Current in vivo models of varicella-zoster virus neurotropism

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Varicella-zoster virus (VZV), an exclusively human herpesvirus, causes chickenpox and establishes a latent infection in ganglia, reactivating decades later to produce zoster and associated neurological complications. An understanding of VZV neurotropism in humans has long been hampered by the lack of an adequate animal model. For example, experimental inoculation of VZV in small animals including guinea pigs and cotton rats results in the infection of ganglia but not a rash. The severe combined immune deficient human (SCID-hu) model allows the study of VZV neurotropism for human neural sub-populations. Simian varicella virus (SVV) infection of rhesus macaques (RM) closely resembles both human primary VZV infection and reactivation, with analyses at early times after infection providing valuable information about the extent of viral replication and the host immune responses. Indeed, a critical role for CD4 T-cell immunity during acute SVV infection as well as reactivation has emerged based on studies using RM. Herein we discuss the results of efforts from different groups to establish an animal model of VZV neurotropism

The latency-associated transcript locus of herpes simplex virus 1 is a virulence determinant in human skin

Herpes simplex virus 1 (HSV-1) infects skin and mucosal epithelial cells and then travels along axons to establish latency in the neurones of sensory ganglia. Although viral gene expression is restricted during latency, the latency-associated transcript (LAT) locus encodes many RNAs, including a 2 kb intron known as the hallmark of HSV-1 latency. Here, we studied HSV-1 infection and the role of the LAT locus in human skin xenografts in vivo and in cultured explants. We sequenced the genomes of our stock of HSV-1 strain 17syn+ and seven derived viruses and found nonsynonymous mutations in many viral proteins that had no impact on skin infection. In contrast, deletions in the LAT locus severely impaired HSV-1 replication and lesion formation in skin. However, skin replication was not affected by impaired intron splicing. Moreover, although the LAT locus has been implicated in regulating gene expression in neurones, we observed only small changes in transcript levels that were unrelated to the growth defect in skin, suggesting that its functions in skin may be different from those in neurones. Thus, although the LAT locus was previously thought to be dispensable for lytic infection, we show that it is a determinant of HSV-1 virulence during lytic infection of human skin

Entrapment of Viral Capsids in Nuclear PML Cages Is an Intrinsic Antiviral Host Defense against Varicella-Zoster Virus

The herpesviruses, like most other DNA viruses, replicate in the host cell nucleus. Subnuclear domains known as promyelocytic leukemia protein nuclear bodies (PML-NBs), or ND10 bodies, have been implicated in restricting early herpesviral gene expression. These viruses have evolved countermeasures to disperse PML-NBs, as shown in cells infected in vitro, but information about the fate of PML-NBs and their functions in herpesvirus infected cells in vivo is limited. Varicella-zoster virus (VZV) is an alphaherpesvirus with tropism for skin, lymphocytes and sensory ganglia, where it establishes latency. Here, we identify large PML-NBs that sequester newly assembled nucleocapsids (NC) in neurons and satellite cells of human dorsal root ganglia (DRG) and skin cells infected with VZV in vivo. Quantitative immuno-electron microscopy revealed that these distinctive nuclear bodies consisted of PML fibers forming spherical cages that enclosed mature and immature VZV NCs. Of six PML isoforms, only PML IV promoted the sequestration of NCs. PML IV significantly inhibited viral infection and interacted with the ORF23 capsid surface protein, which was identified as a target for PML-mediated NC sequestration. The unique PML IV C-terminal domain was required for both capsid entrapment and antiviral activity. Similar large PML-NBs, termed clastosomes, sequester aberrant polyglutamine (polyQ) proteins, such as Huntingtin (Htt), in several neurodegenerative disorders. We found that PML IV cages co-sequester HttQ72 and ORF23 protein in VZV infected cells. Our data show that PML cages contribute to the intrinsic antiviral defense by sensing and entrapping VZV nucleocapsids, thereby preventing their nuclear egress and inhibiting formation of infectious virus particles. The efficient sequestration of virion capsids in PML cages appears to be the outcome of a basic cytoprotective function of this distinctive category of PML-NBs in sensing and safely containing nuclear aggregates of aberrant proteins

Molecular mechanisms of varicella zoster virus pathogenesis. Nat Rev Microbiol 12:197–210. http: //dx.doi.org/10.1038/nrmicro3215

Abstract | Varicella zoster virus (VZV) is the causative agent of varicella (chickenpox) and zoster (shingles). Investigating VZV pathogenesis is challenging as VZV is a human-specific virus and infection does not occur, or is highly restricted, in other species. However, the use of human tissue xenografts in mice with severe combined immunodeficiency (SCID) enables the analysis of VZV infection in differentiated human cells in their typical tissue microenvironment. Xenografts of human skin, dorsal root ganglia or foetal thymus that contains T cells can be infected with mutant viruses or in the presence of inhibitors of viral or cellular functions to assess the molecular mechanisms of VZV-host interactions. In this Review, we discuss how these models have improved our understanding of VZV pathogenesis

Neuronal Subtype and Satellite Cell Tropism Are Determinants of Varicella-Zoster Virus Virulence in Human Dorsal Root Ganglia Xenografts <i>In Vivo</i>

<div><p>Varicella zoster virus (VZV), a human alphaherpesvirus, causes varicella during primary infection. VZV reactivation from neuronal latency may cause herpes zoster, post herpetic neuralgia (PHN) and other neurologic syndromes. To investigate VZV neuropathogenesis, we developed a model using human dorsal root ganglia (DRG) xenografts in immunodeficient (SCID) mice. The SCID DRG model provides an opportunity to examine characteristics of VZV infection that occur in the context of the specialized architecture of DRG, in which nerve cell bodies are ensheathed by satellite glial cells (SGC) which support neuronal homeostasis. We hypothesized that VZV exhibits neuron-subtype specific tropism and that VZV tropism for SGC contributes to VZV-related ganglionopathy. Based on quantitative analyses of viral and cell protein expression in DRG tissue sections, we demonstrated that, whereas DRG neurons had an immature neuronal phenotype prior to implantation, subtype heterogeneity was observed within 20 weeks and SGC retained the capacity to maintain neuronal homeostasis longterm. Profiling VZV protein expression in DRG neurons showed that VZV enters peripherin+ nociceptive and RT97+ mechanoreceptive neurons by both axonal transport and contiguous spread from SGC, but replication in RT97+ neurons is blocked. Restriction occurs even when the SGC surrounding the neuronal cell body were infected and after entry and ORF61 expression, but before IE62 or IE63 protein expression. Notably, although contiguous VZV spread with loss of SGC support would be predicted to affect survival of both nociceptive and mechanoreceptive neurons, RT97+ neurons showed selective loss relative to peripherin+ neurons at later times in DRG infection. Profiling cell factors that were upregulated in VZV-infected DRG indicated that VZV infection induced marked pro-inflammatory responses, as well as proteins of the interferon pathway and neuroprotective responses. These neuropathologic changes observed in sensory ganglia infected with VZV may help to explain the neurologic sequelae often associated with zoster and PHN.</p></div

DRG architecture is maintained in DRG xenografts.

<p>Panels A-C, DRG xenograft, 20 weeks after transplantation, stained with subtype specific markers anti-RT97 (green)/anti-peripherin (red) antibody (A-C). White arrow in (A) denotes the axon hillock at the neuronal cell body. White dotted line in (C) delineates the margin between the DRG xenograft and the murine kidney, arrow shows axons projecting into the murine kidney. Panel D, transmission electron micrograph of DRG xenograft with arrow showing myelinated nerve fiber. Panel E, DRG xenograft 80 weeks after implantation. Black arrow in (E) denotes the nerve root, panel on the right (F) is inset panel (black box) from (E) with arrows showing small, dark neurons (white) and large, light neurons (yellow).</p

VZV restriction in mechanoreceptive neurons occurs after viral entry.

<p>For each panel, one representative image is shown for staining experiments performed on multiple tissue sections (6–10 sections) for each DRG. (A-C) ORF23 capsid protein is a marker of virion entry as well as the later formation of progeny virions; virion entry is indicated by ORF23 capsid puncta at the nuclear rim (green), co-stained with cellular lamin A/C (A, red), and prior to expression of IE62 (B, red) or IE63 (C, red). (D) Dual staining for ORF23 (green) and cellular PML (red). (E) ORF23 nuclear rim staining was not observed when using pre-immune serum (green); red is N-CAM staining. (F) ORF23 capsid protein is not detected in uninfected DRG, as shown by staining for N-CAM (red) and absence of ORF23 (green). (G-H) Staining of adjacent tissue sections stained with antibody for ORF23 (green) and IE63 (G, red) or RT97 (H, red). Two neurons are circled and shown in both panels. The single 488-channel images for Panel G, shown in greyscale for better visualization, are provided for the circled neurons (1 and 2). The contrast of the G, neuron 2, ORF23 B&W panel, is enhanced so that the discrete ORF23 particles are easier to observe.</p

Mechanoreceptive neurons are selectively depleted after the acute phase of DRG infection and SGC infection contributes to VZV-induced neuronal cell loss and ganglionic damage.

<p>DRG xenografts were infected with rOka-delta_gI or rOka-gE/deltaCys (1000 PFU), which are deficient for VZV-induced SGC-neuron membrane fusion and spread in DRG, to assess the impact of infection limited primarily to SGC on neuronal survival. Representative images are shown; for each staining condition 6–10 slides were evaluated. (A) rOka-infected DRG at 70 days after infection, stained for subtype markers peripherin (green) and RT97 (red). White arrows indicate Nageotte nodules. (B) rOka-gE/deltaCys infected DRG at 28 days after infection, stained with mouse anti-RT97 (red) and rabbit anti-IE63 (green) and at 56 days after infection stained with mouse anti-RT97 (red) and rabbit anti-peripherin (green). (C) rOka-gE/deltaCys infected DRG at 56 days after infection stained with mouse anti-RT97 (red) and rabbit anti-IE63 (green). (D) rOka-delta_gI infected DRG at 70 days after infection stained with mouse anti-RT97 (red) and rabbit anti-IE63 (green).</p