Differential requirement for cell fusion and virion formation in the pathogenesis of varicella-zoster virus infection in skin and T cells

Abstract

The protein product of varicella-zoster virus (VZV) ORF47 is a serine/threonine protein kinase and tegument component. Evaluation of two recombinants of the Oka strain, rOka47DeltaC, with a C-terminal truncation of ORF47, and rOka47D-N, with a point mutation in the conserved kinase motif, showed that ORF47 kinase function was necessary for optimal VZV replication in human skin xenografts in SCID mice but not in cultured cells. We now demonstrate that rOka47DeltaC and rOka47D-N mutants do not infect human T-cell xenografts. Differences in the growth of kinase-defective ORF47 mutants allowed an examination of requirements for VZV pathogenesis in skin and T cells in vivo. Although virion assembly was reduced and no virion transport to cell surfaces was observed, epidermal cell fusion persisted, and VZV polykaryocytes were generated by rOka47DeltaC and rOka47D-N in skin. Virion assembly was also impaired in vitro, but VZV-induced cell fusion continued to cause syncytia in cultured cells infected with rOka47DeltaC or rOka47D-N. Intracellular trafficking of envelope glycoprotein E and the ORF47 and IE62 proteins, components of the tegument, was aberrant without ORF47 kinase activity. In summary, normal VZV virion assembly appears to require ORF47 kinase function. Cell fusion was induced by ORF47 mutants in skin, and cell-cell spread occurred even though virion formation was deficient. VZV-infected T cells do not undergo cell fusion, and impaired virion assembly by ORF47 mutants was associated with a complete elimination of T-cell infectivity. These observations suggest a differential requirement for cell fusion and virion formation in the pathogenesis of VZV infection in skin and T cells.

FIG. 1.

Replication of VZV rOka and kinase-defective ORF47 mutants in T-cell xenografts in vivo. T-cell xenografts were infected with rOka, rOka47ΔC, or rOka47D-N grown in human embryonic lung fibroblasts and harvested after 14 and 21 days. The titers of the infected fibroblasts that were used to inoculate the T-cell xenografts are shown on the y axis. Bars represent mean numbers of plaques (± standard errors of the means) by infectious center assay of 4 to 6 implants. Xenografts that did not contain infectious virus were excluded from the averages.

FIG. 2.

Analysis of T-cell xenografts infected with rOka or ORF47 kinase-defective mutants by in situ hybridization for VZV DNA. T-cell xenograft sections were stained with hematoxylin and eosin after inoculation with rOka47ΔC (B), rOka47D-N (D), or rOka (F). Panels A, C, and E show in situ hybridization with a HindIII VZV DNA probe after inoculation with rOka47ΔC (B), rOka47D-N (D), or rOka (F). Insets in panels B, D, and F are controls with the pBR322 vector alone.

FIG. 3.

Imaging of melanoma cells infected with rOka and kinase-defective ORF47 mutants by TEM and SEM. Cells were examined by TEM (A, B, G, H, J, and K) or SEM (C, I, and L). Cells infected with rOka are shown in panels A to C. Schematic representations of panels A to C are shown in panels D to F. Cells infected with rOka47D-N are shown in panels G to I. Cells infected with rOkaΔC are shown in panels J to L. The prototype structures for virions inside cytoplasmic vacuoles and on the cell surface are illustrated in panels A to C, while aberrant viral particles are shown in panels G, H, J, and K.

FIG. 4.

TEM and SEM of skin xenografts infected with rOka or rOka47D-N. Skin sections were examined by TEM (A to D) or SEM (E and F). Skin xenografts infected with rOka showed viral particles on the surface of infected skin cells (A and B). No viral particles but a few spherical structures were observed (arrows) on skin cells infected with rOka47D-N (C and D). TEM showed viral particles in rOka-infected skin cells located in the cytoplasm (E). Viral particles were smaller and less frequent in rOka47D-N-infected skin cells (F).

FIG. 5.

Immunoprecipitation of gE protein with ORF47 antiserum from melanoma cells infected with rOka or kinase-defective ORF47 mutants. Melanoma cells were infected with rOka, rOka47D-N (DN), rOka47ΔC (ΔC), or ROKA47S (47S). Infected cell lysates were incubated with ORF47 antiserum, subjected to SDS-polyacrylamide gel electrophoresis, and probed with polyclonal anti-gE antibody by Western blotting. ROKA47S was kindly provided by J. Cohen, National Institute of Allergy and Infectious Diseases.

FIG. 6.

Expression and intracellular localization of gE in melanoma cells infected with rOka or kinase-defective ORF47 mutants. Melanoma cells were infected with rOka (A, B, and C), rOka47D-N (D, E, and F), or rOka47ΔC (G, H, and I) for 30 h and examined for gE (A, D, and G) and TGN localization (B, E, and H) by confocal microscopy. gE was detected with FITC-labeled anti-rabbit IgG (green). The TGN marker p230 was detected with mouse anti-p230 and secondary Texas Red-conjugated antibody (red). Merged images are shown in panels C, F, and I.

FIG. 7.

Expression and intracellular localization of gI in melanoma cells infected with rOka or kinase-defective ORF47 mutants. Melanoma cells were infected with rOka (A, B, and C), rOka47D-N (D, E, and F), or rOka47ΔC (G, H, and I) for 30 h and examined for gI (A, D, and G) and gE (B, E, and H) by confocal microscopy. gI was detected with gI polyclonal antiserum and Texas Red-labeled anti-rabbit IgG (red). gE was detected with mouse monoclonal anti-gE antibody and secondary FITC-conjugated antibody (green). Merged images are shown in panels C, F, and I. Anti-gI antiserum was kindly provided by S. Silverstein, Columbia University.

FIG. 8.

Confocal analysis of skin xenografts infected with rOka or rOka47ΔC. Skin sections that were infected with rOka (A, C, and E) or rOka47ΔC (B, D, and F) were incubated with anti-gE monoclonal antibody followed by anti-mouse antibody labeled with Texas Red (A, B, E, and F) and ORF47 antiserum followed by anti-rabbit FITC-conjugated antibody (C, D, E, and F). DAPI (4′,6′-diamidino-2-phenylindole) counterstain (A, B, C, and D) was used to demonstrate the contrast between the small, fragmented nuclei (A, arrow) in advanced skin lesions and intact, larger nuclei (A, arrowhead) in areas of newly infected epidermal cells. Enlargements (E and F) demonstrate gE membrane localization and nuclear as well as cytoplasmic localization of ORF47 protein in rOka-infected skin cells (E) and increased cytoplasmic gE and ORF47 protein in rOka47ΔC-infected xenografts (F). Controls are shown in insets: panel A, uninfected skin tissue incubated with anti-gE antibody and ORF47 antiserum; panel E, skin tissue infected with rOka, incubated with mouse IgG and rabbit preimmune serum; panel F, skin tissue infected with rOka47ΔC, incubated with mouse IgG and rabbit preimmune serum.

FIG. 9.

Immunohistochemical analysis of skin xenografts infected with rOka, rOka47ΔC, or rOka47D-N. Sections were prepared from skin implants harvested at day 21 after infection with rOka (A and B), rOka47D-N (C and D), or rOka47ΔC (E and F), stained with monoclonal antibody to gE and counterstained with hematoxylin. Sections in panels A, C, and E are at a magnification of ×10; panels B, D and F are at amagnification of ×63 and are enlargements of the areas indicated with arrows in panels A, C, and E, respectively. Panels G, H, I, and J are controls. Panel G, uninfected skin xenografts, incubated with gE antibody(magnification, ×10); panel H, rOka-infected skin xenografts, incubated with mouse IgG (magnification, ×10); panel I, rOka47D-N-infected skin xenografts, incubated with mouse IgG (magnification, ×63); panel J, rOka47ΔC-infected skin xenografts, incubated with mouse IgG (magnification, ×10).