Lack of strong innate immune reactivity renders macrophages alone unable to control productive Varicella-Zoster Virus infection in an isogenic human iPSC-derived neuronal co-culture model.

With Varicella-Zoster Virus (VZV) being an exclusive human pathogen, human induced pluripotent stem cell (hiPSC)-derived neural cell culture models are an emerging tool to investigate VZV neuro-immune interactions. Using a compartmentalized hiPSC-derived neuronal model allowing axonal VZV infection, we previously demonstrated that paracrine interferon (IFN)-α2 signalling is required to activate a broad spectrum of interferon-stimulated genes able to counteract a productive VZV infection in hiPSC-neurons. In this new study, we now investigated whether innate immune signalling by VZV-challenged macrophages was able to orchestrate an antiviral immune response in VZV-infected hiPSC-neurons. In order to establish an isogenic hiPSC-neuron/hiPSC-macrophage co-culture model, hiPSC-macrophages were generated and characterised for phenotype, gene expression, cytokine production and phagocytic capacity. Even though immunological competence of hiPSC-macrophages was shown following stimulation with the poly(dA:dT) or treatment with IFN-α2, hiPSC-macrophages in co-culture with VZV-infected hiPSC-neurons were unable to mount an antiviral immune response capable of suppressing a productive neuronal VZV infection. Subsequently, a comprehensive RNA-Seq analysis confirmed the lack of strong immune responsiveness by hiPSC-neurons and hiPSC-macrophages upon, respectively, VZV infection or challenge. This may suggest the need of other cell types, like T-cells or other innate immune cells, to (co-)orchestrate an efficient antiviral immune response against VZV-infected neurons.

Nanobody-based retargeting of an oncolytic herpesvirus for eliminating CXCR4+ GBM cells: A proof of principle.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, which remains difficult to cure. The very high recurrence rate has been partly attributed to the presence of GBM stem-like cells (GSCs) within the tumors, which have been associated with elevated chemokine receptor 4 (CXCR4) expression. CXCR4 is frequently overexpressed in cancer tissues, including GBM, and usually correlates with a poor prognosis. We have created a CXCR4-retargeted oncolytic herpesvirus (oHSV) by insertion of an anti-human CXCR4 nanobody in glycoprotein D of an attenuated HSV-1 (ΔICP34.5, ΔICP6, and ΔICP47), thereby describing a proof of principle for the use of nanobodies to target oHSVs toward specific cellular entities. Moreover, this virus has been armed with a transgene expressing a soluble form of TRAIL to trigger apoptosis. In vitro, this oHSV infects U87MG CXCR4+ and patient-derived GSCs in a CXCR4-dependent manner and, when armed, triggers apoptosis. In a U87MG CXCR4+ orthotopic xenograft mouse model, this oHSV slows down tumor growth and significantly improves mice survival. Customizing oHSVs with diverse nanobodies for targeting multiple proteins appears as an interesting approach for tackling the heterogeneity of GBM, especially GSCs. Altogether, our study must be considered as a proof of principle and a first step toward personalized GBM virotherapies to complement current treatments.

Varicella-Zoster Virus ORF9p Binding to Cellular Adaptor Protein Complex 1 Is Important for Viral Infectivity.

ORF9p (homologous to herpes simplex virus 1 [HSV-1] VP22) is a varicella-zoster virus (VZV) tegument protein essential for viral replication. Even though its precise functions are far from being fully described, a role in the secondary envelopment of the virus has long been suggested. We performed a yeast two-hybrid screen to identify cellular proteins interacting with ORF9p that might be important for this function. We found 31 ORF9p interaction partners, among which was AP1M1, the µ subunit of the adaptor protein complex 1 (AP-1). AP-1 is a heterotetramer involved in intracellular vesicle-mediated transport and regulates the shuttling of cargo proteins between endosomes and the trans-Golgi network via clathrin-coated vesicles. We confirmed that AP-1 interacts with ORF9p in infected cells and mapped potential interaction motifs within ORF9p. We generated VZV mutants in which each of these motifs was individually impaired and identified leucine 231 in ORF9p to be critical for the interaction with AP-1. Disrupting ORF9p binding to AP-1 by mutating leucine 231 to alanine in ORF9p strongly impaired viral growth, most likely by preventing efficient secondary envelopment of the virus. Leucine 231 is part of a dileucine motif conserved among alphaherpesviruses, and we showed that VP22 of Marek's disease virus and HSV-2 also interacts with AP-1. This indicates that the function of this interaction in secondary envelopment might be conserved as well.IMPORTANCE Herpesviruses are responsible for infections that, especially in immunocompromised patients, can lead to severe complications, including neurological symptoms and strokes. The constant emergence of viral strains resistant to classical antivirals (mainly acyclovir and its derivatives) pleads for the identification of new targets for future antiviral treatments. Cellular adaptor protein (AP) complexes have been implicated in the correct addressing of herpesvirus glycoproteins in infected cells, and the discovery that a major constituent of the varicella-zoster virus tegument interacts with AP-1 reveals a previously unsuspected role of this tegument protein. Unraveling the complex mechanisms leading to virion production will certainly be an important step in the discovery of future therapeutic targets.

ORF9p phosphorylation by ORF47p is crucial for the formation and egress of varicella-zoster virus viral particles.

The role of the tegument during the herpesvirus lytic cycle is still not clearly established, particularly at the late phase of infection, when the newly produced viral particles need to be fully assembled before being released from the infected cell. The varicella-zoster virus (VZV) protein coded by open reading frame (ORF) 9 (ORF9p) is an essential tegument protein, and, even though its mRNA is the most expressed during the productive infection, little is known about its functions. Using a GalK positive/negative selection technique, we modified a bacterial artificial chromosome (BAC) containing the complete VZV genome to create viruses expressing mutant versions of ORF9p. We showed that ORF9p is hyperphosphorylated during the infection, especially through its interaction with the viral Ser/Thr kinase ORF47p; we identified a consensus site within ORF9p recognized by ORF47p and demonstrated its importance for ORF9p phosphorylation. Strikingly, an ultrastructural analysis revealed that the mutation of this consensus site (glutamate 85 to arginine) strongly affects viral assembly and release, reproducing the ORF47 kinase-dead VZV phenotype. It also slightly diminishes the infectivity toward immature dendritic cells. Taken together, our results identify ORF9p as a new viral substrate of ORF47p and suggest a determinant role of this phosphorylation for viral infectivity, especially during the process of viral particle formation and egress.

Innate immune response and viral interference strategies developed by human herpesviruses.

Viruses are by far the most abundant parasites on earth and they have been found to infect animals, plants and bacteria. However, different types of viruses can only infect a limited range of hosts and many are species-specific. Herpesviruses constitute a large family of DNA viruses that cause diseases in animals, including humans and that are known to undergo lytic or latent infections. Consequently, they developed numerous strategies to counteract host antiviral responses to escape immune surveillance. Innate immune response constitutes the first line of host defence that limits the viral spread and also plays an important role in the activation of adaptive immune response. Viral components are recognized by specific host Pathogen Recognition Receptors (PRRs) which trigger the activation of IRF3, NF-κB and AP-1, three regulators of IFN-ß expression. IFN-ß is responsible for the induction of Interferon-Stimulated Genes (ISGs) that encode antiviral effectors important to limit the viral spread and to establish an antiviral state as well in the infected cells as in the neighbouring non-infected cells. In this review, we will summarize how host cells recognize viral components and activate downstream signalling pathways leading to the production of IFN-ß and ISGs. We will also review the most recent findings in Herpesviruses-encoded proteins involved in host immune evasion.

The varicella-zoster virus immediate-early 63 protein affects chromatin-controlled gene transcription in a cell-type dependent manner.

BACKGROUND: Varicella Zoster Virus Immediate Early 63 protein (IE63) has been shown to be essential for VZV replication, and critical for latency establishment. The activity of the protein as a transcriptional regulator is not fully clear yet. Using transient transfection assays, IE63 has been shown to repress viral and cellular promoters containing typical TATA boxes by interacting with general transcription factors. RESULTS: In this paper, IE63 regulation properties on endogenous gene expression were evaluated using an oligonucleotide-based micro-array approach. We found that IE63 modulates the transcription of only a few genes in HeLa cells including genes implicated in transcription or immunity. Furthermore, we showed that this effect is mediated by a modification of RNA POL II binding on the promoters tested and that IE63 phosphorylation was essential for these effects. In MeWo cells, the number of genes whose transcription was modified by IE63 was somewhat higher, including genes implicated in signal transduction, transcription, immunity, and heat-shock signalling. While IE63 did not modify the basal expression of several NF-kappaB dependent genes such as IL-8, ICAM-1, and IkappaBalpha, it modulates transcription of these genes upon TNFalpha induction. This effect was obviously correlated with the amount of p65 binding to the promoter of these genes and with histone H3 acetylation and HDAC-3 removal. CONCLUSION: While IE63 only affected transcription of a small number of cellular genes, it interfered with the TNF-inducibility of several NF-kappaB dependent genes by the accelerated resynthesis of the inhibitor IkappaBalpha.

Varicella-zoster virus modulates NF-kappaB recruitment on selected cellular promoters.

Intercellular adhesion molecule 1 (ICAM-1) expression is down-regulated in the center of cutaneous varicella lesions despite the expression of proinflammatory cytokines such as gamma interferon and tumor necrosis factor alpha (TNF-alpha). To study the molecular basis of this down-regulation, the ICAM-1 induction of TNF-alpha was analyzed in varicella-zoster virus (VZV)-infected melanoma cells (MeWo), leading to the following observations: (i) VZV inhibits the stimulation of icam-1 mRNA synthesis; (ii) despite VZV-induced nuclear translocation of p65, p52, and c-Rel, p50 does not translocate in response to TNF-alpha; (iii) the nuclear p65 present in VZV-infected cells is no longer associated with p50 and is unable to bind the proximal NF-kappaB site of the icam-1 promoter, despite an increased acetylation and accessibility of the promoter in response to TNF-alpha; and (iv) VZV induces the nuclear accumulation of the NF-kappaB inhibitor p100. VZV also inhibits icam-1 stimulation of TNF-alpha by strongly reducing NF-kappaB nuclear translocation in MRC5 fibroblasts. Taken together, these data show that VZV interferes with several aspects of the immune response by inhibiting NF-kappaB binding and the expression of target genes. Targeting NF-kappaB activation, which plays a central role in innate and adaptive immune responses, leads to obvious advantages for the virus, particularly in melanocytes, which are a site of viral replication in the skin.

Varicella-zoster virus IE63 protein phosphorylation by roscovitine-sensitive cyclin-dependent kinases modulates its cellular localization and activity.

During the first stage of Varicella-Zoster virus (VZV) infection, IE63 (immediate early 63 protein) is mostly expressed in the nucleus and also slightly in the cytoplasm, and during latency, IE63 localizes in the cytoplasm quite exclusively. Because phosphorylation is known to regulate various cellular mechanisms, we investigated the impact of phosphorylation by roscovitine-sensitive cyclin-dependent kinase (RSC) on the localization and functional properties of IE63. We demonstrated first that IE63 was phosphorylated on Ser-224 in vitro by CDK1 and CDK5 but not by CDK2, CDK7, or CDK9. Furthermore, by using roscovitine and CDK1 inhibitor III (CiIII), we showed that CDK1 phosphorylated IE63 on Ser-224 in vivo. By mutagenesis and the use of inhibitors, we demonstrated that phosphorylation on Ser-224 was important for the correct localization of the protein. Indeed, the substitution of these residues by alanine led to an exclusive nuclear localization of the protein, whereas mutations into glutamic acid did not modify its subcellular distribution. When transfected or VZV-infected cells were treated with roscovitine or CiIII, an exclusive nuclear localization of IE63 was also observed. By using a transfection assay, we also showed that phosphorylation on Ser-224 and Thr-222 was essential for the down-regulation of the basal activity of the VZV DNA polymerase gene promoter. Similarly, roscovitine and CiIII impaired these properties of the wild-type form of IE63. These observations clearly demonstrated the importance of CDK1-mediated IE63 phosphorylation for a correct distribution of IE63 between both cellular compartments and for its repressive activity toward the promoter tested.

Varicella-zoster virus IE63 protein represses the basal transcription machinery by disorganizing the pre-initiation complex.

Using transient transfection assays, regulation properties of varicella-zoster virus (VZV)-encoded IE63 protein were analyzed on several VZV immediate early (ORF4), early (ORF28) and late (ORF67) promoters. IE63 was shown to repress the basal activity of most of the promoters tested in epithelial (Vero) and neuronal (ND7) cells to various extents. Trans-repressing activities were also observed on heterologous viral and cellular promoters. Since a construct carrying only a TATA box sequence and a series of wild-type or mutated interleukin (IL)-8 promoters was also repressed by IE63, the role of upstream regulatory elements was ruled out. Importantly, the basal activity of a TATA-less promoter was not affected by IE63. Using a series of IE63 deletion constructs, amino acids 151-213 were shown to be essential to the trans-repressing activity in Vero cells, while in ND7 cells the essential region extended to a much larger carboxy-terminal part of the protein. We also demonstrate that IE63 is capable of disrupting the transcriptional pre-initiation complex and of interacting with several general transcription factors. The central and carboxy-terminal domains of IE63 are important for these effects. Altogether, these results demonstrate that IE63 protein is a transcriptional repressor whose activity is directed towards general transcription factors.

Regions of the varicella-zoster virus open reading frame 63 latency-associated protein important for replication in vitro are also critical for efficient establishment of latency.

Varicella-zoster virus (VZV) open reading frame 63 (ORF63) is one of the most abundant transcripts expressed during VZV latency in humans, and ORF63 protein has been detected in human ganglia by several laboratories. Deletion of over 90% of the ORF63 gene showed that the protein is required for efficient establishment of latency in rodents. We have constructed viruses with a series of mutations in ORF63. While prior experiments showed that transfection of cells with a plasmid expressing ORF63 but lacking the putative nuclear localization signal of the protein resulted in increased expression of the protein in the cytoplasm, we found that ORF63 protein remained in the nucleus in cells infected with a VZV ORF63 nuclear localization signal deletion mutant. This mutant was not impaired for growth in cell culture or for latency in rodents. Replacement of five serine or threonine phosphorylation sites in ORF63 with alanines resulted in a virus that was impaired for replication in vitro and for latency. A series of ORF63 carboxy-terminal mutants showed that the last 70 amino acids do not affect replication in vitro or latency in rodents; however, the last 108 amino acids are important for replication and latency. Thus, regions of ORF63 that are important for replication in vitro are also required for efficient establishment of latency.

Varicella-Zoster virus proteins encoded by open reading frames 14 and 67 are both dispensable for the establishment of latency in a rat model.

A rat model of Varicella-Zoster virus (VZV) provides a system in which to investigate the molecular determinants of viral latency in dorsal root ganglia (DRG). In this study, we determined whether the VZV glycoproteins gC and gI, corresponding to VZV open reading frames (ORFs) 14 and 67, respectively, were required for the establishment of latency in this model. A VZV gI deletion mutant (DeltagI) derived from a recombinant Oka (rOka) cosmid and a gC null mutant obtained from a clinical isolate were inoculated into the footpads of 6-week-old rats, and the presence of viral DNA and eight different VZV RNA transcripts corresponding to the three classes of genes was investigated by in situ RT-PCR amplification and in situ hybridization (ISH) in the DRG at 1 week, 1 month, and 18-24 months after infection. VZV DNA and restricted RNA expression was established with both deletion mutants as well as the parental rOka virus. Both VZV DNA and RNA were detected in neurons and non-neuronal cells. The pattern of viral RNA expression detected with both gC and gI mutants was restricted with transcripts for VZV genes 62 and 63 most frequently expressed 18-24 months after infection. Transcripts for VZV genes 18, 28, and 29 were also detected at these time points but at a slightly lower frequency. Transcripts for the late gene 40 were never detected. We conclude that VZV ORFs 14 and 67 are dispensable for the establishment of a latent infection in this model.

Absence of intercellular adhesion molecule 1 expression in varicella zoster virus-infected keratinocytes during herpes zoster: another immune evasion strategy?

Downregulation of major histocompatibility complex (MHC) class I, MHC-II, and intercellular adhesion molecule 1 (ICAM-1) expression in infected cell lines allows some viruses to escape host immunity. In skin lesions of varicella zoster virus (VZV), MHC-II transcripts were demonstrated in keratinocytes around vesicles, but not in VZV-infected cells. Whether other immunoevasive mechanisms are present during herpes zoster (HZ) is not yet elucidated. The aim of the study was to disclose the temporal immunohistochemical expression of immune escape mechanisms during HZ. Sequential skin biopsies were performed in 5 HZ patients. VZV IE63, CD1a, CD3, CD4, CD8, CD56, CD68, L1, HLA-DR, HLA-ABC, interleukin (IL)-6, IL-10, interferon gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), and ICAM-1 expressions were assessed on frozen sections using immunohistochemistry. Controls consisted of normal skin, herpes simplex virus (HSV) skin infections, and other distinct bullous skin diseases. HLA-DR and ICAM-1 expressions were not observed in VZV- and HSV-infected keratinocytes, contrasting with their upregulation in the surrounding epidermis and inside nonviral blisters. However, HLA-ABC expressions were not inhibited in VZV-infected keratinocytes. Furthermore, the CD4/CD8 ratio remained unmodified during the infection evolution, and this ratio was variable among patients. Increased IFNgamma, TNFalpha, and IL-6 expressions were present, but IL-10 expression only increased in later stages. In contrast to in vitro MHC-I and MHC-II downregulation, VZV infection is related to MHC-II but not MHC-I expression on infected keratinocytes. The absence of ICAM-1 expression on infected keratinocytes may reduce their antigen presentation capacities to LFA-1 ligand-bearing T cells. This may represent another VZV-associated immune escape mechanism. Increased IFNgamma, TNFalpha, and IL-6 expressions suggest a TH1 profile.

Atypical recurrent varicella in 4 patients with hemopathies.

Relapsing varicella may occur in children with HIV infection and more rarely in younger adults. Our aim was to report unusual clinical, histologic, and virologic aspects of 4 elderly patients with malignant hemopathies who had an unusual form of recurrent varicella develop. Conventional microscopy, immunohistochemistry, and in situ hybridization were applied to smears and skin biopsy specimens. The patients presented a few dozen, scattered, large, papulovesicular lesions with central crusting. No zoster-associated pain or dermatomal distribution of the lesions was noted. Conventional microscopy revealed vascular changes and epidermal alterations typical for alpha-herpes virus infection. The varicella zoster virus major viral envelope glycoproteins gE and gB, and the immediate-early varicella zoster virus IE63 protein and the corresponding genome sequence for gE were detected on Tzanck smears; they were localized in endothelial cells and keratinocytes on skin biopsy specimens. The varicella zoster virus infection in endothelial cells, the vascular involvement, and the widespread distribution of the lesions suggest that the reported eruptions are vascular rather than neural in origin. These findings invalidate the diagnosis of herpes zoster but strongly support the diagnosis of recurrent varicella in an indolent and yet unreported presentation. Furthermore, these eruptions differ from relapsing varicella in children and young adults by the age of the patients, the paucity of clinical lesions, the larger diameter of the lesions and their peculiar clinical aspect, the significantly longer time interval between primary varicella and the recurrence, the prolonged healing time of the lesions, their mild disease course, and the fact that all the lesions are in the same stage of development.

Phosphorylation of varicella-zoster virus IE63 protein by casein kinases influences its cellular localization and gene regulation activity.

During the early phase of varicella-zoster virus (VZV) infection, Immediate Early protein 63 (IE63) is expressed rapidly and abundantly in the nucleus, while during latency, this protein is confined mostly to the cytoplasm. Because phosphorylation is known to regulate many cellular events, we investigated the importance of this modification on the cellular localization of IE63 and on its regulatory properties. We demonstrate here that cellular casein kinases I and II are implicated in the in vitro and in vivo phosphorylation of IE63. A mutational approach also indicated that phosphorylation of the protein is important for its correct cellular localization in a cell type-dependent fashion. Using an activity test, we demonstrated that IE63 was able to repress the gene expression driven by two VZV promoters and that phosphorylation of the protein was required for its full repressive properties. Finally, we showed that IE63 was capable of exerting its repressive activity in the cytoplasm, as well as in the nucleus, suggesting a regulation at the transcriptional and/or post-transcriptional level.