Varicella Zoster Virus disrupts MAIT cell polyfunctional effector responses.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells that respond to riboflavin biosynthesis and cytokines through TCR-dependent and -independent pathways, respectively. MAIT cell activation plays an immunoprotective role against several pathogens, however the functional capacity of MAIT cells following direct infection or exposure to infectious agents remains poorly defined. We investigated the impact of Varicella Zoster Virus (VZV) on blood-derived MAIT cells and report virus-mediated impairment of activation, cytokine production, and altered transcription factor expression by VZV infected (antigen+) and VZV exposed (antigen-) MAIT cells in response to TCR-dependent and -independent stimulation. Furthermore, we reveal that suppression of VZV exposed (antigen-) MAIT cells is not mediated by a soluble factor from neighbouring VZV infected (antigen+) MAIT cells. Finally, we demonstrate that VZV impairs the cytolytic potential of MAIT cells in response to riboflavin synthesising bacteria. In summary, we report a virus-mediated immune-evasion strategy that disarms MAIT cell responses.

Impaired endocytosis and accumulation in early endosomal compartments defines herpes simplex virus-mediated disruption of the nonclassical MHC class I-related molecule MR1.

Presentation of metabolites by the major histocompatibility complex class I-related protein 1 (MR1) molecule to mucosal-associated invariant T cells is impaired during herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections. This is surprising given these viruses do not directly synthesise MR1 ligands. We have previously identified several HSV proteins responsible for rapidly downregulating the intracellular pool of immature MR1, effectively inhibiting new surface antigen presentation, while preexisting ligand-bound mature MR1 is unexpectedly upregulated by HSV-1. Using flow cytometry, immunoblotting, and high-throughput fluorescence microscopy, we demonstrate that the endocytosis of surface MR1 is impaired during HSV infection and that internalized molecules accumulate in EEA1-labeled early endosomes, avoiding degradation. We establish that the short MR1 cytoplasmic tail is not required for HSV-1-mediated downregulation of immature molecules; however it may play a role in the retention of mature molecules on the surface and in early endosomes. We also determine that the HSV-1 US3 protein, the shorter US3.5 kinase and the full-length HSV-2 homolog, all predominantly target mature surface rather than total MR1 levels. We propose that the downregulation of intracellular and cell surface MR1 molecules by US3 and other HSV proteins is an immune-evasive countermeasure to minimize the effect of impaired MR1 endocytosis, which might otherwise render infected cells susceptible to MR1-mediated killing by mucosal-associated invariant T cells.

Modulation of MHC and MHC-Like Molecules by Varicella Zoster Virus.

Varicella zoster virus (VZV) is a medically important human herpesvirus that has co-evolved with the human host to become a highly successful and ubiquitous pathogen. Whilst it is clear the innate and adaptive arms of the immune response play key roles in controlling this virus during both primary and reactivated infections, it is also apparent that VZV "fights back" by encoding multiple functions that impair a wide range of immune molecules. This capacity to manipulate the immune response is likely to be important in underpinning the success of VZV as a human pathogen. In this review, we will focus on the plethora of mechanisms that VZV has evolved to prevent and/or delay immune functions via regulating the expression of major histocompatibility complex (MHC) class I and MHC class II molecules, as well as several MHC-like molecules. In doing so, we will highlight both established and newly emerged VZV-encoded immunomodulatory capabilities and provide context to new avenues of research that seek to build the most comprehensive understanding of how this virus interfaces with these aspects of host immunity.

Modulation of Apoptosis and Cell Death Pathways by Varicella-Zoster Virus.

Like other herpesviruses, varicella-zoster virus (VZV) evolved a wide range of functions to modulate a broad array of host defences, presumably as a means to provide a survival advantage to the virus during infection. In addition to control of components of the adaptive immune response, VZV also modulates a range of innate responses. In this context, it has become increasingly apparent that VZV encodes specific functions that interfere with programmed cell death (PCD) pathways. This review will overview the current understanding of VZV-mediated control of PCD pathways, focussing on the three most well-defined PCD pathways: apoptosis, necroptosis and pyroptosis. We will also discuss future directions about these PCD pathways that are yet to be explored in the context of VZV infection.

Varicella zoster virus downregulates expression of the non-classical antigen presentation molecule CD1d.

BACKGROUND: The non-classical antigen presentation molecule CD1d presents lipid antigens to invariant natural killer T (iNKT) cells. Activation of these cells triggers a rapid cytokine response providing an interface between innate and adaptive immune responses. The importance of CD1d and iNKT cells in varicella zoster virus (VZV) infection has been emphasised by clinical reports of individuals with CD1d or iNKT cell deficiencies experiencing severe, disseminated varicella post-vaccination. METHODS: Three strains of VZV, VZV-S, rOka, and VZV rOka-66S were used to infect Jurkat cells. Flow cytometry of VZV- and mock-infected cells assessed the modulatory impact of VZV on CD1d. Infected cell-supernatant and transwell coculture experiments explored the role of soluble factors in VZV-mediated immunomodulation. CD1d transcripts were assessed by RT-qPCR. RESULTS: Surface and intracellular flow cytometry demonstrated CD1d was strikingly downregulated by VZV-S and rOka in both infected and VZV antigen-negative cells compared to mock. CD1d downregulation is cell-contact-dependant and CD1d transcripts are targeted by VZV. Mechanistic investigations using rOka-66S (unable to express the viral kinase ORF66), implicate this protein in CD1d modulation in infected cells. CONCLUSIONS: VZV implements multiple mechanisms targeting both CD1d transcript and protein. This provides evidence of VZV interaction with and manipulation of the CD1d-iNKT cell axis.

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.

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.

Viral Impacts on MR1 Antigen Presentation to MAIT Cells.

Mucosal-associated invariant T (MAIT) cells are abundant innate-like T cells important in antimicrobial immunity. These cells express a semi-invariant T cell receptor that recognizes the Major Histocompatibility Complex (MHC) class I-related protein 1 (MR1) in complex with small metabolite antigens derived from a range of commensal and pathogenic bacteria and yeasts, but not other pathogens such as viruses. Thus, MR1 stimulation of MAIT cells was thought to act as a sensor of bacterial infection and was not directly involved in anti-viral immunity. Surprisingly, viruses have recently been shown to directly impair MR1 antigen presentation by targeting the intracellular pool of MR1 for degradation. In this review, we summarize our current understanding of viral evasion of MR1 presentation pathway, and contrast this to evasion of other related MHC molecules. We examine MAIT cell activity in viral infection with a focus on the role of TCR-mediated activation of these innate-like cells and speculate on the selective pressure for viral evasion of MR1 antigen presentation. Overall, viral evasion of MR1 presentation uncovers a new avenue of research and implies that the MR1-MAIT cell axis is more important in viral immunity than was previously appreciated.

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.

Infection and Functional Modulation of Human Monocytes and Macrophages by Varicella-Zoster Virus.

Varicella-zoster virus (VZV) is associated with viremia during primary infection that is presumed to stem from infection of circulating immune cells. While VZV has been shown to be capable of infecting a number of different subsets of circulating immune cells, such as T cells, dendritic cells, and NK cells, less is known about the interaction between VZV and monocytes. Here, we demonstrate that blood-derived human monocytes are permissive to VZV replication in vitro VZV-infected monocytes exhibited each temporal class of VZV gene expression, as evidenced by immunofluorescent staining. VZV virions were observed on the cell surface and viral nucleocapsids were observed in the nucleus of VZV-infected monocytes by scanning electron microscopy. In addition, VZV-infected monocytes were able to transfer infectious virus to human fibroblasts. Infected monocytes displayed impaired dextran-mediated endocytosis, and cell surface immunophenotyping revealed the downregulation of CD14, HLA-DR, CD11b, and the macrophage colony-stimulating factor (M-CSF) receptor. Analysis of the impact of VZV infection on M-CSF-stimulated monocyte-to-macrophage differentiation demonstrated the loss of cell viability, indicating that VZV-infected monocytes were unable to differentiate into viable macrophages. In contrast, macrophages differentiated from monocytes prior to exposure to VZV were highly permissive to infection. This study defines the permissiveness of these myeloid cell types to productive VZV infection and identifies the functional impairment of VZV-infected monocytes.IMPORTANCE Primary VZV infection results in the widespread dissemination of the virus throughout the host. Viral transportation is known to be directly influenced by susceptible immune cells in the circulation. Moreover, infection of immune cells by VZV results in attenuation of the antiviral mechanisms used to control infection and limit spread. Here, we provide evidence that human monocytes, which are highly abundant in the circulation, are permissive to productive VZV infection. Furthermore, monocyte-derived macrophages were also highly permissive to VZV infection, although VZV-infected monocytes were unable to differentiate into macrophages. Exploring the relationships between VZV and permissive immune cells, such as human monocytes and macrophages, elucidates novel immune evasion strategies and provides further insight into the control that VZV has over the immune system.

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.

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.

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.

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.

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.

Nuclear domain 10 components upregulated via interferon during human cytomegalovirus infection potently regulate viral infection.

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that causes life-threatening disease in immunocompromised and immunonaïve individuals. Type I interferons (IFNs) are crucial molecules in the innate immune response to HCMV and are also known to upregulate several components of the interchromosomal multiprotein aggregates collectively referred to as nuclear domain 10 (ND10). In the context of herpesvirus infection, ND10 components are known to restrict gene expression. This raises the question as to whether key ND10 components (PML, Sp100 and hDaxx) act as anti-viral IFN-stimulated genes (ISGs) during HCMV infection. In this study, analysis of ND10 component transcription during HCMV infection demonstrated that PML and Sp100 were significantly upregulated whilst hDaxx expression remained unchanged. In cells engineered to block the production of, or response to, type I IFNs, upregulation of PML and Sp100 was not detected during HCMV infection. Furthermore, pre-treatment with an IFN-ß neutralizing antibody inhibited upregulation of PML and Sp100 during both infection and treatment with HCMV-infected cell supernatant. The significance of ND10 components functioning as anti-viral ISGs during HCMV infection was determined through knockdown of PML, Sp100 and hDaxx. ND10 knockdown cells were significantly more permissive to HCMV infection, as previously described but, in contrast to control cells, could support HCMV plaque formation following IFN-ß pre-treatment. This ability of HCMV to overcome the potently anti-viral effects of IFN-ß in ND10 expression deficient cells provides evidence that ND10 component upregulation is a key mediator of the anti-viral activity of IFN-ß.

Human Cytomegalovirus-Encoded Human Interleukin-10 (IL-10) Homolog Amplifies Its Immunomodulatory Potential by Upregulating Human IL-10 in Monocytes.

UNLABELLED: The human cytomegalovirus (HCMV) gene UL111A encodes cytomegalovirus-encoded human interleukin-10 (cmvIL-10), a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). This viral homolog exhibits a range of immunomodulatory functions, including suppression of proinflammatory cytokine production and dendritic cell (DC) maturation, as well as inhibition of major histocompatibility complex (MHC) class I and class II. Here, we present data showing that cmvIL-10 upregulates hIL-10, and we identify CD14(+)monocytes and monocyte-derived macrophages and DCs as major sources of hIL-10 secretion in response to cmvIL-10. Monocyte activation was not a prerequisite for cmvIL-10-mediated upregulation of hIL-10, which was dose dependent and controlled at the transcriptional level. Furthermore, cmvIL-10 upregulated expression of tumor progression locus 2 (TPL2), which is a regulator of the positive hIL-10 feedback loop, whereas expression of a negative regulator of the hIL-10 feedback loop, dual-specificity phosphatase 1 (DUSP1), remained unchanged. Engagement of the hIL-10 receptor (hIL-10R) by cmvIL-10 led to upregulation of heme oxygenase 1 (HO-1), an enzyme linked with suppression of inflammatory responses, and this upregulation was required for cmvIL-10-mediated upregulation of hIL-10. We also demonstrate an important role for both phosphatidylinositol 3-kinase (PI3K) and STAT3 in the upregulation of HO-1 and hIL-10 by cmvIL-10. In addition to upregulating hIL-10, cmvIL-10 could exert a direct immunomodulatory function, as demonstrated by its capacity to upregulate expression of cell surface CD163 when hIL-10 was neutralized. This study identifies a mechanistic basis for cmvIL-10 function, including the capacity of this viral cytokine to potentially amplify its immunosuppressive impact by upregulating hIL-10 expression. IMPORTANCE: Human cytomegalovirus (HCMV) is a large, double-stranded DNA virus that causes significant human disease, particularly in the congenital setting and in solid-organ and hematopoietic stem cell transplant patients. A prominent feature of HCMV is the wide range of viral gene products that it encodes which function to modulate host defenses. One of these is cmvIL-10, which is a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). In this study, we report that, in addition to exerting a direct biological impact, cmvIL-10 upregulates the expression of hIL-10 by primary blood-derived monocytes and that it does so by modulating existing cellular pathways. This capacity of cmvIL-10 to upregulate hIL-10 represents a mechanism by which HCMV may amplify its immunomodulatory impact during infection.

Varicella-Zoster Virus and Herpes Simplex Virus 1 Differentially Modulate NKG2D Ligand Expression during Productive Infection.

UNLABELLED: Natural killer (NK) cell-deficient patients are particularly susceptible to severe infection with herpesviruses, especially varicella-zoster virus (VZV) and herpes simplex virus 1 (HSV-1). The critical role that NK cells play in controlling these infections denotes an intricate struggle for dominance between virus and NK cell antiviral immunity; however, research in this area has remained surprisingly limited. Our study addressed this absence of knowledge and found that infection with VZV was not associated with enhanced NK cell activation, suggesting that the virus uses specific mechanisms to limit NK cell activity. Analysis of viral regulation of ligands for NKG2D, a potent activating receptor ubiquitously expressed on NK cells, revealed that VZV differentially modulates expression of the NKG2D ligands MICA, ULBP2, and ULBP3 by upregulating MICA expression while reducing ULBP2 and ULBP3 expression on the surface of infected cells. Despite being closely related to VZV, infection with HSV-1 produced a remarkably different effect on NKG2D ligand expression. A significant decrease in MICA, ULBP2, and ULBP3 was observed with HSV-1 infection at a total cellular protein level, as well as on the cell surface. We also demonstrate that HSV-1 differentially regulates expression of an additional NKG2D ligand, ULBP1, by reducing cell surface expression while total protein levels are unchanged. Our findings illustrate both a striking point of difference between two closely related alphaherpesviruses, as well as suggest a powerful capacity for VZV and HSV-1 to evade antiviral NK cell activity through novel modulation of NKG2D ligand expression. IMPORTANCE: Patients with deficiencies in NK cell function experience an extreme susceptibility to infection with herpesviruses, in particular, VZV and HSV-1. Despite this striking correlation, research into understanding how these two alphaherpesviruses interact with NK cells is surprisingly limited. Through examination of viral regulation of ligands to the activating NK cell receptor NKG2D, we reveal patterns of modulation by VZV, which were unexpectedly varied in response to regulation by HSV-1 infection. Our study begins to unravel the undoubtedly complex interactions that occur between NK cells and alphaherpesvirus infection by providing novel insights into how VZV and HSV-1 manipulate NKG2D ligand expression to modulate NK cell activity, while also illuminating a distinct variation between two closely related alphaherpesviruses.