Varicella-zoster virus retains major histocompatibility complex class I proteins in the Golgi compartment of infected cells

Abstract

We sought to examine the effects of varicella-zoster virus (VZV) infection on the expression of major histocompatibility complex class I (MHC I) molecules by human fibroblasts and T lymphocytes. By flow cytometry, VZV infection reduced the cell surface expression of MHC I molecules on fibroblasts significantly, yet the expression of transferrin receptor was not affected. Importantly, when human fetal thymus/liver implants in SCID-hu mice were inoculated with VZV, cell surface MHC I expression was downregulated specifically on VZV-infected human CD3+ T lymphocytes, a prominent target that sustains VZV viremia. The stage in the MHC I assembly process that was disrupted by VZV in fibroblasts was examined in pulse-chase and immunoprecipitation experiments in the presence of endoglycosidase H. MHC I complexes continued to be assembled in VZV-infected cells and were not retained in the endoplasmic reticulum. In contrast, immunofluorescence and confocal microscopy showed that VZV infection resulted in an accumulation of MHC I molecules which colocalized to the Golgi compartment. Inhibition of late viral gene expression by treatment of infected fibroblasts with phosphonoacetic acid did not influence the modulation of MHC I expression, nor did transfection of cells with plasmids expressing immediate early viral proteins. However, cells transfected with a plasmid carrying the early gene ORF66 did result in a significant downregulation of MHC I expression, suggesting that this gene encodes a protein with an immunomodulatory function. Thus, VZV downregulates MHC I expression by impairing the transport of MHC I molecules from the Golgi compartment to the cell surface; this effect may enable the virus to evade CD8+ T-cell immune recognition during VZV pathogenesis, including the critical phase of T-lymphocyte-associated viremia.

FIG. 1

FACS analysis of MHC I molecules, transferrin receptor (CD71), and VZV proteins on VZV-infected cells. HFFs were either infected with VZV for 24 h (B and E) or mock infected (A and D), and cell preparations were stained with antibodies and fluorescent conjugates to MHC I and VZV proteins (A and B) or to transferrin receptor and VZV proteins (D and E). The percentages of VZV⁺ and VZV⁻ cell populations expressing cell surface MHC I molecules (C) and transferrin receptor (F) are shown. (G) Percentage of VZV⁺ and VZV⁻ cell populations expressing cell surface MHC I molecules. HFF, MeWo, and MRC-5 cells were infected with VZV for 24 h and stained with antibodies and fluorescent conjugates to MHC I and VZV proteins and analyzed by flow cytometry.

FIG. 2

FACS analysis of MHC I and VZV proteins on VZV-infected human T lymphocytes. SCID-hu thymus/liver implants were inoculated with VZV- or mock-infected cells, and 7 days later cell preparations were stained with antibodies and fluorescent conjugates to MHC I and VZV proteins and CD3. CD3⁺ T lymphocytes were gated and analyzed for MHC I and VZV antigen expression. A typical FACS plot of MHC I and VZV antigens from mock- (A) and VZV-infected (B) thymus/liver implants is shown. The percentage of CD3⁺/VZV⁺ cells was determined for each mouse (C). The percentage of VZV⁺ and VZV⁻ human CD3⁺ T-cell populations expressing cell surface MHC I molecules is shown (D).

FIG. 3

Immunofluorescent staining of intracellular MHC I and VZV antigens in VZV-infected cells. VZV-infected cells were fixed, permeabilized, and incubated with VZV-immune human serum and an anti-MHC I MAb, which were detected using Texas red-conjugated anti-human IgG and FITC-conjugated anti-mouse IgG antibodies. VZV antigen staining was detected in the majority of VZV-infected cells (A). Intense perinuclear MHC I staining was detected in VZV-infected cells (B, arrow), whereas diffuse cytoplasmic MHC I staining was detected in VZV-negative cells (B, arrowhead). The yellow color indicates colocalization of VZV and MHC I proteins in VZV-infected cells (C).

FIG. 4

Biochemical analysis of the synthesis and transport of MHC I molecules in VZV-infected cells. VZV- and mock-infected cells were labeled with [³⁵S]methionine-cysteine and then chased for 0, 1, 2, and 3 h. Total cell lysates were immunoprecipitated with W6/32, and immune complexes were treated either with (+) or without (−) endo H. The MHC I endo H-resistant (R) and -sensitive (S) forms are indicated.

FIG. 5

ConA, ceramide, and MHC I immunofluorescent staining of VZV-infected cells. VZV-infected cells were labeled with Texas red-conjugated ConA (A to C) or Texas red-conjugated BODIPY-ceramide (D to F), fixed, permeabilized, and stained with anti-MHC I MAb and FITC-conjugated anti-mouse IgG antibody. Fluorescent images were overlayed to assess colocalization (C and F). The yellow color represents colocalization of MHC I molecules and the fluorescent marker of the Golgi compartment (F). No colocalization was detected with the fluorescent ER marker (C).

FIG. 6

Biochemical analysis of the synthesis of MHC I molecules in VZV-infected cells. VZV- and mock-infected cells were labeled with [³⁵S]methionine-cysteine and then chased for 1 h. Total cell lysates were immunoprecipitated with rabbit anti-heavy chain (αHC) or W6/32 antibody, and immune complexes were treated either with (+) or without (−) endo H. The MHC I endo H-resistant (R) and -sensitive (S) forms are indicated. β₂m and the coimmunoprecipitating protein seen in VZV-infected cells are indicated.

FIG. 7

Analysis of MHC I downregulation in VZV- and mock-infected cells treated with the viral DNA inhibitor PAA. (A) Western blot of viral gC was performed on total cell lysates from mock-infected cells (lane 1), mock-infected cells with PAA (lane 2), VZV-infected cells at the time of inoculation onto uninfected cells (lane 3), and VZV-infected cells in the absence (lane 4) or presence (lane 5) of PAA at 24 h postinfection. (B) The percentage of VZV⁺ and VZV⁻ cell populations expressing cell surface MHC I molecules. Cells were infected with VZV in the absence or presence of PAA for 24 h and stained with antibodies and fluorescent conjugates to MHC I and VZV proteins and analyzed by flow cytometry.

FIG. 8

Analysis of MHC I downregulation in cells transfected with plasmids expressing VZV proteins. HFFs were transiently transfected with plasmids expressing VZV ORF4, ORF10, ORF47, ORF61, ORF62, ORF63, and ORF66 or a parental control plasmid (control). At 48 h posttransfection cell preparations were stained for MHC I expression. Data are shown as the mean fluorescence intensities of specific cell surface MHC I staining.