas orthologous transcripts in other alphaherpesviruses are typically expressed during the immediate early phase. Therefore, our work both establishes the architecture of the VZV transcriptome and provides insight into regulation of alphaherpesvirus gene expression.

Epstein-Barr Virus (EBV) Tegument Protein BGLF2 Suppresses Type I Interferon Signaling To Promote EBV Reactivation.

Interferon alpha (IFN-α) and IFN-ß are type I IFNs that are induced by virus infection and are important in the host's innate antiviral response. EBV infection activates multiple cell signaling pathways, resulting in the production of type I IFN which inhibits EBV infection and virus-induced B-cell transformation. We reported previously that EBV tegument protein BGLF2 activates p38 and enhances EBV reactivation. To further understand the role of BGLF2 in EBV infection, we used mass spectrometry to identify cellular proteins that interact with BGLF2. We found that BGLF2 binds to Tyk2 and confirmed this interaction by coimmunoprecipitation. BGLF2 blocked type I IFN-induced Tyk2, STAT1, and STAT3 phosphorylation and the expression of IFN-stimulated genes (ISGs) IRF1, IRF7, and MxA. In contrast, BGLF2 did not inhibit STAT1 phosphorylation induced by IFN-γ. Deletion of the carboxyl-terminal 66 amino acids of BGLF2 reduced the ability of the protein to repress type I IFN signaling. Treatment of gastric carcinoma and Raji cells with IFN-α blocked BZLF1 expression and EBV reactivation; however, expression of BGLF2 reduced the ability of IFN-α to inhibit BZLF1 expression and enhanced EBV reactivation. In summary, EBV BGLF2 interacts with Tyk2, inhibiting Tyk2, STAT1, and STAT3 phosphorylation and impairs type I IFN signaling; BGLF2 also counteracts the ability of IFN-α to suppress EBV reactivation.IMPORTANCE Type I interferons are important for controlling virus infection. We have found that the Epstein-Barr virus (EBV) BGLF2 tegument protein binds to a protein in the type I interferon signaling pathway Tyk2 and inhibits the expression of genes induced by type I interferons. Treatment of EBV-infected cells with type I interferon inhibits reactivation of the virus, while expression of EBV BGLF2 reduces the ability of type I interferon to inhibit virus reactivation. Thus, a tegument protein delivered to cells during virus infection inhibits the host's antiviral response and promotes virus reactivation of latently infected cells. Therefore, EBV BGLF2 might protect virus-infected cells from the type I interferon response in cells undergoing lytic virus replication.

Direct RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen.

Characterizing complex viral transcriptomes by conventional RNA sequencing approaches is complicated by high gene density, overlapping reading frames, and complex splicing patterns. Direct RNA sequencing (direct RNA-seq) using nanopore arrays offers an exciting alternative whereby individual polyadenylated RNAs are sequenced directly, without the recoding and amplification biases inherent to other sequencing methodologies. Here we use direct RNA-seq to profile the herpes simplex virus type 1 (HSV-1) transcriptome during productive infection of primary cells. We show how direct RNA-seq data can be used to define transcription initiation and RNA cleavage sites associated with all polyadenylated viral RNAs and demonstrate that low level read-through transcription produces a novel class of chimeric HSV-1 transcripts, including a functional mRNA encoding a fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L. Thus, direct RNA-seq offers a powerful method to characterize the changing transcriptional landscape of viruses with complex genomes.

Molecular Aspects of Varicella-Zoster Virus Latency.

Primary varicella-zoster virus (VZV) infection causes varicella (chickenpox) and the establishment of a lifelong latent infection in ganglionic neurons. VZV reactivates in about one-third of infected individuals to cause herpes zoster, often accompanied by neurological complications. The restricted host range of VZV and, until recently, a lack of suitable in vitro models have seriously hampered molecular studies of VZV latency. Nevertheless, recent technological advances facilitated a series of exciting studies that resulted in the discovery of a VZV latency-associated transcript (VLT) and provide novel insights into our understanding of VZV latency and factors that may initiate reactivation. Deducing the function(s) of VLT and the molecular mechanisms involved should now be considered a priority to improve our understanding of factors that govern VZV latency and reactivation. In this review, we summarize the implications of recent discoveries in the VZV latency field from both a virus and host perspective and provide a roadmap for future studies.

Vaccine Development for Varicella-Zoster Virus.

Varicella-zoster virus (VZV) is the first and only human herpesvirus for which a licensed live attenuated vaccine, vOka, has been developed. vOka has highly safe and effective profiles; however, worldwide herd immunity against VZV has not yet been established and it is far from eradication. Despite the successful reduction in the burden of VZV-related illness by the introduction of the vaccine, some concerns about vOka critically prevent worldwide acceptance and establishment of herd immunity, and difficulties in addressing these criticisms often relate to its ill-defined mechanism of attenuation. Advances in scientific technologies have been applied in the VZV research field and have contributed toward uncovering the mechanism of vOka attenuation as well as VZV biology at the molecular level. A subunit vaccine targeting single VZV glycoprotein, rationally designed based on the virological and immunological research, has great potential to improve the strategy for eradication of VZV infection in combination with vOka.

A spliced latency-associated VZV transcript maps antisense to the viral transactivator gene 61.

Varicella-zoster virus (VZV), an alphaherpesvirus, establishes lifelong latent infection in the neurons of >90% humans worldwide, reactivating in one-third to cause shingles, debilitating pain and stroke. How VZV maintains latency remains unclear. Here, using ultra-deep virus-enriched RNA sequencing of latently infected human trigeminal ganglia (TG), we demonstrate the consistent expression of a spliced VZV mRNA, antisense to VZV open reading frame 61 (ORF61). The spliced VZV latency-associated transcript (VLT) is expressed in human TG neurons and encodes a protein with late kinetics in productively infected cells in vitro and in shingles skin lesions. Whereas multiple alternatively spliced VLT isoforms (VLTly) are expressed during lytic infection, a single unique VLT isoform, which specifically suppresses ORF61 gene expression in co-transfected cells, predominates in latently VZV-infected human TG. The discovery of VLT links VZV with the other better characterized human and animal neurotropic alphaherpesviruses and provides insights into VZV latency.

Human Embryonic Stem Cell-Derived Neurons Are Highly Permissive for Varicella-Zoster Virus Lytic Infection.

Varicella-zoster virus (VZV) is highly cell associated when grown in culture and has a much higher (4,000- to 20,000-fold increased) particle-to-PFU ratio in vitro than herpes simplex virus (HSV). In contrast, VZV is highly infectious in vivo by airborne transmission. Neurons are major targets for VZV in vivo; in neurons, the virus can establish latency and reactivate to produce infectious virus. Using neurons derived from human embryonic stem cells (hESC) and cell-free wild-type (WT) VZV, we demonstrated that neurons are nearly 100 times more permissive for WT VZV infection than very-early-passage human embryonic lung cells or MRC-5 diploid human fibroblasts, the cells used for vaccine production or virus isolation. The peak titers achieved after infection were ∼10-fold higher in human neurons than in MRC-5 cells, and the viral genome copy number-to-PFU ratio for VZV in human neurons was 500, compared with 50,000 for MRC-5 cells. Thus, VZV may not necessarily have a higher particle-to-PFU ratio than other herpesviruses; instead, the cells previously used to propagate virus in vitro may have been suboptimal. Furthermore, based on electron microscopy, neurons infected with VZV produced fewer defective or incomplete viral particles than MRC-5 cells. Our data suggest that neurons derived from hESC may have advantages compared to other cells for studies of VZV pathogenesis, for obtaining stocks of virus with high titers, and for isolating VZV from clinical specimens.IMPORTANCE Varicella-zoster virus (VZV) causes chickenpox and shingles. Cell-free VZV has been difficult to obtain, both for in vitro studies and for vaccine production. While numerous cells lines have been tested for their ability to produce high titers of VZV, the number of total virus particles relative to the number of viral particles that can form plaques in culture has been reported to be extremely high relative to that in other viruses. We show that VZV grows to much higher titers in human neurons than in other cell types in vitro and that the number of total virus genomes relative to the number of viral particles that can form plaques in culture is much lower in human neurons than other cultured cells. These findings indicate that human neurons may be useful for studying VZV in vitro, for growing preparations of virus with high titers, and for isolating the virus from human samples.

Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation.

Mechanisms of neuronal infection by varicella-zoster virus (VZV) have been challenging to study due to the relatively strict human tropism of the virus and the paucity of tractable experimental models. Cellular mitogen-activated protein kinases (MAPKs) have been shown to play a role in VZV infection of nonneuronal cells, with distinct consequences for infectivity in different cell types. Here, we utilize several human neuronal culture systems to investigate the role of one such MAPK, the c-Jun N-terminal kinase (JNK), in VZV lytic infection and reactivation. We find that the JNK pathway is specifically activated following infection of human embryonic stem cell-derived neurons and that this activation of JNK is essential for efficient viral protein expression and replication. Inhibition of the JNK pathway blocked viral replication in a manner distinct from that of acyclovir, and an acyclovir-resistant VZV isolate was as sensitive to the effects of JNK inhibition as an acyclovir-sensitive VZV isolate in neurons. Moreover, in a microfluidic-based human neuronal model of viral latency and reactivation, we found that inhibition of the JNK pathway resulted in a marked reduction in reactivation of VZV. Finally, we utilized a novel technique to efficiently generate cells expressing markers of human sensory neurons from neural crest cells and established a critical role for the JNK pathway in infection of these cells. In summary, the JNK pathway plays an important role in lytic infection and reactivation of VZV in physiologically relevant cell types and may provide an alternative target for antiviral therapy.IMPORTANCE Varicella-zoster virus (VZV) has infected over 90% of people worldwide. While primary infection leads to the typically self-limiting condition of chickenpox, the virus can remain dormant in the nervous system and may reactivate later in life, leading to shingles or inflammatory diseases of the nervous system and eye with potentially severe consequences. Here, we take advantage of newer stem cell-based technologies to study the mechanisms by which VZV infects human neurons. We find that the c-Jun N-terminal kinase (JNK) pathway is activated by VZV infection and that blockade of this pathway limits lytic replication (as occurs during primary infection). In addition, JNK inhibition limits viral reactivation, exhibiting parallels with herpes simplex virus reactivation. The identification of the role of the JNK pathway in VZV infection of neurons reveals potential avenues for the development of alternate antiviral drugs.

In vitro system using human neurons demonstrates that varicella-zoster vaccine virus is impaired for reactivation, but not latency.

Varicella-zoster virus (VZV) establishes latency in human sensory and cranial nerve ganglia during primary infection (varicella), and the virus can reactivate and cause zoster after primary infection. The mechanism of how the virus establishes and maintains latency and how it reactivates is poorly understood, largely due to the lack of robust models. We found that axonal infection of neurons derived from hESCs in a microfluidic device with cell-free parental Oka (POka) VZV resulted in latent infection with inability to detect several viral mRNAs by reverse transcriptase-quantitative PCR, no production of infectious virus, and maintenance of the viral DNA genome in endless configuration, consistent with an episome configuration. With deep sequencing, however, multiple viral mRNAs were detected. Treatment of the latently infected neurons with Ab to NGF resulted in production of infectious virus in about 25% of the latently infected cultures. Axonal infection of neurons with vaccine Oka (VOka) VZV resulted in a latent infection similar to infection with POka; however, in contrast to POka, VOka-infected neurons were markedly impaired for reactivation after treatment with Ab to NGF. In addition, viral transcription was markedly reduced in neurons latently infected with VOka compared with POka. Our in vitro system recapitulates both VZV latency and reactivation in vivo and may be used to study viral vaccines for their ability to establish latency and reactivate.

Identification of a novel mutation in MAGT1 and progressive multifocal leucoencephalopathy in a 58-year-old man with XMEN disease.

XMEN disease (X-linked immunodeficiency with Magnesium defect, Epstein-Barr virus infection and Neoplasia) is a novel primary immune deficiency caused by mutations in MAGT1 and characterised by chronic infection with Epstein-Barr virus (EBV), EBV-driven lymphoma, CD4 T-cell lymphopenia, and dysgammaglobulinemia [1]. Functional studies have demonstrated roles for magnesium as a second messenger in T-cell receptor signalling [1], and for NKG2D expression and consequently NK- and CD8 T-cell cytotoxicity [2]. 7 patients have been described in the literature; the oldest died at 45 years and was diagnosed posthumously [1-3]. We present the case of a 58-year-old Caucasian gentleman with a novel mutation in MAGT1 with the aim of adding to the phenotype of this newly described disease by detailing his clinical course over more than 20 years.

Characterization of the human herpesvirus 6A U23 gene.

Human herpesvirus 6 (HHV-6), which replicates abundantly in T cells, belongs to the Roseolovirus genus within the betaherpesvirus subfamily. Members of the Roseolovirus genus encode seven unique genes, U20, U21, U23, U24, U24A, U26, and U100. The present study focused on one of these, U23, by analyzing the characteristics of its gene product in HHV-6A-infected cells. The results indicated that the U23 protein was expressed at the late phase of infection as a glycoprotein, but was not incorporated into virions, and mostly stayed within the trans Golgi network (TGN) in HHV-6A-infected cells. Furthermore, analysis using a U23-defective mutant virus showed that the gene is nonessential for viral replication in vitro.

Varicella-zoster virus ORF49 functions in the efficient production of progeny virus through its interaction with essential tegument protein ORF44.

The ORF49 tegument protein of varicella-zoster virus (VZV) is one of the core gene products that is conserved among herpesvirus family members. Although ORF49 is known to be a cell-tropic factor, its detailed functions remain elusive. ORF44 is another core gene product reported to be essential, although its characterization and detailed functional analysis have not been reported. These two core gene products form a complex in other herpesviruses beyond the host species and herpesvirus subfamilies. Here, we show that complex formation between ORF44 and ORF49 is conserved in VZV. We serendipitously found that binding is eliminated by an amino acid substitution at position 129 (phenylalanine 129), and four amino acids in the carboxyl-terminal half of the acidic cluster in ORF49 (i.e., aspartate-phenylalanine-aspartate-glutamate from positions 41 to 44 [41DFDE44]) were identified as its binding motif. Alanine substitutions in each domain rendered the ORF44F129A mutation lethal for VZV, similar to deletion of the entire ORF44. The phenotype of the ORF49-41AAAA44 mutation was comparable to that of the ORF49-defective virus, including small-plaque formation, impaired growth, and low infectious virus production. These results suggest that the interaction between ORF44 and ORF49 is essential for their role in VZV infection and that ORF49 is required for the efficient production of infectious progeny virus mediated by the conserved interaction between the two proteins.

Varicella-zoster virus glycoprotein M.

Glycoprotein M (gM) is conserved among herpesviruses. Important features are its 6-8 transmembrane domains without a large extracellular domain, localization to the virion envelope, complex formation with another envelope glycoprotein, glycoprotein N (gN), and role in virion assembly and egress. In varicella-zoster virus (VZV), the gM homolog is encoded by ORF50. VZV gM is predicted to be an eight-transmembrane envelope glycoprotein with a complex N-linked oligosaccharide. It mainly localizes to the trans-Golgi network, where final virion envelopment occurs. Studies in which VZV gM or its partner gN were disrupted suggest that the gM/gN complex plays an important role in cell-to-cell spread. Here, we summarize the biological features of VZV gM, including our recent findings on its characterization and function.

Characterization of the varicella-zoster virus ORF50 gene, which encodes glycoprotein M.

The ORF50 gene of the varicella-zoster virus (VZV) encodes glycoprotein M (gM), which is conserved among all herpesviruses and is important for the cell-to-cell spread of VZV. However, few analyses of ORF50 gene expression or its posttranscriptional and translational modifications have been published. Here we found that in VZV-infected cells, ORF50 encoded four transcripts: a full-size transcript, which was translated into the gM, and three alternatively spliced transcripts, which were not translated. Using a splicing-negative mutant virus, we showed that the alternative transcripts were nonessential for viral growth in cell culture. In addition, we found that two amino acid mutations of gM, V42P and G301M, blocked gM's maturation and transport to the trans-Golgi network, which is generally recognized as the viral assembly complex. We also found that the mutations disrupted gM's interaction with glycoprotein N (gN), revealing their interaction through a bond that is otherwise unreported for herpesviruses. Using this gM maturation-negative virus, we found that immature gM and gN were incorporated into intracellularly isolated virus particles and that mature gM was required for efficient viral growth via cell-to-cell spread but not for virion morphogenesis. The virus particles were more abundant at the abnormally enlarged perinuclear cisternae than those of the parental virus, but they were also found at the cell surface and in the culture medium. Additionally, in the gM maturation-negative mutant virus-infected melanoma cells, typical syncytium formation was rarely seen, again indicating that mature gM functions in cell-to-cell spread via enhancement of syncytium formation.