A Novel Role for PX, a Structural Protein of Fowl Adenovirus Serotype 4 (FAdV4), as an Apoptosis-Inducer in Leghorn Male Hepatocellular Cell

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  Figure 1. Fowl adenovirus serotype 4 (FAdV4) induced apoptosis in Leghorn male hepatocellular (LMH) cells. (a) FAdV4 infection caused a cytopathic effect (CPE) in LMH cells. LMH cells were mock-infected or infected with FAdV4 at an increased MOI of 0.01, 0.1, 1 and 10. Thirty-six hours after infection, LMH cells were visualized under a microscope. The scale bar represents 100μm. (b) Examination of the hexon viral protein in FAdV4-infected cells by Western Blot. Forty-eight hours after FAdV4 infection, structural protein hexon was examined with a Western Blot using an anti-hexon specific antibody. (c,d) FAdV4 infection caused apoptosis in LMH cells. LMH cells were infected with FAdV4 at an increased MOI of 0.01, 0.1, 1 and 10. At different time points (12, 24 and 48h) after infection, cells were harvested, stained with Annexin V-PE and 7-AAD, and analyzed by flow cytometry. Mock-infected cells were used as controls. The percentage of apoptotic cells in (c) was quantified (d). (e) Inhibition of FAdV4-induced apoptosis by caspase inhibitors. LMH cells were seeded on 12-well plates and cultured overnight. Caspase inhibitors (z-VAD-fmk, z-DEVD-fmk, z-IETD-fmk and z-LEHD-fmk) were applied to…

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  Figure 2. Ectopic expression of PX protein in LMH cells induced apoptosis. (a) Schematic diagram of recombinant eukaryotic expression vector pRK5-FLAG-viral genes. We constructed a total of 11 structural proteins into the eukaryotic expression vector pRK5-FLAG. (b) Ectopic expressions of FLAG-viral protein recombinants in LMH cells. Recombinant plasmids (pRK5-FLAG-hexon, -fiber1, -fiber2, -pIIIa, -IVa2, -pTP, -pVI, -pVII, -pVIII, -pX and -penton base) were individually transfected into LMH cells using the jetPRIMETM transfection reagent. Forty-eight hours after transfection, cells were harvested and examined with Western Blot using anti-FLAG antibodies. (c,d) Twenty-four or 48 h after transfection as above described, cells were harvested, stained with Annexin V-PE and 7-AAD, and analyzed by flow cytometry. Empty plasmid-transfected cells were used as controls (c). The percentage of apoptotic cells 48 h post-transfection (as in c) was quantified (d). (e) Inhibition of PX-induced apoptosis by caspase inhibitors. LMH cells were seeded on 12-well plates and cultured overnight. Caspase inhibitors (z-VAD-fmk, z-DEVD-fmk, z-IETD-fmk and z-LEHD-fmk) were added to cell culture for 2 h prior to…

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  Figure 3. Localization of PX in the nucleus is required for PX-induced apoptosis. (a) LMH cells were seeded on 12-well plates and cultured overnight, followed by infection with FAdV4 at a MOI of 1 or transfected with pRK5-FLAG-pX at 1 μg per well. Twenty-four hours after treatment, cells were fixed and probed with an anti-PX monoclonal antibody, followed by incubation with FITC-conjugated goat anti-mouse antibodies (green). Cell nuclei were counterstained with DAPI (blue). Cells were observed with a fluorescence microscope. The scale bar represents 10 μm. (b) Schematics represent the genes encoding the full-length or truncated PX molecules (from Δ1 to Δ6). (c) Distribution of PX or PX-truncated mutants in LMN cells. LMH cells were transfected with indicated plasmids as described in (a). Twenty-four hours after transfection, cells were fixed and probed with mouse anti-FLAG antibodies, followed by incubation with FITC-conjugated goat anti-mouse secondary antibodies (green). Cell nuclei were counterstained with DAPI (blue). LMH cells were observed with a fluorescence microscope. The scale bar represents 10 μm. (d,e) Examination of apoptosis in LMH cells expressing PX or PX-truncated mutants.…

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  Figure 4. Mutation of nuclear localization sequences (NLS) on both sides of -FSTKQ- within the amino acids 14-25 of PX affected its nuclear translocation associated with inhibition of PX-induced apoptosis. (a) Schematics represent the genes encoding the wild-type pX and nuclear localization sequence (NLS) mutant pX molecules (pX-wt, pX-nls1m, pX-nls2m, and pX-nls1/2m). (b) Distribution of PX or PX-nls-mutants in LMN cells. LMH cells were transfected with indicated plasmids as described in (a). Twenty-four hours after transfection, cells were fixed and probed with mouse anti-FLAG antibodies, followed by incubation with FITC-conjugated goat anti-mouse secondary antibodies (green). Nuclei were counterstained with DAPI (blue). LMH cells were observed with a fluorescence microscope. The scale bar represents 10μm. (c,d) Examination of apoptosis in LMH cells expressing PX or PX-NLS mutants. LMH cells were transfected with indicated plasmids as described in (a). Forty-eight hours after transfection, cells were harvested, stained with Annexin V-PE and 7-AAD, and analyzed by flow cytometry (c). The percentage of apoptotic cells in (c) was quantified and shown in (d). Results are representative of…

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  Figure 5. Amino acids 11 and 129 Alanines of PX are crucial to PX-induced apoptosis. (a) Alignment of PX amino acid sequences of virulent FAdV4 strains isolated in China with that of apathogenic FAdV ON1 strain. (b) Substitution of aa 11 and 129 alanines by threonines did not affect the localization of PX. LMH cells were seeded on 12-well plates and cultured overnight, followed by transfection with vectors pRK5-FLAG-pXA11T, pRK5-FLAG-pXA129T, and pRK5-FLAG-pXA11/129T, respectively. Twenty-four hours after transfection, cells were fixed and probed with mouse anti-FLAG antibodies, followed by incubation with FITC-conjugated goat anti-mouse antibodies (green). Cell nuclei were counterstained with DAPI (blue). Cells were observed with a fluorescence microscope. The scale bar represents 10μm. (c and d) Examination of apoptosis in LMH cells expressing PX or PX-mutants. LMH cells were transfected with indicated plasmids as described above. Forty-eight hours after transfection, cells were harvested, stained with Annexin V-PE and 7-AAD, and analyzed by flow cytometry (c); empty plasmid-transfected cells were used as controls. The percentage of apoptotic cells in (c) was quantified and shown as in…

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  Figure 6. Inhibition of FAdV4-induced apoptosis by caspase-3 inhibitor restricted viral replication. LMH cells treated with z-DEVD-fmk (caspase-3 inhibitor) or DMSO or medium only as controls were infected with FAdV4 at a MOI of 0.1, respectively. At different time points (12, 24, 36, and 48h) post FAdV4 infection, the viral titers in cell cultures (a) and supernatants (b) were determined with TCID50 using 96-well plates. Results are representative of three independent experiments. Data are presented as mean ± SD, n = 3. The significance of the differences between the groups was performed by one-way ANOVA (* stands for p < 0.05, and ** for p < 0.01).