Leigh Zerboni

Varicella-Zoster Virus Transfer to Skin by T Cells and Modulation of Viral Replication by Epidermal Cell Interferon-α

Primary infection with varicella-zoster virus (VZV) causes the characteristic syndrome of varicella, or chickenpox. Experiments in severe combined immunodeficiency mice with human skin grafts (SCIDhu mice) indicate that VZV infection of T cells can mediate transfer of infectious virus to skin. VZV-infected T cells reached epithelial sites of replication within 24 h after entering the circulation. Memory CD4+ T cells were the predominant population recovered from skin in SCIDhu mice given uninfected or infected mononuclear cells, suggesting that immune surveillance by memory T cells may facilitate VZV transfer. The increased susceptibility of memory T cells to VZV infection may further enhance their role in VZV pathogenesis. During VZV skin infection, viral gene products down-regulated interferon-α to permit focal replication, whereas adjacent epidermal cells mounted a potent interferon-α response against cell–cell spread. Interleukin-1α, although activated in VZV-infected cells, did not trigger expression of endothelial adhesion molecules, thereby avoiding early recruitment of inflammatory cells. The prolonged varicella incubation period appears to represent the time required for VZV to overcome antiviral responses of epidermal cells and generate vesicles at the skin surface. Modulation of VZV replication by cutaneous innate immunity may avoid an incapacitating infection of the host that would limit opportunities for VZV transmission.

Early Reconstitution of Immunity and Decreased Severity of Herpes Zoster in Bone Marrow Transplant Recipients Immunized with Inactivated Varicella Vaccine

Varicella-zoster virus (VZV) causes herpes zoster after bone marrow transplantation (BMT). The immunogenicity of heat-inactivated varicella vaccine and effects on VZV pathogenesis were evaluated in 75 BMT patients randomized to receive vaccine or no intervention. Among 14 patients given a single dose at 1 month after transplantation, the mean (+/-SE) stimulation index (SI) was 12.20 +/- 3.13 compared with 4.83 +/- 2.74 (P = .036) in 14 unvaccinated patients, but clinical disease was not altered. Among 24 patients vaccinated at 1, 2, and 3 months, mean SI was 8.43 +/- 3.89 versus 2.00 +/- 0.33 (P = .014) in 23 unvaccinated patients at 4 months and 8.56 +/- 2.81 versus 5.30 +/- 2.47 (P = .043) at 5 months. Disease severity associated with VZV reactivation was decreased dramatically in vaccinees given three doses; severity scores were 6.4 +/- 1.0 versus 11.8 +/- 1.1 (P = .007). This experience with varicella vaccine in BMT patients is the first evidence that active immunization can reduce morbidity due to herpesvirus reactivation in high-risk populations.

Molecular mechanisms of varicella zoster virus pathogenesis

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.

Mechanisms of varicella-zoster virus neuropathogenesis in human dorsal root ganglia.

ABSTRACT Varicella-zoster virus (VZV) is a human alphaherpesvirus that infects sensory ganglia and reactivates from latency to cause herpes zoster. VZV replication was examined in human dorsal root ganglion (DRG) xenografts in mice with severe combined immunodeficiency using multiscale correlative immunofluorescence and electron microscopy. These experiments showed the presence of VZV genomic DNA, viral proteins, and virion production in both neurons and satellite cells within DRG. Furthermore, the multiscale analysis of VZV-host cell interactions revealed virus-induced cell-cell fusion and polykaryon formation between neurons and satellite cells during VZV replication in DRG in vivo. Satellite cell infection and polykaryon formation in neuron-satellite cell complexes provide mechanisms to amplify VZV entry into neuronal cell bodies, which is necessary for VZV transfer to skin in the affected dermatome during herpes zoster. These mechanisms of VZV neuropathogenesis help to account for the often severe neurologic consequences of herpes zoster.

Construction of varicella-zoster virus recombinants from parent Oka cosmids and demonstration that ORF65 protein is dispensable for infection of human skin and T cells in the SCID-hu mouse model.

ABSTRACT We generated an ORF65 deletion mutant by using a cosmid system constructed from the genome of a low-passage clinical isolate (P-Oka). Using the SCID-hu mouse model, we demonstrated that the ORF65 protein is dispensable for viral replication in skin and T cells, which are critical host cell targets during primary varicella-zoster virus infection.

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.

Analysis of the Persistence of Humoral and Cellular Immunity in Children and Adults Immunized with Varicella Vaccine

The persistence of humoral and cellular immunity to varicella-zoster virus (VZV) was evaluated in 60 children and 18 adults immunized with live attenuated VZV vaccine. At a mean of 5 years after vaccination, 93% of children and 94% of adults had IgG antibodies to VZV as determined by ELISA. VZV antibody concentrations were significantly higher at 5 years than at 1 year after immunization in children and adults. Cell-mediated immunity to VZV was detected in 87% of children and 94% of adults at 5 years. The mean stimulation index was significantly higher at 5 years than at 1 year among children and adults. Cytokine responses to VZV, including interleukin-2, interferon-gamma, and interleukin-10 were equivalent between children and adults at 5 years. In summary, varicella immunization induced long-term humoral and cellular immunity, and initial differences between cell-mediated responses in children and adults diminished over time.

Mutational Analysis of the Repeated Open Reading Frames, ORFs 63 and 70 and ORFs 64 and 69, of Varicella-Zoster Virus

ABSTRACT Varicella-zoster virus (VZV) open reading frame 63 (ORF63), located between nucleotides 110581 and 111417 in the internal repeat region, encodes a nuclear phosphoprotein which is homologous to herpes simplex virus type 1 (HSV-1) ICP22 and is duplicated in the terminal repeat region as ORF70 (nucleotides 118480 to 119316). We evaluated the role of ORFs 63 and 70 in VZV replication, using recombinant VZV cosmids and PCR-based mutagenesis to make single and dual deletions of these ORFs. VZV was recovered within 8 to 10 days when cosmids with single deletions were transfected into melanoma cells along with the three intact VZV cosmids. In contrast, VZV was not detected in transfections carried out with a dual deletion cosmid. Infectious virus was recovered when ORF63 was cloned into a nonnative AvrII site in this cosmid, confirming that failure to generate virus was due to the dual ORF63/70 deletion and that replication required at least one gene copy. This requirement may be related to our observation that ORF63 interacts directly with ORF62, the major immediate-early transactivating protein of VZV. ORF64 is located within the inverted repeat region between nucleotides 111565 and 112107; it has some homology to the HSV-1 Us10 gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64 and ORF69 were deleted individually or simultaneously using the VZV cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques with the same kinetics and morphology as viruses generated with the parental cosmids. The dual deletion of ORF64 and ORF69 was associated with an abnormal plaque phenotype characterized by very large, multinucleated syncytia. Finally, all of the deletion mutants that yielded recombinants retained infectivity for human T cells in vitro and replicated efficiently in human skin in the SCIDhu mouse model of VZV pathogenesis.

T-Cell Tropism and the Role of ORF66 Protein in Pathogenesis of Varicella-Zoster Virus Infection

ABSTRACT The pathogenesis of varicella-zoster virus (VZV) involves a cell-associated viremia during which infectious virus is carried from sites of respiratory mucosal inoculation to the skin. We now demonstrate that VZV infection of T cells is associated with robust virion production and modulation of the apoptosis and interferon pathways within these cells. The VZV serine/threonine protein kinase encoded by ORF66 is essential for the efficient replication of VZV in T cells. Preventing ORF66 protein expression by stop codon insertion (pOka66S) impaired the growth of the parent Oka (pOka) strain in T cells in SCID-hu T-cell xenografts in vivo and reduced formation of VZV virions. The lack of ORF66 protein also increased the susceptibility of infected T cells to apoptosis and reduced the capacity of the virus to interfere with induction of the interferon (IFN) signaling pathway following exposure to IFN-γ. However, preventing ORF66 protein expression only slightly reduced growth in melanoma cells in culture and did not diminish virion formation in these cells. The pOka66S virus showed only a slight defect in growth in SCID-hu skin implants compared with intact pOka. These observations suggest that the ORF66 kinase plays a unique role during infection of T cells and supports VZV T-cell tropism by contributing to immune evasion and enhancing survival of infected T cells.

The Immediate-Early 63 Protein of Varicella-Zoster Virus: Analysis of Functional Domains Required for Replication In Vitro and for T-Cell and Skin Tropism in the SCIDhu Model In Vivo

ABSTRACT The immediate-early 63-kDa (IE63) protein of varicella-zoster virus (VZV) is a phosphoprotein encoded by open reading frame (ORF) ORF63/ORF70. To identify functional domains, 22 ORF63 mutations were evaluated for effects on IE63 binding to the major VZV transactivator, IE62, and on IE63 phosphorylation and nuclear localization in transient transfections, and after insertion into the viral genome with VZV cosmids. The IE62 binding site was mapped to IE63 amino acids 55 to 67, with R59/L60 being critical residues. Alanine substitutions within the IE63 center region showed that S165, S173, and S185 were phosphorylated by cellular kinases. Four mutations that changed two putative nuclear localization signal (NLS) sequences altered IE63 distribution to a cytoplasmic/nuclear pattern. Only three of 22 mutations in ORF63 were compatible with recovery of infectious VZV from our cosmids, but infectivity was restored by inserting intact ORF63 into each mutated cosmid. The viable IE63 mutants had a single alanine substitution, altering T171, S181, or S185. These mutants, rOKA/ORF63rev[T171], rOKA/ORF63rev[S181], and rOKA/ORF63rev[S185], produced less infectious virus and had a decreased plaque phenotype in vitro. ORF47 kinase protein and glycoprotein E (gE) synthesis was reduced, indicating that IE63 contributed to optimal expression of early and late gene products. The three IE63 mutants replicated in skin xenografts in the SCIDhu mouse model, but virulence was markedly attenuated. In contrast, infectivity in T-cell xenografts was not altered. Comparative analysis suggested that IE63 resembled the herpes simplex virus type 1 US1.5 protein, which is expressed colinearly with ICP22 (US1). In summary, most mutations of ORF63 made with our VZV cosmid system were lethal for infectivity. The few IE63 changes that were tolerated resulted in VZV mutants with an impaired capacity to replicate in vitro. However, the IE63 mutants were attenuated in skin but not T cells in vivo, indicating that the contribution of the IE63 tegument/regulatory protein to VZV pathogenesis depends upon the differentiated human cell type which is targeted for infection within the intact tissue microenvironment.