An optimised protocol for the expression and purification of adenovirus core protein VII.

Adenovirus protein VII (pVII) is a highly basic core protein, bearing resemblance to mammalian histones. Despite its diverse functions, a comprehensive understanding of its structural intricacies and the mechanisms underlying its functions remain elusive, primarily due to the complexity of producing a good amount of soluble pVII. This study aimed to optimise the expression and purification of recombinant pVII from four different adenoviruses with a simple vector construct. This study successfully determined the optimal conditions for efficiently purifying pVII across four adenovirus species, revealing the differential preference for bacterial expression systems. The One Shot BL21 Star (DE3) proved favourable over Rosetta 2 (DE3) pLysS with consistent levels of expression between IPTG-induced and auto-induction. We demonstrated that combining chemical and mechanical cell lysis is possible and highly effective. Other noteworthy benefits were observed in using RNase during sample processing. The addition of RNase has significantly improved the quality and quantity of the purified protein as confirmed by chromatographic and western blot analyses. These findings established a solid groundwork for pVII purification methodologies and carry the significant potential to assist in unveiling the core structure of pVII, its arrangement within the core, DNA condensation intricacies, and potential pathways for nuclear transport.

Multi-targeted loss of the antigen presentation molecule MR1 during HSV-1 and HSV-2 infection.

The major histocompatibility complex (MHC), Class-I-related (MR1) molecule presents microbiome-synthesized metabolites to Mucosal-associated invariant T (MAIT) cells, present at sites of herpes simplex virus (HSV) infection. During HSV type 1 (HSV-1) infection there is a profound and rapid loss of MR1, in part due to expression of unique short 3 protein. Here we show that virion host shutoff RNase protein downregulates MR1 protein, through loss of MR1 transcripts. Furthermore, a third viral protein, infected cell protein 22, also downregulates MR1, but not classical MHC-I molecules. This occurs early in the MR1 trafficking pathway through proteasomal degradation. Finally, HSV-2 infection results in the loss of MR1 transcripts, and intracellular and surface MR1 protein, comparable to that seen during HSV-1 infection. Thus HSV coordinates a multifaceted attack on the MR1 antigen presentation pathway, potentially protecting infected cells from MAIT cell T cell receptor-mediated detection at sites of primary infection and reactivation.

Structural and functional characterization of siadenovirus core protein VII nuclear localization demonstrates the existence of multiple nuclear transport pathways.

Adenovirus protein VII (pVII) plays a crucial role in the nuclear localization of genomic DNA following viral infection and contains nuclear localization signal (NLS) sequences for the importin (IMP)-mediated nuclear import pathway. However, functional analysis of pVII in adenoviruses to date has failed to fully determine the underlying mechanisms responsible for nuclear import of pVII. Therefore, in the present study, we extended our analysis by examining the nuclear trafficking of adenovirus pVII from a non-human species, psittacine siadenovirus F (PsSiAdV). We identified a putative classical (c)NLS at pVII residues 120-128 (120PGGFKRRRL128). Fluorescence polarization and electrophoretic mobility shift assays demonstrated direct, high-affinity interaction with both IMPα2 and IMPα3 but not IMPß. Structural analysis of the pVII-NLS/IMPα2 complex confirmed a classical interaction, with the major binding site of IMPα occupied by K124 of pVII-NLS. Quantitative confocal laser scanning microscopy showed that PsSiAdV pVII-NLS can confer IMPα/ß-dependent nuclear localization to GFP. PsSiAdV pVII also localized in the nucleus when expressed in the absence of other viral proteins. Importantly, in contrast to what has been reported for HAdV pVII, PsSiAdV pVII does not localize to the nucleolus. In addition, our study demonstrated that inhibition of the IMPα/ß nuclear import pathway did not prevent PsSiAdV pVII nuclear targeting, indicating the existence of alternative pathways for nuclear localization, similar to what has been previously shown for human adenovirus pVII. Further examination of other potential NLS signals, characterization of alternative nuclear import pathways, and investigation of pVII nuclear targeting across different adenovirus species is recommended to fully elucidate the role of varying nuclear import pathways in the nuclear localization of pVII.

Suppression of MR1 by human cytomegalovirus inhibits MAIT cell activation.

Introduction: The antigen presentation molecule MHC class I related protein-1 (MR1) is best characterized by its ability to present bacterially derived metabolites of vitamin B2 biosynthesis to mucosal-associated invariant T-cells (MAIT cells). Methods: Through in vitro human cytomegalovirus (HCMV) infection in the presence of MR1 ligand we investigate the modulation of MR1 expression. Using coimmunoprecipitation, mass spectrometry, expression by recombinant adenovirus and HCMV deletion mutants we investigate HCMV gpUS9 and its family members as potential regulators of MR1 expression. The functional consequences of MR1 modulation by HCMV infection are explored in coculture activation assays with either Jurkat cells engineered to express the MAIT cell TCR or primary MAIT cells. MR1 dependence in these activation assays is established by addition of MR1 neutralizing antibody and CRISPR/Cas-9 mediated MR1 knockout. Results: Here we demonstrate that HCMV infection efficiently suppresses MR1 surface expression and reduces total MR1 protein levels. Expression of the viral glycoprotein gpUS9 in isolation could reduce both cell surface and total MR1 levels, with analysis of a specific US9 HCMV deletion mutant suggesting that the virus can target MR1 using multiple mechanisms. Functional assays with primary MAIT cells demonstrated the ability of HCMV infection to inhibit bacterially driven, MR1-dependent activation using both neutralizing antibodies and engineered MR1 knockout cells. Discussion: This study identifies a strategy encoded by HCMV to disrupt the MR1:MAIT cell axis. This immune axis is less well characterized in the context of viral infection. HCMV encodes hundreds of proteins, some of which regulate the expression of antigen presentation molecules. However the ability of this virus to regulate the MR1:MAIT TCR axis has not been studied in detail.

Varicella Zoster Virus Impairs Expression of the Nonclassical Major Histocompatibility Complex Class I-Related Gene Protein (MR1).

The antigen presentation molecule MR1 (major histocompatibility complex, class I-related) presents ligands derived from the riboflavin (vitamin B) synthesis pathway, which is not present in mammalian species or viruses, to mucosal-associated invariant T (MAIT) cells. In this study, we demonstrate that varicella zoster virus (VZV) profoundly suppresses MR1 expression. We show that VZV targets the intracellular reservoir of immature MR1 for degradation, while preexisting, ligand-bound cell surface MR1 is protected from such targeting, thereby highlighting an intricate temporal relationship between infection and ligand availability. We also identify VZV open reading frame (ORF) 66 as functioning to suppress MR1 expression when this viral protein is expressed during transient transfection, but this is not apparent during infection with a VZV mutant virus lacking ORF66 expression. This indicates that VZV is likely to encode multiple viral genes that target MR1. Overall, we identify an immunomodulatory function of VZV whereby infection suppresses the MR1 biosynthesis pathway.

Adenoviruses in Avian Hosts: Recent Discoveries Shed New Light on Adenovirus Diversity and Evolution.

While adenoviruses cause infections in a wide range of vertebrates, members of the genus Atadenovirus, Siadenovirus, and Aviadenovirus predominantly infect avian hosts. Several recent studies on avian adenoviruses have encouraged us to re-visit previously proposed adenovirus evolutionary concepts. Complete genomes and partial DNA polymerase sequences of avian adenoviruses were extracted from NCBI and analysed using various software. Genomic analyses and constructed phylogenetic trees identified the atadenovirus origin from an Australian native passerine bird in contrast to the previously established reptilian origin. In addition, we demonstrated that the theories on higher AT content in atadenoviruses are no longer accurate and cannot be considered as a species demarcation criterion for the genus Atadenovirus. Phylogenetic reconstruction further emphasised the need to reconsider siadenovirus origin, and we recommend extended studies on avian adenoviruses in wild birds to provide finer evolutionary resolution.

Developing student codesigned immersive virtual reality simulations for teaching of challenging concepts in molecular and cellular biology.

Molecular biology theory represents a critical scaffold, which underpins multiple disciplines within life sciences education. However, it is well-documented that undergraduate students can struggle to achieve deeper understanding of key concepts and/or their application. One challenging, contributory aspect is the "invisible" nature of molecular biology processes compounded by critical 3D spatial orientations of the principal components and their interactions. Molecular theory specifically requires students to construct accurate, mental spatial models to develop their understanding. However, much of the traditional teaching and examination of such theory is limited to 2D representations. Technology-enhanced, complementary teaching and examination approaches, which engage students with spatial aspects of theoretical concepts, offer an exciting opportunity to support student learning in this area. In this study, we have explored the integration of an immersive virtual reality simulation based on a challenging molecular biology concept within an existing module taught at University College Cork. A mixed methods approach, grounded in learning theory, was undertaken to assess the student user and learning experience. The consensus response from students was one of enhanced learning, understanding, engagement, and motivation. Student partnership in the process of simulation design and integration was key to delivering the fully integrated experience.

Targeting Aryl Hydrocarbon Receptor Signaling Enhances Type I Interferon-Independent Resistance to Herpes Simplex Virus.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcript factor that plays an important role in regulating immunity and cell differentiation. However, its role in cell-autonomous antiviral resistance has not been fully elucidated. Here, we show that interruption of AHR signaling in human cells by a chemical antagonist or genetic targeting led to significant reductions in the replication of herpes simplex virus 1 (HSV-1) and cytomegalovirus (CMV), revealing an unexpected proviral function of AHR. Interestingly, the enhanced viral control in the absence of AHR is independent of type I interferon (IFN) signaling. Together, these results reveal a previously unknown function of AHR in promoting viral replication in vitro and suggest a potential intervention point for treating viral disease. IMPORTANCE This study describes how a virus might utilize host aryl hydrocarbon receptor signaling to promote its replication, even in the presence of type I interferons.

Varicella zoster virus encodes a viral decoy RHIM to inhibit cell death.

Herpesviruses are known to encode a number of inhibitors of host cell death, including RIP Homotypic Interaction Motif (RHIM)-containing proteins. Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily and is responsible for causing chickenpox and shingles. We have identified a novel viral RHIM in the VZV capsid triplex protein, open reading frame (ORF) 20, that acts as a host cell death inhibitor. Like the human cellular RHIMs in RIPK1 and RIPK3 that stabilise the necrosome in TNF-induced necroptosis, and the viral RHIM in M45 from murine cytomegalovirus that inhibits cell death, the ORF20 RHIM is capable of forming fibrillar functional amyloid complexes. Notably, the ORF20 RHIM forms hybrid amyloid complexes with human ZBP1, a cytoplasmic sensor of viral nucleic acid. Although VZV can inhibit TNF-induced necroptosis, the ORF20 RHIM does not appear to be responsible for this inhibition. In contrast, the ZBP1 pathway is identified as important for VZV infection. Mutation of the ORF20 RHIM renders the virus incapable of efficient spread in ZBP1-expressing HT-29 cells, an effect which can be reversed by the inhibition of caspases. Therefore we conclude that the VZV ORF20 RHIM is important for preventing ZBP1-driven apoptosis during VZV infection, and propose that it mediates this effect by sequestering ZBP1 into decoy amyloid assemblies.

Virus-Mediated Suppression of the Antigen Presentation Molecule MR1.

The antigen-presenting molecule MR1 presents microbial metabolites related to vitamin B2 biosynthesis to mucosal-associated invariant T cells (MAIT cells). Although bacteria and fungi drive the MR1 biosynthesis pathway, viruses have not previously been implicated in MR1 expression or its antigen presentation. We demonstrate that several herpesviruses inhibit MR1 cell surface upregulation, including a potent inhibition by herpes simplex virus type 1 (HSV-1). This virus profoundly suppresses MR1 cell surface expression and targets the molecule for proteasomal degradation, whereas ligand-induced cell surface expression of MR1 prior to infection enables MR1 to escape HSV-1-dependent targeting. HSV-1 downregulation of MR1 is dependent on de novo viral gene expression, and we identify the Us3 viral gene product as functioning to target MR1. Furthermore, HSV-1 downregulation of MR1 disrupts MAIT T cell receptor (TCR) activation. Accordingly, virus-mediated targeting of MR1 defines an immunomodulatory strategy that functionally disrupts the MR1-MAIT TCR axis.

Manipulation of the Innate Immune Response by Varicella Zoster Virus.

Varicella zoster virus (VZV) is the causative agent of chickenpox (varicella) and shingles (herpes zoster). VZV and other members of the herpesvirus family are distinguished by their ability to establish a latent infection, with the potential to reactivate and spread virus to other susceptible individuals. This lifelong relationship continually subjects VZV to the host immune system and as such VZV has evolved a plethora of strategies to evade and manipulate the immune response. This review will focus on our current understanding of the innate anti-viral control mechanisms faced by VZV. We will also discuss the diverse array of strategies employed by VZV to regulate these innate immune responses and highlight new knowledge on the interactions between VZV and human innate immune cells.

Granzyme B Cleaves Multiple Herpes Simplex Virus 1 and Varicella-Zoster Virus (VZV) Gene Products, and VZV ORF4 Inhibits Natural Killer Cell Cytotoxicity.

Immune regulation of alphaherpesvirus latency and reactivation is critical for the control of virus pathogenesis. This is evident for herpes simplex virus 1 (HSV-1), where cytotoxic T lymphocytes (CTLs) prevent viral reactivation independent of apoptosis induction. This inhibition of HSV-1 reactivation has been attributed to granzyme B cleavage of HSV infected cell protein 4 (ICP4); however, it is unknown whether granzyme B cleavage of ICP4 can directly protect cells from CTL cytotoxicity. Varicella zoster virus (VZV) is closely related to HSV-1; however, it is unknown whether VZV proteins contain granzyme B cleavage sites. Natural killer (NK) cells play a central role in VZV and HSV-1 pathogenesis and, like CTLs, utilize granzyme B to kill virally infected target cells. However, whether alphaherpesvirus granzyme B cleavage sites could modulate NK cell-mediated cytotoxicity has yet to be established. This study aimed to identify novel HSV-1 and VZV gene products with granzyme B cleavage sites and assess whether they could protect cells from NK cell-mediated cytotoxicity. We have demonstrated that HSV ICP27, VZV open reading frame 62 (ORF62), and VZV ORF4 are cleaved by granzyme B. However, in an NK cell cytotoxicity assay, only VZV ORF4 conferred protection from NK cell-mediated cytotoxicity. The granzyme B cleavage site in ORF4 was identified via site-directed mutagenesis and, surprisingly, the mutation of this cleavage site did not alter the ability of ORF4 to modulate NK cell cytotoxicity, suggesting that ORF4 has a novel immunoevasive function that is independent from the granzyme B cleavage site.IMPORTANCE HSV-1 causes oral and genital herpes and establishes life-long latency in sensory ganglia. HSV-1 reactivates multiple times in a person's life and can cause life-threatening disease in immunocompromised patients. VZV is closely related to HSV-1, causes chickenpox during primary infection, and establishes life-long latency in ganglia, from where it can reactivate to cause herpes zoster (shingles). Unlike HSV-1, VZV only infects humans, and there are limited model systems; thus, little is known concerning how VZV maintains latency and why VZV reactivates. Through studying the link between immune cell cytotoxic functions, granzyme B, and viral gene products, an increased understanding of viral pathogenesis will be achieved.

Functional paralysis of human natural killer cells by alphaherpesviruses.

Natural killer (NK) cells are implicated as important anti-viral immune effectors in varicella zoster virus (VZV) infection. VZV can productively infect human NK cells, yet it is unknown how, or if, VZV can directly affect NK cell function. Here we demonstrate that VZV potently impairs the ability of NK cells to respond to target cell stimulation in vitro, leading to a loss of both cytotoxic and cytokine responses. Remarkably, not only were VZV infected NK cells affected, but VZV antigen negative NK cells that were exposed to virus in culture were also inhibited. This powerful impairment of function was dependent on direct contact between NK cells and VZV infected inoculum cells. Profiling of the NK cell surface receptor phenotype by multiparameter flow cytometry revealed that functional receptor expression is predominantly stable. Furthermore, inhibited NK cells were still capable of releasing cytotoxic granules when the stimulation signal bypassed receptor/ligand interactions and early signalling, suggesting that VZV paralyses NK cells from responding. Phosflow examination of key components in the degranulation signalling cascade also demonstrated perturbation following culture with VZV. In addition to inhibiting degranulation, IFN-γ and TNF production were also repressed by VZV co-culture, which was most strongly regulated in VZV infected NK cells. Interestingly, the closely related virus, herpes simplex virus type 1 (HSV-1), was also capable of efficiently infecting NK cells in a cell-associated manner, and demonstrated a similar capacity to render NK cells unresponsive to target cell stimulation-however HSV-1 differentially targeted cytokine production compared to VZV. Our findings progress a growing understanding of pathogen inhibition of NK cell function, and reveal a previously unreported strategy for VZV to manipulate the immune response.

Interferon-Independent Upregulation of Interferon-Stimulated Genes during Human Cytomegalovirus Infection is Dependent on IRF3 Expression.

The antiviral activity of type I interferons (IFNs) is primarily mediated by interferon-stimulated genes (ISGs). Induction of ISG transcription is achieved when type I IFNs bind to their cognate receptor and activate the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathways. Recently it has become clear that a number of viruses are capable of directly upregulating a subset of ISGs in the absence of type I IFN production. Using cells engineered to block either the response to, or production of type I IFN, the regulation of IFN-independent ISGs was examined in the context of human cytomegalovirus (HCMV) infection. Several ISGs, including IFIT1, IFIT2, IFIT3, Mx1, Mx2, CXCL10 and ISG15 were found to be upregulated transcriptionally following HCMV infection independently of type I IFN-initiated JAK-STAT signaling, but dependent on intact IRF3 signaling. ISG15 protein regulation mirrored that of its transcript with IFNß neutralization failing to completely inhibit ISG15 expression post HCMV infection. In addition, no detectable ISG15 protein expression was observed following HCMV infection in IRF3 knockdown CRISPR/Cas-9 clones indicating that IFN-independent control of ISG expression during HCMV infection of human fibroblasts is absolutely dependent on IRF3 expression.

Gal power: the diverse roles of galectins in regulating viral infections.

Viruses, as a class of pathogenic microbe, remain a significant health burden globally. Viral infections result in significant morbidity and mortality annually and many remain in need of novel vaccine and anti-viral strategies. The development of effective novel anti-viral therapeutics, in particular, requires detailed understanding of the mechanism of viral infection, and the host response, including the innate and adaptive arms of the immune system. In recent years, the role of glycans and lectins in pathogen-host interactions has become an increasingly relevant issue. This review focuses on the interactions between a specific lectin family, galectins, and the broad range of viral infections in which they play a role. Discussed are the diverse activities that galectins play in interacting directly with virions or the cells they infect, to promote or inhibit viral infection. In addition we describe how galectin expression is regulated both transcriptionally and post-transcriptionally by viral infections. We also compare the contribution of known galectin-mediated immune modulation, across a range of innate and adaptive immune anti-viral responses, to the outcome of viral infections.

Restriction of Human Cytomegalovirus Infection by Galectin-9.

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus. While HCMV infection is generally asymptomatic in the immunocompetent, it can have devastating consequences in those with compromised or underdeveloped immune systems, including transplant recipients and neonates. Galectins are a widely expressed protein family that have been demonstrated to modulate both antiviral immunity and regulate direct host-virus interactions. The potential for galectins to directly modulate HCMV infection has not previously been studied, and our results reveal that galectin-9 (Gal-9) can potently inhibit HCMV infection. Gal-9-mediated inhibition of HCMV was dependent upon its carbohydrate recognition domains and thus dependent on glycan interactions. Temperature shift studies revealed that Gal-9 specific inhibition was mediated primarily at the level of virus-cell fusion and not binding. Additionally, we found that during reactivation of HCMV in hematopoietic stem cell transplant (HSCT) patients soluble Gal-9 is upregulated. This study provides the first evidence for Gal-9 functioning as a potent antiviral defense effector molecule against HCMV infection and identifies it as a potential clinical candidate to restrict HCMV infections.IMPORTANCE Human cytomegalovirus (HCMV) continues to cause serious and often life-threatening disease in those with impaired or underdeveloped immune systems. This virus is able to infect and replicate in a wide range of human cell types, which enables the virus to spread to other individuals in a number of settings. Current antiviral drugs are associated with a significant toxicity profile, and there is no vaccine; these factors highlight a need to identify additional targets for the development of anti-HCMV therapies. We demonstrate for the first time that secretion of a member of the galectin family of proteins, galectin-9 (Gal-9), is upregulated during natural HCMV-reactivated infection and that this soluble cellular protein possesses a potent capacity to block HCMV infection by inhibiting virus entry into the host cell. Our findings support the possibility of harnessing the antiviral properties of Gal-9 to prevent HCMV infection and disease.

Interferon-Independent Innate Responses to Cytomegalovirus.

The critical role of interferons (IFNs) in mediating the innate immune response to cytomegalovirus (CMV) infection is well established. However, in recent years the functional importance of the IFN-independent antiviral response has become clearer. IFN-independent, IFN regulatory factor 3 (IRF3)-dependent interferon-stimulated gene (ISG) regulation in the context of CMV infection was first documented 20 years ago. Since then several IFN-independent, IRF3-dependent ISGs have been characterized and found to be among the most influential in the innate response to CMV. These include virus inhibitory protein, endoplasmic reticulum-associated IFN-inducible (viperin), ISG15, members of the interferon inducible protein with tetratricopeptide repeats (IFIT) family, interferon-inducible transmembrane (IFITM) proteins and myxovirus resistance proteins A and B (MxA, MxB). IRF3-independent, IFN-independent activation of canonically IFN-dependent signaling pathways has also been documented, such as IFN-independent biphasic activation of signal transducer and activator of transcription 1 (STAT1) during infection of monocytes, differential roles of mitochondrial and peroxisomal mitochondrial antiviral-signaling protein (MAVS), and the ability of human CMV (HCMV) immediate early protein 1 (IE1) protein to reroute IL-6 signaling and activation of STAT1 and its associated ISGs. This review examines the role of identified IFN-independent ISGs in the antiviral response to CMV and describes pathways of IFN-independent innate immune response induction by CMV.

Varicella zoster virus productively infects human natural killer cells and manipulates phenotype.

Varicella zoster virus (VZV) is a ubiquitous human alphaherpesvirus, responsible for varicella upon primary infection and herpes zoster following reactivation from latency. To establish lifelong infection, VZV employs strategies to evade and manipulate the immune system to its advantage in disseminating virus. As innate lymphocytes, natural killer (NK) cells are part of the early immune response to infection, and have been implicated in controlling VZV infection in patients. Understanding of how VZV directly interacts with NK cells, however, has not been investigated in detail. In this study, we provide the first evidence that VZV is capable of infecting human NK cells from peripheral blood in vitro. VZV infection of NK cells is productive, supporting the full kinetic cascade of viral gene expression and producing new infectious virus which was transmitted to epithelial cells in culture. We determined by flow cytometry that NK cell infection with VZV was not only preferential for the mature CD56dim NK cell subset, but also drove acquisition of the terminally-differentiated maturity marker CD57. Interpretation of high dimensional flow cytometry data with tSNE analysis revealed that culture of NK cells with VZV also induced a potent loss of expression of the low-affinity IgG Fc receptor CD16 on the cell surface. Notably, VZV infection of NK cells upregulated surface expression of chemokine receptors associated with trafficking to the skin -a crucial site in VZV disease where highly infectious lesions develop. We demonstrate that VZV actively manipulates the NK cell phenotype through productive infection, and propose a potential role for NK cells in VZV pathogenesis.

Varicella-Zoster Virus ORF63 Protects Human Neuronal and Keratinocyte Cell Lines from Apoptosis and Changes Its Localization upon Apoptosis Induction.

There are many facets of varicella-zoster virus (VZV) pathogenesis that are not fully understood, such as the mechanisms involved in the establishment of lifelong latency, reactivation, and development of serious conditions like postherpetic neuralgia (PHN). Virus-encoded modulation of apoptosis has been suggested to play an important role in these processes. VZV open reading frame 63 (ORF63) has been shown to modulate apoptosis in a cell-type-specific manner, but the impact of ORF63 on cell death pathways has not been examined in isolation in the context of human cells. We sought to elucidate the effect of VZV ORF63 on apoptosis induction in human neuron and keratinocyte cell lines. VZV ORF63 was shown to protect differentiated SH-SY5Y neuronal cells against staurosporine-induced apoptosis. In addition, VZV infection did not induce high levels of apoptosis in the HaCaT human keratinocyte line, highlighting a delay in apoptosis induction. VZV ORF63 was shown to protect HaCaT cells against both staurosporine- and Fas ligand-induced apoptosis. Confocal microscopy was utilized to examine VZV ORF63 localization during apoptosis induction. In VZV infection and ORF63 expression alone, VZV ORF63 became more cytoplasmic, with aggregate formation during apoptosis induction. Taken together, this suggests that VZV ORF63 protects both differentiated SH-SY5Y cells and HaCaT cells from apoptosis induction and may mediate this effect through its localization change during apoptosis. VZV ORF63 is a prominent VZV gene product in both productive and latent infection and thus may play a critical role in VZV pathogenesis by aiding neuron and keratinocyte survival.IMPORTANCE VZV, a human-specific alphaherpesvirus, causes chicken pox during primary infection and establishes lifelong latency in the dorsal root ganglia (DRG). Reactivation of VZV causes shingles, which is often followed by a prolonged pain syndrome called postherpetic neuralgia. It has been suggested that the ability of the virus to modulate cell death pathways is linked to its ability to establish latency and reactivate. The significance of our research lies in investigating the ability of ORF63, a VZV gene product, to inhibit apoptosis in novel cell types crucial for VZV pathogenesis. This will allow an increased understanding of critical enigmatic components of VZV pathogenesis.