Creating the "Dew Drop on a Rose Petal": the Molecular Pathogenesis of Varicella-Zoster Virus Skin Lesions

Abstract

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chicken pox) as the primary infection in a susceptible host. Varicella is very contagious through its transmission by direct contact with vesicular skin lesions that contain high titers of infectious virus and respiratory droplets. While the clinical manifestations of primary VZV infection are well recognized, defining the molecular mechanisms that drive VZV pathogenesis in the naive host before adaptive antiviral immunity is induced has been a challenge due to species specificity. This review focuses on advances made in identifying the differentiated human host cells targeted by VZV to cause varicella, the processes involved in viral takeover of these heterogenous cell types, and the host cell countermeasures that typically culminate in a benign illness. This work has revealed many unexpected and multifaceted mechanisms used by VZV to achieve its high prevalence and persistence in the human population.

Keywords: herpesvirus; varicella-zoster virus; viral pathogenesis.

FIG 1

Events in the pathogenesis of primary varicella-zoster virus (VZV) infection. Primary VZV infection—varicella, commonly called chickenpox—is acquired by inoculation of mucosal epithelial cells of the upper respiratory tract when a susceptible person is exposed to infectious virus transmitted by respiratory droplets from an infected person or by direct contact with VZV in skin lesions. Replication at the mucosal site of entry allows VZV transfer into tonsils and other local lymphoid tissues, where T cells become infected. The process of transfer to T cells may be facilitated by the infection of epithelial cells that penetrate deep into the tonsils. Infected dendritic cells may help transfer the virus to regional lymph nodes, enhancing T cell-mediated viremia. VZV-infected T cells can then transport the virus to skin sites of replication via a cell-associated viremia followed by exiting of infected T cells from capillaries in proximity to hair follicle cells that support the initial stage of skin infection. After a 10- to 21-day interval, the dew drop on a rose petal vesicular rash of varicella appears and VZV can spread from the newly infected person to susceptible contacts, initiating the stages of primary infection again.

FIG 2

The dew drop on a rose petal presentation of varicella vesicles and models of virion structure and VZV replication. (A) The earliest varicella vesicles are fluid-filled vesicles with a surrounding erythematous base, described as resembling a dew drop on a rose petal. (B) VZV particles have linear VZV genomes packaged into an icosahedral nucleocapsid core that is formed from proteins encoded by orf20, orf21, orf23, orf33, orf40, and orf41. Capsids are surrounded by a tegument layer, which is a less well-defined structure that is made up of proteins with known or predicted regulatory functions, including the immediate early (IE) viral transactivating factors that are encoded by orf4, orf62, and orf63; those that are encoded by the orf9-orf12 gene cluster; the two viral kinases ORF47 and ORF66; and others. The outer virion component is a lipid membrane envelope that is derived from cellular membranes with incorporated viral glycoproteins, including gB/gH-gL, which form the minimal fusion complex. During replication, enveloped VZV particles attach to cell membranes, fuse, and release tegument proteins. Uncoated capsids dock at nuclear pores, where genomic DNA is injected into the nucleus and circularizes. Immediate-early genes are expressed, followed by early and late genes. Nucleocapsids are assembled and package newly synthesized genomic DNA, move to the inner nuclear membrane, and bud across the nuclear membrane. Capsids enter the cytoplasm, virion glycoproteins mature in the trans-Golgi region, and tegument proteins assemble in vesicles; capsids undergo secondary envelopment and are transported to cell surfaces, where newly assembled virus particles are released. (Reproduced from reference 30). ER, endoplasmic reticulum.

FIG 3

Varicella-zoster virus (VZV) infection of skin. VZV trafficking from mucosal sites of entry through the vasculature to skin is promoted by the capacity of VZV to remodel T cells. VZV manipulates T cell biology through activation of the cellular ZAP70 and Akt signaling pathways from within the infected cell, which results in the enhancement of skin homing by infected T cells. T cell surface proteins are both upregulated and downregulated to produce a constellation of cell surface proteins that would typically support their migration to the skin for immune surveillance functions. Formation of the dew drop on a rose petal at the skin surface requires an interval of 10 to 21 days after exposure of a susceptible person to varicella because VZV transfer by infected T cells into skin triggers potent innate antiviral responses of cells within the skin tissue microenvironment. Visible lesions are formed only when VZV has overcome the innate barrier caused by upregulation of IFN-α, pSTAT, and PML bodies in surrounding uninfected cells. To support the process of lesion formation, VZV takes over dermal and epidermal cells by activation of the STAT3 pathway, which upregulates the antiapoptotic protein survivin while also downregulating IFN-α, STAT1, and KRT1/10 while upregulating KRT15. Uninfected T cells that traffic through sites of VZV lesion formation may amplify VZV viremia, resulting in later waves of skin lesions that are common in varicella.

FIG 4

VZV infection of T cells, trafficking to skin xenografts, and the innate interferon barrier against skin lesion formation. (A) CD3⁺ T cells from tonsils cocultured with VZV-infected fibroblasts for 48 h. Pleomorphic virus particles (120 to 200 nm) distributed between microvilli on the T cell surface (top); a representative high-magnification (×50,000) scanning electron micrograph of two viral particles; arrows indicate anti-VZV gE immunogold label (center) (adapted from reference 41); complete virions (white arrowheads) are shown in the cytoplasm (Cyt) of infected T cells (adapted from reference 26). Nuc, nucleus. (B) CD3⁺ T cells detected around hair follicles (top) and along basement membranes (bottom) at 24 h after tail vein injection (arrows) (×400); VZV lesion formation was detected 21 days after T cell transfer by anti-VZV IgG (brown; DAB) (top) compared with nonimmune IgG (lower) (×100). (C) IFN-α and phosphorylated Stat1 were detected in sections stained with anti-VZV IgG (brown); anti-IFN-α or anti-phospho-Stat1antibody are shown (purple; Vector VIP) (×200); IFN-α and nuclear translocation of pStat1 were prominent in adjacent uninfected cells but not VZV-infected cells. (D) Enlarged skin lesions (anti-VZV IgG, purple) at day 7 in animals given anti-IFN-α/β receptor blocking antibody (lower) compared with controls (top) (×100). (B to D were reproduced from reference with permission of the publisher.).

FIG 5

Viral determinants of VZV T cell tropism. (A) Comparison of the percentage of infected T cells by flow cytometry at day 7 after inoculation of T cell (thymus/liver) xenografts with wild-type (WT) VZV or varicella vaccine using antibodies to CD4, CD8, and VZV proteins (top) and viral titers at 7, 14, and 21 days (bottom; orange, wild type; blue, vaccine; mean ± SEM; dotted line, level of detection). (Adapted from reference 30). (B) Effects of mutations of the gE N terminus in T cell xenografts inoculated with rOka, rOka-Y51, rOkaS31A, or rOka-ΔY51-P187 evaluated for the percentage of infected T cells and titers (mean for those yielding virus ± SD). (Adapted from reference 50). (C) Effects of gI deletion in T cell xenografts inoculated with rOka (orange), rOkaΔgI (0), and rOka-gI@Avr rescue (pink) (mean ± SD) are shown (adapted from reference 55). Effects of mutations of the gI promoter in T cell xenografts inoculated with rOKA (blue), rOKAΔgI (orange), rOKAgI-Sp1 (green), rOKAgI-AP1 (light blue), rOKAgI-USF (pink), or rOKAgI-Sp1/USF (gray) are shown; titers were compared to rOKa; *, P < 0.05; ***, P < 0.001 (bottom) (adapted from reference 104). (D) Effects of ORF47 kinase mutations in T cell xenografts inoculated with rOka, rOka47C, or rOka47D-N; titers are shown (mean for those yielding virus ± SEM). (Adapted from reference 58). (E) Effects of ORF66 kinase mutations in T cell xenografts inoculated pOka, pOka66S, or pOka66G102A are shown; titers were compared with those of pOKa; effects of ORF66 kinase mutations on apoptosis of tonsil T cells infected with pOka, pOka66S, pOka66G102A, or pOka66S250P are shown and were stained with antibodies to VZV proteins, CD3, and active caspase-3 for the percentage of caspase-3-positive cells in each category; *, P < 0.05 compared with pOka (right). (Adapted from reference 61).

FIG 6

VZV modulation of T cell surface proteins to support skin trafficking, verification of VZV remodeling of naive T cells, and dependence on Zap70 and Akt pathway activation by VZV “from within.” (A) VZV modulation of the phenotypic hierarchy of tonsil T cell populations. Shown is the hierarchical relationship of pooled UI, Bys, and V+T cells based on surface phenotypes by principal-component analysis (PCA; left) and agglomerative clustering (right) of equal numbers of cells from each population. V+T cells were iteratively compared with a randomly selected equal number of UI and Bys T cells; a representative iteration is shown. Hierarchical cluster heatmap of V+T cells; each row denotes a T cell and each column represents a protein; color scale represents protein expression levels. Dendrograms denote the Euclidean distance between clustered cells. (B) Modulation of surface proteins on VZV-infected T cells associated with activation and migration. Boxplot of changes in cell surface protein and VZV gE protein expression (x axis) on all V+T cells (green) compared with UI (red) and Bys (blue) T cells (representative experiment). Each box depicts the 25th and 75th percentile and median (center line) expression values (y axis); whiskers indicate 1.5 × the interquartile range. Fold change in the median expression of the proteins was determined between V+ versus UI, V+ versus Bys, and Bys versus UI T cells. Bonferroni correction adjusted P values from 1-sided, 2-sample Wilcoxon tests corresponding to the alternative hypotheses of more than 20% change in protein expressions (in arcsinh scale) in the V+T cells were determined. (C) SPADE trees showing the differential expression of cell surface proteins indicated on UI and V+T cells across phenotypic hierarchies, shown for CD45RO, CD69, and CCR7. Node color represents the intensity of expression; blue, minimum; red, maximum. (D) Fold change in the expression of pZap70, pSLP76, and pErk1/2 in V+ and Bys T cells at 48 and 72 hpi relative to UI T cells by SPADE (left). (E) Graphic depiction of T cell signaling pathway activation in VZV-infected T cells, indicating the hierarchical locations of the phosphoproteins that were tested (bold font). VZV particles and green arrows indicate the point at which the activation or downregulation of the signaling pathway was detected. The activation index (AI) was calculated for each phosphoprotein in V+ and UI T cells and is represented as circles on the pathway diagram. Basal activation in UI T cells is shown as red circles on the left branch, with circle sizes corresponding to the AI for each protein. The right branches show the fold changes in AI for each protein in V+T cells relative to UI T cells, green circles indicate an increase, and yellow circles indicate a decrease in AI. —, denotes activation;- -, denotes inhibition. Proteins noted in small font are intermediate pathway components, not tested. (Reproduced from reference with permission of the publisher).

FIG 7

VZV infection and modulation of peripheral blood NK cells. (A) VZV infects human peripheral blood NK cells, CD3⁺CD56⁺ lymphocytes, and T cells. Representative flow cytometry plots are shown of mock or VZV-S infection, examining surface VZV gE:gI expression on live T cells (CD3⁺CD56^–), CD3⁺CD56⁺ lymphocytes, and NK cells (CD3^–CD56⁺). (B) Representative flow cytometry plots are shown of vOka infection, examining surface gE:gI expression on live T cells (CD3⁺CD56^–), CD3⁺CD56⁺ lymphocytes, and NK cells (CD3^–CD56⁺) (n = 3). (C) VZV infects both CD57^– and CD57^bright NK cells and drives CD57 expression. Plots show CD57 expression between mock, bystander, and VZV⁺ CD57^– NK cells (left) and CD57 versus gE:gI expression for VZV cultured CD57^– NK cells (middle), from one representative donor. Graphs show frequency of CD57 expression on mock, bystander and VZV⁺ CD57^– NK cells for three donors. Bars indicate mean; *, P < 0.05 (two-tailed paired t test). (D) VZV upregulates expression of skin-homing chemokine receptors on NK cells. Representative plots show CCR4 or CLA expression against CD56 expression for mock, bystander, and VZV⁺ NK cells. (Reproduced from reference with permission).

FIG 8

Viral determinants of VZV skin tropism. (A) Skin xenograft in SCID mouse and productive infection with release of enveloped virus from skin cells at 21 days (TEM; ×24,000), virions with intact lipid bilayer on cell surface (×108,000) (adapted from reference with permission), and polykaryocyte formation at 21 days shown by staining for gE (brown) (adapted from reference 58). (B) Infected skin organ culture with VZV-positive cells (red) at day 6 in the epidermis and hair follicles (black arrows). (Adapted from reference 27). (C) Comparison of viral titers in skin xenografts inoculated with pOka, varicella vaccine, and gC defective Ellen strain (mean ± SEM). (Adapted from reference 73). (D) Skin lesions shown by staining for gE (brown) at 21 days after inoculation with rOka, rOka with deletion of ORF7 (v7D), and vOka (ATCC); titers (mean ± SD) at 10 and 21 for those yielding virus and number from which virus was recovered per number inoculated. (Reproduced from reference with permission). (E) Effects of mutations of the gE N terminus in skin xenografts inoculated with rOka, rOka-Dp27-Y51, rOka-Δp27-G90, or rOka-ΔY51-G90; titers were compared with rOka mean for those yielding virus ± SEM. (Reproduced from reference with permission). *, P < 0.05; **, P < 0.01. (F) Effects of mutations of the gI promoter in skin xenografts inoculated with rOKA (black), rOKAΔgI (gray green), rOKAgI-Sp1 (pink), rOKAgI-AP1 (blue), rOKAgI-USF (green), or rOKAgI-Sp1/USF (orange), and rOKAgI: rep-Sp1/USF (orange), or rOKA ΔgI-gI@Avr (gray); titers compared with rOKa; *, P < 0.05; **, P < 0.01. (Adapted from reference 104).

FIG 9

Effects on skin pathogenesis of the disruption of glycoprotein B-mediated regulation of syncytium formation and inhibition of glycoprotein H functions. (A) Viral titers in skin xenografts at 10 and 21 dpi after inoculation with pOka, gB-Y881F, gB-Y920F, or gB-Y881/920F and frequency of virus-positive xenografts/number inoculated. (B) Viral genome copies/ng human DNA at 10 and 21 dpi and frequency of VZV DNA-positive xenografts/number inoculated. *, P < 0.05; ***, P < 0.001 by two-way analysis of variance (ANOVA). (C and D) Composite images of hematoxylin and eosin-stained 5-μm sections of infected skin xenografts at 21 dpi. Dotted lines outline the extent of virus penetration. Black boxes indicate where confocal microscopy images were captured on serial sections. Virus titers and genome copies/ng human DNA for each implant are indicated (red) in each panel. (E and F) Confocal microscopy of pOka- and Y881/920F-infected skin xenografts (21 dpi) stained for gE (cyan), capsid protein ORF23 (pink), TGN46 (brown), and nuclei (violet). Black boxes are shown in higher-magnification images. (Scale bar, 100 μm). (Reproduced from reference 81). (G) Mice with skin xenografts treated with anti-gH MAb 206 for 0 to 12 days or 4 to 12 days postinoculation with pOka; VZV gE (red) and hematoxylin (blue). Representative lesions at 7 dpi, A, G, and M; 14 dpi, B, H, and N; 21 dpi, C, I, and O; 28 dpi, D, J, and P; 35 dpi, E, K, and Q; and 42 dpi, F, L, and R; the number of xenografts with lesions is indicated at the bottom left of each panel. Magnification, ×50. (Adapted from reference 83). (H) gH DI mutants disrupt skin pathogenesis. Viral replication at 10 and 21 dpi; positive xenografts of total inoculated are indicated. **, P < 0.01; ***, P < 0.001. (I) gH DIII mutants reduce skin pathogenesis. Viral replication at 10 and 21 dpi; positive xenografts of total inoculated are indicated. ***, P < 0.001. (Reproduced from reference 57).

FIG 10

Protein kinases and other viral determinants of skin tropism. (A) Small lesions in skin xenografts inoculated with rOka47C and rOka47D-N (anti-VZV IgG, brown) with intact basement membrane (arrowheads) and hair follicles not infected (arrow, D-N) compared with extensive rOka lesions with dissolution of the basement membrane layer (arrowhead) and infected hair follicles (arrow); titers are shown of rOka, rOka47C, and rOka47D-N (mean ± SD) for xenografts yielding virus and percentage that were infected. (Adapted from reference 87). (B) Titers are shown of pOka and pOka66S for skin xenografts yielding virus (*, P < 0.01; **, P < 0.001). (Adapted from reference 26). (C) Titers (mean ± SD) are shown of pOKA-BAC, pOKA-BAC23mRFP-R, and pOKA-BACmRFP23 for xenografts yielding virus (SD); *, no growth in any xenografts. (Adapted from reference 88). (D) Titers (mean ± SD) of rOka, rOkaΔ35/35@Avr, rOka35#1, or rOka35#2 in skin xenografts (*, P < 0.05). (Adapted from reference 89). (E) Lesions in skin xenografts inoculated with ORF10-ORF12 cluster mutants (anti-VZV IgG, purple); large lesions with POKA (A) and POKA12 (F); small epidermal lesions (arrows) with POKA11 (B), POKA10/11 (C), and POKA11/12 (D); no lesions with POKA10/11/12 (E); titers (mean + SD) for ORF10-to-ORF12 cluster gene mutants yielding virus (*, P < 0.05). (Adapted from reference 92).

FIG 11

VZV restriction by PML nuclear bodies and their disruption by ORF61 in skin. (A) PML expression in uninfected cells in mock-infected and pOka-infected skin sections stained for PML (green). (Reproduced from reference 101). (B) PML cages sequester VZV nucleocapsids (NC) at 21 days after infection, shown by confocal microscopy after staining for PML (green) and ORF23 capsid protein (red) in a skin cell nucleus; scale, 5 mm. (Reproduced from reference 107). (C) PML cages sequester mature and immature VZV nucleocapsids, shown in by immunogold-electron microscopy (EM) in fibroblasts at 48 h after infection; clusters of nucleocapsids (arrows) are sequestered in a fibrous PML cage (arrowheads, PML labeled with 10-nm gold particles). Percentage of the total number of PML-specific gold particles associated with VZV capsids (black bar) or free within the nuclear area excluding PML-NBs (gray bar). (Reproduced from reference 107). (D) Schematic representation of the ORF61 protein sequence (amino acids 1 to 467). The diagonal shaded box indicates the RING domain; filled boxes indicate the three conserved SIMs; the sequences of the SIM hydrophobic core are highlighted by italics and underlined. The numbers in superscript show the amino acid positions of the SIMs in ORF61. (E) Lesions in xenografts infected with pOka- or pOka-mSIM-N&C for 21 days were identified by VZV gE expression. (F) Confocal microscopy of pOka- or pOka-mSIM-N&C-infected skin cells stained for ORF61 (red), PML (green), and nuclei (blue) showing the association of ORF61-positive puncta with PML-NBs in newly infected cells. Scale bars, 2.5 mm. (G) Quantitation of ORF61/PML-NB association in pOka- or pOka-mSIM-N&C-infected cells that had ORF61-positive puncta shown as percentage of ORF61-positive puncta colocalized/not colocalized with PML-NBs (pOka, n = 167; mSIM-N&C, n = 202; ***, P < 0.0001 by chi-square test). (H) Replication of pOka and ORF61 SIM mutant viruses in skin xenografts; mean titer ± SEM of xenografts that yielded virus at 10 and 21 days. *, P < 0.05; ***, P < 0.001; versus pOka (two-way ANOVA). (D to H were reproduced from reference 101).

FIG 12

IRF9 and autophagy responses in the pathogenesis of VZV skin infection. (A) Immunofluorescence detection of IRF9 in uninfected (top) and outside and within the lesion area of VZV-infected (bottom) skin xenografts. Sections were stained for IRF9 (Alexa Fluor 488; green) and gE (Alexa Fluor 594; red). (Reproduced from reference 109). (B) Autophagosomes were observed in infected keratinocytes recovered from varicella lesions using fluorescent probes to LC3B and VZV IE62; a keratinocyte with a bifid nucleus exhibited LC3B puncta characteristic of LC3B-II in autophagosomes and nuclear IE62 (left) and a second more typical infected keratinocyte showed both punctate LC3B and IE62 in the cytoplasm. (Adapted from reference with permission).

FIG 13

Signal transducer and activator of transcription 3 (STAT3) and survivin induction by VZV promote skin pathogenesis. (A) Skin sections at 21 days after infection were stained with anti-VZV-IE63 to identify infected cells and anti-pSTAT3, detected with DAB (brown); sections were treated with calf intestinal phosphatase to confirm the specificity of pSTAT3 detection. (B) Skin sections were stained with antibodies to ORF61 (Alexa Fluor 488; green), pSTAT3 (Alexa Fluor 594; red), and Hoechst nuclear stain and were examined by confocal microscopy. (C) VZV-infected and uninfected skin sections were stained with antibodies to gE (Alexa Fluor 594; red) and survivin (Alexa Fluor 488; green) and examined by confocal microscopy; white arrow, nuclear survivin; yellow arrow, no survivin; pink arrow, cytoplasmic surviving. (Reproduced from reference 116).

FIG 14

Effects of inhibition of STAT3 and CREB activation on VZV skin pathogenesis. (A) Skin xenografts were inoculated with VZV expressing firefly luciferase; mice were treated with dimethyl sulfoxide (DMSO) (group I, 1 to 5) or S3I-201 (group II, 6 to 10) from day 0 to 16; mouse 8 xenografts were uninfected; mice were imaged for 21 days; graphs show average bioluminescence from skin xenografts that had a positive signal (y axis) versus days postinfection (x axis) in DMSO- and S3I-201-treated mice; arrows indicate the discontinuation of treatment on day 16; bars show the number of positive xenografts. (Reproduced from reference 116). (B) Inhibition of pCREB interaction with p300/CBP interferes with skin infection as shown by in vivo imaging of xenografts inoculated with VZV expressing firefly luciferase DMSO or XX-650-23 from day 0 to 14; mice were imaged for 21 days; graphs show the bioluminescence signal quantified by measuring the average radiance inside a region of interest standardized for size over each skin xenograft with average radiance plotted against the day postinfection; asterisks indicate significant differences. (Reproduced from reference 117).

FIG 15

VZV effects on keratinocyte differentiation. (A) VZV downregulation of keratin KRT1 and KRT 10 expression in varicella lesion biopsy samples. KRT10 (red, top) and KRT1 (red, bottom) expression in the spinous layer; loss of both KRTs in VZV-positive areas (green); scale bars, 50 mm (left, DAPI nuclear stain); mean fluorescent intensity (MFI) of KRT10 and KRT1 from infected and uninfected cells from the spinous layer; P values determined with Student’s t test. (Reproduced from reference with permission). (B) Restoration of keratin 10 expression and reduction of kallikrein (KLK) 6 and VZV propagation by Nutlin-3 treatment of skin explants: KLK6 and gE and KRI10 and gE in uninfected, pOka infected, or infected and treated with Nutlin-3 at 14 days; dotted lines indicate the dermal-epidermal junction; *, regions of epidermal disruption. Bar, 50 μm. (Reproduced from reference with permission).