Article Figures & Data
Figures
- Fig. 1.
HCMV pp65 is a nuclear protein that is detected in subnuclear structures. HF were grown on coverslips and infected with AD169. Three to five days postinfection (100% cytopathic effect), cells were fixed and stained in immunofluorescence assays with the pp65-specific MAb 65-8 (A), MAb 28-19 (B), or polyclonal guinea pig serum against bacterially expressed pp65 (C). Cos7 cells transfected with a vector expressing pp65 (D) or an EGFP-pp65 fusion protein (E and F) were fixed and stained with MAb 28-19 (D only). Magnifications: (A to C) ×400; (D to F) ×1,000.
- Fig. 2.
HCMV-encoded structural and nonstructural proteins are retained in the nuclear matrix of HCMV AD169-infected human fibroblasts. Nuclei from AD169-infected HF were isolated by treatment with nonionic detergent and then treated with DNase and high salt to remove soluble components from the nuclear matrix fraction. (A) Nuclear matrix-containing filters were probed with MAbs specific for gB, IE1, IE2, ppUL44, ppUL69, pp28, MCP, and pp65. (B) Filters containing proteins from nuclear matrix fractions washed with 1 M guanidine hydrochloride prior to electrophoretic separation were probed with the antibodies listed above. Sizes are indicated in kilodaltons.
- Fig. 3.
Quantitative Western blots of nuclear matrix preparations from AD169-infected HF. HCMV-infected cells were fractionated, and protein content in each fraction was determined as described in Materials and Methods. Twenty-five micrograms of each of the fractions was loaded into the lanes (C, cytoplasm, soluble protein; N, detergent-treated nuclei; NM, nuclear matrix pellet; GW, guanidine hydrochloride-washed NM) and transferred to nitrocellulose filters. Filters were reacted with MAb p63-27 against IE-1 (A) or MAb 28-19 against pp65 (B) and processed for autoradiography. Counts for each fraction were determined on a PhosphorImager and were as follows: IE-1, 449,874.4, 13,951.6, 9,332.5, and 9,514.3 for C, N, NM, and GW fractions, respectively; pp65 (68 kDa), 239,720.7, 2,048,547.0, 1,206,468.7, and 1,172,115.9 for C, N, NM, and GW fractions, respectively; pp65 (50-kDa form), 33,155.2, 1,003,560.2, 774,609.0, and 188,991.4 for C, N, NM, and GW fractions, respectively. Sizes are indicated in kilodaltons.
- Fig. 4.
Immunofluorescence assays of in situ-extracted, AD169-infected HF. HF grown on glass coverslips were infected with HCMV AD169 5 days prior to harvesting. Infected cells were untreated (A, C, E, G, and I) or extracted with detergent, DNase, and high salt (B, D, F, H, and J) before fixation with 2.5% paraformaldehyde. Coverslips were then reacted with MAbs specific for IE-1 (A and B), ppUL44 (C and D), MCP (E and F), ppUL69 (G and H), or pp65 (I and J). Antibody binding was detected with FITC-conjugated goat anti-mouse IgG antibody and recorded by conventional fluorescence microscopy. Magnification for all frames is ×348.
- Fig. 5.
pp65 expressed by recombinant vaccinia virus vv-pp65 is retained in the nuclear matrix of infected BSC-1 monkey cells. Cytoplasmic (C), nuclear (N), nuclear matrix (NM), and guanidine-washed nuclear matrix (GW) fractions were prepared from recombinant vaccinia virus vv-Eco V gb (A)- or vv-pp65 (B)-infected BSC-1 cells as described in Materials and Methods. The samples (10 μg per lane) were analyzed by Western blotting using a gB-specific or pp65-specific MAb and developed with 125I-protein A. Sizes are indicated in kilodaltons.
- Fig. 6.
pp65 binds to a nuclear matrix protein which comigrates with lamin B1 in vitro. (A) Nuclear matrix material was isolated from the human carcinoma cell line HEp-2 as described in Materials and Methods. Proteins were electrophoretically separated and then blotted onto nitrocellulose. Filters were then reacted with pp65-specific MAb 28-19 (lane C), purified pp65 followed by anti-pp65 MAb 28-19 (lane FW), or anti-lamin B1 MAb NA12 (lane NA12). Antibody binding was detected by addition of anti-mouse IgG antibody followed by125I-protein A and autoradiography. Migration of molecular mass markers is shown in kilodaltons at the left. (B) Coprecipitation of pp65 and lamins from soluble nuclear matrix extracts. Soluble nuclear matrix extracts prepared from AD169-infected HF were immunoprecipitated with MAbs against pp65. Immunoprecipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose for Western blotting with antibodies specific for pp65 (28-19), lamins (237), and a control MAb specific for ppUL44 (28-21). Antibody binding was detected by addition of rabbit anti-mouse IgG antibody (lanes 28-19 and 28-21 only) followed by 125I-protein A and autoradiography. Immunoglobulin heavy chains were detected with a rabbit anti-mouse immunoglobulin antibody followed by addition of125I-protein A and autoradiography (lane C). Migration of the molecular mass marker (in kilodaltons) and immunoglobulin heavy chains (Hc) is shown in the margins.
- Fig. 7.
Colocalization of pp65 and lamins. Nuclear matrix-extracted (A to C) or unextracted (D to F) HCMV-infected cells were stained with a murine MAb against pp65 (green; A and D) and a rabbit antiserum against lamins a and c (red; B) or against lamin b (red; E). Colocalization (blue) of pp65 and lamins is shown in panels C and F. Magnification for all panels, ×890.
Tables
- Table 1.
Enrichment of pp65 on the nuclear matrix
Fractiona % Total cellular proteinb % of protein in fractionc IE-1 pp65 (68 kDa) pp65 (50 kDa) Cytoplasm/soluble protein 86.3 93.2 (1.08d) 5.1 (0.06) 1.7 (0.02) Detergent-treated nuclei 8.9 2.9 (0.33) 43.9 (4.93) 50.2 (5.64) Nuclear matrix 3.0 1.9 (0.63) 25.9 (8.63) 38.7 (12.94) Guanidine-HCl-washed nuclear matrix 1.8 2.0 (1.11) 25.1 (13.94) 9.4 (5.22) ↵a HCMV-infected HF were fractionated into the indicated cellular fractions as described in Materials and Methods.
↵b The amount of total protein represented in each fraction was determined as described in Materials and Methods, and the percentage of total cellular protein was calculated as follows: % = (total protein in fraction/total cellular protein) × 100.
↵c The amount of IE-1, pp65 (68 kDa) or pp65 (50 kDa) in each fraction was determined by phosphorimaging. The results are presented as the percentage of total IE-1, pp65 (68 kDa), or pp65 (50 kDa) in each fraction as calculated by the following formula: % = (counts in fraction/total counts for protein) × 100.
↵d The specific enrichment of each protein in a specific fraction, determined by the following formula: enrichment = % of protein determined in footnote c/% of total cellular protein determined in footnote b.
