Disruption of PML Nuclear Bodies Is Mediated by ORF61 SUMO-Interacting Motifs and Required for Varicella-Zoster Virus Pathogenesis in Skin

Promyelocytic leukemia protein (PML) has antiviral functions and many viruses encode gene products that disrupt PML nuclear bodies (PML NBs). However, evidence of the relevance of PML NB modification for viral pathogenesis is limited and little is known about viral gene functions required for PML NB disruption in infected cells in vivo. Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes cutaneous lesions during primary and recurrent infection. Here we show that VZV disrupts PML NBs in infected cells in human skin xenografts in SCID mice and that the disruption is achieved by open reading frame 61 (ORF61) protein via its SUMO-interacting motifs (SIMs). Three conserved SIMs mediated ORF61 binding to SUMO1 and were required for ORF61 association with and disruption of PML NBs. Mutation of the ORF61 SIMs in the VZV genome showed that these motifs were necessary for PML NB dispersal in VZV-infected cells in vitro. In vivo, PML NBs were highly abundant, especially in basal layer cells of uninfected skin, whereas their frequency was significantly decreased in VZV-infected cells. In contrast, mutation of the ORF61 SIMs reduced ORF61 association with PML NBs, most PML NBs remained intact and importantly, viral replication in skin was severely impaired. The ORF61 SIM mutant virus failed to cause the typical VZV lesions that penetrate across the basement membrane into the dermis and viral spread in the epidermis was limited. These experiments indicate that VZV pathogenesis in skin depends upon the ORF61-mediated disruption of PML NBs and that the ORF61 SUMO-binding function is necessary for this effect. More broadly, our study elucidates the importance of PML NBs for the innate control of a viral pathogen during infection of differentiated cells within their tissue microenvironment in vivo and the requirement for a viral protein with SUMO-binding capacity to counteract this intrinsic barrier

Regulation of the ORF61 Promoter and ORF61 Functions in Varicella-Zoster Virus Replication and Pathogenesis▿

Varicella-zoster virus (VZV) open reading frame 61 (ORF61) encodes a protein that transactivates viral and cellular promoters in transient-transfection assays and is the ortholog of herpes simplex virus ICP0. In this report, we mapped the ORF61 promoter and investigated its regulation by viral and cellular proteins in transient-expression experiments and by mutagenesis of the VZV genome (parent Oka strain). The 5′ boundary of the minimal ORF61 promoter required for IE62 transactivation was mapped to position −95 relative to the mRNA start site, and three noncanonical GT-rich Sp1-binding sites were documented to occur within the region comprising positions −95 to −45. Contributions of the three Sp1-binding-site motifs, designated Sp1a, Sp1b, and Sp1c, to ORF61 expression and viral replication were varied despite their similar sequences. Two sites, Sp1a and Sp1c, functioned synergistically. When both sites were mutated in the pOka genome to produce pOka-61proΔSp1ac, the mutant virus expressed significantly less ORF61 protein. Using this mutant to investigate ORF61 functions resulted in reductions in the expression levels of IE proteins, viral kinases ORF47 and ORF66, and the major glycoprotein gE, with the most impact on gE. Virion morphogenesis appeared to be intact despite minimal ORF61 expression. Pretreating melanoma cells with sodium butyrate enhanced titers of pOka-61proΔSp1ac but not pOka, suggesting that ORF61 has a role in histone deacetylase inhibition. Growth of pOka-61proΔSp1ac was impaired in SCIDhu skin xenografts, indicating that the regulation of the ORF61 promoter by Sp1 family proteins is important for ORF61 expression in vivo and that ORF61 contributes to VZV virulence at skin sites of replication

Facile Synthesis and Characterization of Chitosan Functionalized Silver Nanoparticles for Antibacterial and Anti-Lung Cancer Applications

In the treatment of bacterial contamination, the problem of multi-drug resistance is becoming an increasingly pressing concern. Nanotechnology advancements enable the preparation of metal nanoparticles that can be assembled into complex systems to control bacterial and tumor cell growth. The current work investigates the green production of chitosan functionalized silver nanoparticles (CS/Ag NPs) using Sida acuta and their inhibition efficacy against bacterial pathogens and lung cancer cells (A549). Initially, a brown color formation confirmed the synthesis, and the chemical nature of the synthesized NPs were examined by UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) coupled with energy dispersive spectroscopy (EDS), and transmission electron microscopy (TEM). FTIR demonstrated the occurrence of CS and S. acuta functional groups in the synthesized CS/Ag NPs. The electron microscopy study exhibited CS/Ag NPs with a spherical morphology and size ranges of 6–45 nm, while XRD analysis demonstrated the crystallinity of Ag NPs. Further, the bacterial inhibition property of CS/Ag NPs was examined against K. pneumoniae and S. aureus, which showed clear inhibition zones at different concentrations. In addition, the antibacterial properties were further confirmed by a fluorescent AO/EtBr staining technique. Furthermore, prepared CS/Ag NPs exhibited a potential anti-cancer character against a human lung cancer cell line (A549). In conclusion, our findings revealed that the produced CS/Ag NPs could be used as an excellent inhibitory material in industrial and clinical sectors

Origin and instability of GAA repeats: insights from Alu elements

Expansion of GAA repeats in the intron of the frataxin gene is involved in the autosomal recessive Friedreich's ataxia (FRDA). The GAA repeats arise from a stretch of adenine residues of an Alu element. These repeats have a size ranging from 7–38 in the normal population, and expand to thousands in the affected individuals. The mechanism of origin of GAA repeats, their polymorphism and stability are not well understood. In this study, we have carried out an extensive analysis of GAA repeats at several loci in the humans. This analysis indicates the association of a majority of GAA repeats with the 3′ end of an “A” stretch present in the Alu repeats. Further, the prevalence of GAA repeats correlates with the evolutionary age of Alu subfamilies as well as with their relative frequency in the genome. Our study on GAA repeat polymorphism at some loci in the normal population reveals that the length of the GAA repeats is determined by the relative length of the flanking A stretch. Based on these observations, a possible mechanism for origin of GAA repeats and modulatory effects of flanking sequences on repeat instability mediated by DNA triplex is proposed

Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry▿

Varicella-zoster virus (VZV) glycoprotein E (gE) is the most abundant glycoprotein in infected cells and, in contrast to those of other alphaherpesviruses, is essential for viral replication. The gE ectodomain contains a unique N-terminal region required for viral replication, cell-cell spread, and secondary envelopment; this region also binds to the insulin-degrading enzyme (IDE), a proposed VZV receptor. To identify new functional domains of the gE ectodomain, the effect of mutagenesis of the first cysteine-rich region of the gE ectodomain (amino acids 208 to 236) was assessed using VZV cosmids. Deletion of this region was compatible with VZV replication in vitro, but cell-cell spread of the rOka-ΔCys mutant was reduced significantly. Deletion of the cysteine-rich region abolished the binding of the mutant gE to gI but not to IDE. Preventing gE binding to gI altered the pattern of gE expression at the plasma membrane of infected cells and the posttranslational maturation of gI and its incorporation into viral particles. In contrast, deletion of the first cysteine-rich region did not affect viral entry into human tonsil T cells in vitro or into melanoma cells infected with cell-free VZV. These experiments demonstrate that gE/gI heterodimer formation is essential for efficient cell-cell spread and incorporation of gI into viral particles but that it is dispensable for infectious varicella-zoster virion formation and entry into target cells. Blocking gE binding to gI resulted in severe impairment of VZV infection of human skin xenografts in SCIDhu mice in vivo, documenting the importance of cell fusion mediated by this complex for VZV virulence in skin

Functions of the ORF9-to-ORF12 Gene Cluster in Varicella-Zoster Virus Replication and in the Pathogenesis of Skin Infection ▿

The gene cluster composed of varicella-zoster virus (VZV) open reading frame 9 (ORF9) to ORF12 encodes four putative tegument proteins and is highly conserved in most alphaherpesviruses. In these experiments, the genes within this cluster were deleted from the VZV parent Oka (POKA) individually or in combination, and the consequences for VZV replication were evaluated with cultured cells in vitro and with human skin xenografts in SCID mice in vivo. As has been reported for ORF10, ORF11 and ORF12 were dispensable for VZV replication in melanoma and human embryonic fibroblast cells. In contrast, deletion of ORF9 was incompatible with the recovery of infectious virus. ORF9 localized to the virion tegument and formed complexes with glycoprotein E, which is an essential protein, in VZV-infected cells. Recombinants lacking ORF10 and ORF11 (POKAΔ10/11), ORF11 and ORF12 (POKAΔ11/12), or ORF10, ORF11 and ORF12 (POKAΔ10/11/12) were viable in cultured cells. Their growth kinetics did not differ from those of POKA, and nucleocapsid formation and virion assembly were not disrupted. In addition, these deletion mutants showed no differences compared to POKA in infectivity levels for primary human tonsil T cells. Deletion of ORF12 had no effect on skin infection, whereas replication of POKAΔ11, POKAΔ10/11, and POKAΔ11/12 was severely reduced, and no virus was recovered from skin xenografts inoculated with POKAΔ10/11/12. These results indicate that with the exception of ORF9, the individual genes within the ORF9-to-ORF12 gene cluster are dispensable and can be deleted simultaneously without any apparent effect on VZV replication in vitro but that the ORF10-to-ORF12 cluster is essential for VZV virulence in skin in vivo

Varicella-Zoster Virus Glycoprotein E Is a Critical Determinant of Virulence in the SCID Mouse-Human Model of Neuropathogenesis ▿

Varicella-zoster virus (VZV) is a neurotropic alphaherpesvirus. VZV infection of human dorsal root ganglion (DRG) xenografts in immunodeficient mice models the infection of sensory ganglia. We examined DRG infection with recombinant VZV (recombinant Oka [rOka]) and the following gE mutants: gEΔ27-90, gEΔCys, gE-AYRV, and gE-SSTT. gEΔ27-90, which lacks the gE domain that interacts with a putative receptor insulin-degrading enzyme (IDE), replicated as extensively as rOka, producing infectious virions and significant cytopathic effects within 14 days of inoculation. Since neural cells express IDE, the gE/IDE interaction was dispensable for VZV neurotropism. In contrast, gEΔCys, which lacks gE/gI heterodimer formation, was significantly impaired at early times postinfection; viral genome copy numbers increased slowly, and infectious virus production was not detected until day 28. Delayed replication was associated with impaired cell-cell spread in ganglia, similar to the phenotype of a gI deletion mutant (rOkaΔgI). However, at later time points, infection of satellite cells and other supportive nonneuronal cells resulted in extensive DRG tissue damage and cell loss such that cytopathic changes observed at day 70 were more severe than those for rOka-infected DRG. The replication of gE-AYRV, which is impaired for trans-Golgi network (TGN) localization, and the replication of gE-SSTT, which contains mutations in an acidic cluster, were equivalent to that of rOka, causing significant cytopathic effects and infectious virus production by day 14; genome copy numbers were equivalent to those of rOka. These experiments suggest that the gE interaction with cellular IDE, gE targeting to TGN sites of virion envelopment, and phosphorylation at SSTT are dispensable for VZV DRG infection, whereas the gE/gI interaction is critical for VZV neurovirulence

Expression of Varicella-Zoster Virus Immediate-Early Regulatory Protein IE63 in Neurons of Latently Infected Human Sensory Ganglia ▿

Varicella-zoster virus (VZV) causes varicella and establishes latency in sensory nerve ganglia, but the characteristics of VZV latency are not well defined. Immunohistochemical detection of the VZV immediate-early 63 (IE63) protein in ganglion neurons has been described, but there are significant discrepancies in estimates of the frequency of IE63-positive neurons, varying from a rare event to abundant expression. We examined IE63 expression in cadaver ganglia using a high-potency rabbit anti-IE63 antibody and corresponding preimmune serum. Using standard immunohistochemical techniques, we evaluated 10 ganglia that contained VZV DNA from seven individuals. These experiments showed that neuronal pigments were a confounding variable; however, by examining sections coded to prevent investigator bias and applying statistical analysis, we determined that IE63 protein, if present, is in a very small proportion of neurons (<2.8%). To refine estimates of IE63 protein abundance, we modified our protocol by incorporating a biological stain to exclude the pigment signal and evaluated 27 ganglia from 18 individuals. We identified IE63 protein in neurons within only one ganglion, in which VZV glycoprotein E and an immune cell infiltrate were also demonstrated. Antigen preservation was shown by detection of neuronal synaptophysin. These data provide evidence that the expression of IE63 protein, which has been referred to as a latency-associated protein, is rare. Refining estimates of VZV protein expression in neurons is important for developing a hypothesis about the mechanisms by which VZV latency may be maintained