Fragment of tegument protein pp65 of human cytomegalovirus induces autoantibodies in BALB/c mice

Abstract

Introduction: Human cytomegalovirus (HCMV) infection has been implicated in the development of autoimmunity, including systemic lupus erythematosus (SLE). Previously we reported that HCMV phosphoprotein 65 (pp65) could induce early onset of autoantibody and glomerulonephritis on lupus-prone NZB/W mice. This study further examined whether the B cell epitope(s) in pp65 is able to drive the development of autoantibody.

Methods: Sera from SLE patients or HCMVpp65-immunized mice were analyzed for anti-nuclear antibody by immunoblotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescent stain and Crithidia luciliae stain. The deposition of immunoglobulin to the kidney was also examined by immunofluorescent stain. The interactions between pp65 sub-fragment to cellular proteins were revealed by yeast two-hybrid analyses.

Results: Our results showed that most SLE patients possessed antibodies to the C-terminal half of the HCMVpp65 antigen. Of these positive sera, 73% were also positive to the pp65336-439 sub-fragment. The immunization of pp65336-439 induced formation of multiple anti-nuclear antibodies, including anti-chromatin, anti-centriole, anti-mitotic spindle type I/II (MSA I/II) and a significant elevation of anti-double-stranded DNA (anti-dsDNA) antibodies on BALB/c mice. Yeast two-hybrid analyses revealed the binding of pp65336-439 sub-fragment to cellular proteins. Immunoglobulin deposition on glomeruli was also detected on pp65336-439-immunized mice.

Conclusions: Our data suggested that HCMVpp65336-439 sub-fragment may induce cross-reactive antibodies to several nuclear antigens, which could contribute to the development of autoimmunity in genetic-suspected individuals.

Figure 1

Schematic representation of truncated HCMVpp65 His-tag fusion proteins (Swiss-Prot: P06725). The full-length of HCMVpp65 is given in the top figure. Below that, six pp65 sub-fragments, pp65_1-167, pp65_167-336, pp65_336-561, pp65_336-379, pp65_379-455, pp65_455-561, and four C terminal truncated peptides, pp65_336-422, pp65_336-439, pp65_336-448, pp65_336-455, are shown. The name of the plasmids which encoded pp65 sub-fragment proteins are given at the right. HCMVpp65, human cytomegalovirus phosphoprotein 65 (65 kD).

Figure 2

Detection of anti-HCMVpp65 antibody by ELISA and immunoblot assay in immunized BALB/c mice. The IgG against HCMVpp65 or HeLa extract from pp65_1-167 (n = 11), pp65_336-439 (n = 17), SA-C3d (n = 5) or PBS (n = 2) immunized mice. (a) ELISA assays for anti-HCMV reactivity against purified HCMV virions. Sera were 500X diluted and positivity was defined by mean + 3 s.e.m. of SA-C3d-immunized sera. O.D.₄₅₀ > 0.50 was considered to be positive. (b) Immunoblot analysis on anti-HCMV reactivity against purified HCMV virions. Sera were 500X diluted. Top panel: pp65_1-167 (n = 5), pp65_336-439 (n = 5) and PBS (n = 2). Lane 1 to 2 and 3 to 4, PBS-immunized sera at 8 and 12 weeks, lane 5 to 9 and 15 to 19, pp65 to _1-167-immunized sera, lane 10 to 14 and 20 to 24, pp65_336-439-immunized sera, lane 25, N: 1,000X diluted healthy control serum, lane 26, P: 1,000X diluted SLE patient's serum. Bottom panel: SA-C3d (n = 5), pp65_1-167 (n = 6) and pp65_336-439 (n = 12). Lane 1 to 5, SA-C3d-immunized sera, lane 6, N: 1,000X diluted healthy control serum, lane 7 to 8, P: 1,000X diluted SLE patients' sera, lane 9 to 14, pp65_1-167-immunized sera, lane 15 to 26, pp65_336-439-immunized sera. (c) ELISA assays for anti-HeLa reactivity against total HeLa lysate. Sera were 500X diluted. O.D.₄₅₀ > 0.48 was considered to be positive. (d) Immunoblot analysis with mouse sera at eight weeks post-immunization against HCMV and total HeLa lysate. Top panel: purified HCMV virion blot. Bottom panel: total HeLa lysate blot. Lane1 to 4, C3d-immunized sera, lane 5 to 8, pp65_1-167-immunized sera, lane 9 to 12, pp65_167-336-immunized sera, lane 13 to 16, pp65_336-439-immunized sera. Molecular mass markers (kD) are shown on the left. MW: molecular weight. w: weeks of post-immunization. Graphs depict mean ± s.e.m. values. Unpaired Student t test was performed. Results with a P-value of < 0.05 were considered to be significant. These results are representative of triplicated experiments.

Figure 3

Detection of anti-nuclear reactivity in pp65_1-167, pp65_336-439, SA-C3d or PBS immunized sera. HEp-2 substrate slides were used for detection of anti-nuclear antibodies. Sera from eight weeks post-immunization were 100X diluted for ANA stains. Patterns of speckle (a, i), chromatin (b, j), nucleosome (c, k), MSA I (d, l), MSA II (e, m) and centriole (f, n) were revealed with sera from pp65_336-439-immunized mice. Nucleolar response (g, o) was detected in sera from pp65_1-167 or pp65_336-439 immunized mice. Nuclear reactivity was not found in SA-C3d or PBS immunized mice (h, p). White arrowheads indicate the pattern of nuclear responses. MSA I/II: mitotic spindle type I/II.

Figure 4

Detection of anti-dsDNA antibody in pp65_1-167, pp65_336-439, SA-C3d or PBS immunized sera. (a) ELISA assays for anti-dsDNA activity at 1:100 serum dilution. The dsDNA positivity is defined by mean + 3 s.e.m. of SA-C3d-immunized sera. O.D.₄₅₀ > 0.49 was considered to be positive. (b) Isotyping of anti-dsDNA antibody for pp65_336-439-immunized mice (n = 17) at eight weeks post-immunization. Tests were performed on ELISA at 1:100 dilution. dsDNA(+): dsDNA seropositive, dsDNA(-): dsDNA seronegative. Graphs depict mean ± s.e.m. values. Unpaired Student t test was performed. Results with a P-value of < 0.05 were considered to be significant. (c) Representations of Crithidia luciliae stain by sera from (c1) pp65_336-439, (c2) pp65_1-167, (c3) SA-C3d or (c4) PBS immunized animals at eight weeks post-immunization at 1:40 dilution. White arrowheads indicate dsDNA positive stains. (d) The summarized results of ELISA assays and Crithidia luciliae stains. These results are representative of triplicated experiments.

Figure 5

Detection of anti-nuclear reactivity in pp65_1-167 or pp65_336-439 specific antibodies from eight weeks post-immunization sera. (a) ELISA assays of affinity purified anti-pp65_336-439 antibody (pp65_336-439), anti-pp65_1-167 antibody (pp65_1-167), SLE patients' sera (SLE) and flow through against purified HCMV virions. Graphs depict mean ± s.e.m. values. Unpaired Student t test was performed. Results with a P-value of < 0.05 were considered to be significant. (b) Immunoblot assays on affinity purified anti-pp65_336-439 antibody (pp65_336-439), anti-pp65_1-167 antibody (pp65_1-167), SLE patients' sera (SLE) and flow through against purified HCMV virions. Molecular mass markers (kD) are shown on the left. MW: molecular weight. (c) ANA stains with HEp-2 substrate slides were performed with HCMV affinity-purified antibody. Patterns of speckle (c₁, c₇), chromatin (c₂, c₈), centriole (c₃, c₉) and MSA II (c₄, c₁₀) were revealed with affinity purified anti-pp65_336-439 antibodies. Nuclear pattern was not found in flow through (c₅, c₁₁) or affinity-purified anti-pp65_1-167 antibody (c₆, c₁₂) stains. White arrowheads indicate the patterns of nuclear response. MSA II: mitotic spindle type II.

Figure 6

The cross-reactivity of purified anti-pp65_336-439 or anti-pp65_1-167 antibody to dsDNA. (a) ELISA assays for affinity-purified anti-pp65_336-439 antibody (pp65_336-439), anti-pp65_1-167 antibody (pp65_1-167), flow through and SLE patients' sera (SLE) against dsDNA. Graphs depict mean ± s.e.m. values. Unpaired Student t test was performed. Results with a P-value of < 0.05 were considered to be significant. (b) Representations of Crithidia luciliae stain by affinity-purified anti-pp65_336-439 antibody (b₁, pp65_336-439), anti-pp65_1-167 antibody (b₂, pp65_1-167), flow through (b₃) and SLE patients' sera (b₄, SLE) against dsDNA. White arrowheads indicate the positive stains. (c) Representations of Immunofluorescent stain for immunoglobulin deposition on glomerular by pp65_336-439 (c₁) immunized mice, pp65_1-167 (c₂) immunized mice, SA-C3d (c₃) immunized mice or PBS (c₄) controls. Kidneys were collected at 20 weeks of age. White arrowheads indicate antibody deposition on glomerular. dsDNA: double-stranded DNA.

Figure 7

Detection of protein-to-protein interaction between pp65_336-439 and HeLa lysate. Whole HeLa lysate was separated by SDS-PAGE and transferred to nitrocellulose paper. The pp65_336-439 or pp65_1-167 His-tag fusion peptide was incubated with blot at concentrations of 20, 10, 5, 2.5, 1.25, 0.625 mg/ml (No.1 to 6) or at concentrations of 20, 10, 5, 2.5 mg/ml (No.7 to 10), respectively. Asterisk indicates protein-free negative control. Molecular mass markers (kD) are shown on the left. MW: molecular weight (kD).