Influenza A Virus Dysregulates Host Histone Deacetylase 1 That Inhibits Viral Infection in Lung Epithelial Cells
- R. M. Sandri-Goldin, Editor
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FIG 1IAV downregulates the expression of HDAC1. (A) A549 cells (8 × 105) were infected with PR8 at an MOI of 0.5 for 24 h. The uninfected (UNI) and infected (INF) cells were harvested and processed, and HDAC1 mRNAs and beta-actin mRNAs were detected by quantitative real-time PCR. The level of HDAC1 mRNA was normalized to beta-actin mRNA. The normalized value of HDAC1 mRNA in UNI sample was considered 100% for comparison to the INF sample. Error bars represents the means ± the standard errors of the means of three independent experiments; the P value was calculated using a t test. (B) A549 cells were infected as described above and harvested at the indicated times. Total cell lysates were prepared, and HDAC1 (62 kDa), PDI (57 kDa), and viral NP (56 kDa) were detected in uninfected and infected (2, 6, 12, and 24 h) cell lysates by WB. PDI was detected as the loading control, and NP was detected as the infection marker. (C) The HDAC1 and PDI protein bands were quantified using Image Studio Lite V4.0 software (Li-Cor), and the amount of HDAC1 was normalized to PDI. The normalized amount of HDAC1 in the UNI sample or the 2-h sample was considered 100% for comparisons to the 6-, 12-, and 24-h samples. Data presented are means ± the standard errors of the means of three independent experiments; the P value was calculated using one-way ANOVA. (D) A549 cells were infected with IAV WSN strain at an MOI of 0.5, 3.0 and 5.0 for 24 h. Total cell lysates were prepared, and HDAC1, PDI, and NP were detected in UNI and infected (0.5, 3.0, and 5.0) cell lysates by WB. (E) A549 cells were infected with live or UV-irradiated PR8 at an MOI of 0.5 for 24 h. Total cell lysates were prepared, and HDAC1, actin (42 kDa, as loading control), and NP were detected in UNI, INF, and INF-UV cell lysates by WB. (F) A549 cells were infected with live or UV-irradiated influenza virus A/New Caledonia/20/1999 (H1N1) strain at an MOI of 0.5 for 24 h. Total cell lysates were prepared, and HDAC1, actin, and NP were detected in UNI, INF, and INF-UV cell lysates by WB. (G) A549 cells were infected with PR8 as described above and subsequently treated with NH4Cl (20 mM) or MG132 (10 μM) for 24 h. Total cell lysates were prepared, and HDAC1, actin, and NP were detected in UNI and INF cell lysates by WB. (H) The HDAC1 and actin protein bands were quantified as described above, and the amount of HDAC1 was normalized to actin. The normalized amount of HDAC1 in respective UNI samples was considered 100% for comparisons to INF samples. The data presented are means ± the standard errors of the means of three independent experiments; the P value was calculated by using one-way ANOVA. (I) Ubiquitin (Ubiq) and actin were detected in the cell lysates described above by WB. U, uninfected; I, infected. MW, molecular weight.
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FIG 2The knockdown of HDAC1 expression promotes IAV infection. (A) A549 cells (2 × 105) were transfected with indicated concentrations of nontargeting control (CT) siRNA or HDAC1-targeting (HD1) siRNA for 72 h. Total cell lysates were prepared, and HDAC1 and PDI were detected by WB. (B to D) A549 cells were transfected with 10 nM CT siRNA or HD1 siRNA for 72 h. The cells were then infected with PR8 at an MOI of 0.5, and the culture medium and the cells were harvested separately after 2, 6, 12, and 24 h of infection. The virion yield in the culture medium was measured by WB of NP (B) and by microplaque assay (C). The data presented are means ± the standard errors of the means of three independent experiments; the P value was calculated by using two-way ANOVA. (D) Total lysates of the cells were prepared, and HDAC1, PDI, and NP were detected by WB. (E) A549 cells transfected with CT or HD1 siRNAs, as described above, were infected with PR8 at an MOI of 0.1 in the presence of 0.1 μg of trypsin/ml, and the virion yield in the culture medium was measured by microplaque assay after 24 and 48 h. The virion yield from respective CT siRNA samples was considered to be 1-fold for comparisons to HD1 siRNA samples. The data presented are means ± the standard errors of the means of three independent experiments; the P values were calculated using one-way ANOVA. (F) A549 cells were transfected with no siRNA, CT siRNA, or HD1 siRNA for 72 h. The cell viability was determined using an MTT assay. MW, molecular weight; ns, not significant.
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FIG 3The overexpression of HDAC1 inhibits IAV infection. (A, B, and D) A549 cells (8 × 105) were transfected with empty plasmid pcDNA3 (pc) or pcDNA3 containing HDAC1 (HD1) for 48 h. Cells were then infected with PR8 at an MOI of 0.5, and the culture medium and the cells were harvested separately after 24 h. The virion yield in the culture medium was measured by WB of NP (A) and by microplaque assay (B). The data presented are means ± standard errors of the means of three independent experiments; the P value was calculated using a t test. (C) A549 cells transfected with pc or HD1 plasmids, as described above, were infected with PR8 at an MOI of 0.1 in the presence of 0.1 μg of trypsin/ml, and the virion yield in the culture medium was measured by microplaque assay after 24, 48, and 72 h. The virion yield from “pc” samples was considered 100% for comparisons to HD1 samples. The data presented are means ± the standard errors of the means of three independent experiments; the P value was calculated by using one-way ANOVA. (D) Total lysates of the cells were prepared, and HDAC1, acetylated-histone H3 (Lys9) (Acet H3; 17 kDa), total histone H3 (Total H3, 17 kDa), and NP were detected by WB. Acet H3 was detected as the HDAC1 substrate, and total H3 was detected as the loading control. (E) Transfection efficiency of A549 cells. Cells were transfected with a plasmid expressing green fluorescent protein as above. The cells were viewed, and an image was acquired on an inverted fluorescence microscope (Olympus) under a magnification of ×10. MW, molecular weight.
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FIG 4IAV downregulates the activity of HDAC1. (A) MDCK cells (8 × 105) were infected with PR8 at an MOI of 0.5 and harvested after 2, 6, 12, and 24 h of infection. Total cell lysates were prepared, and acetylated-histone H3 (Lys9) (Acet H3), total histone H3 (Total H3), PDI, HDAC1, actin, and NP were detected in uninfected (UNI) and infected (2, 6, 12, and 24 h) cell lysates by WB. Acet H3, an HDAC1 substrate, was detected as a marker of HDAC1 activity, and total H3, PDI, and actin were detected as loading controls. (B) Acet H3 and total H3 protein bands were quantified as Fig. 1C, and the amount of Acet H3 was normalized to the total H3. The normalized amount of Acet H3 in UNI sample was considered 1-fold for comparisons to 2-, 6-, 12-, and 24-h samples. The data presented are means ± the standard errors of the means of three independent experiments; the P value was calculated by using one-way ANOVA. MW, molecular weight.
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FIG 5The inhibition of HDAC activity promotes IAV infection. (A to C) A549 cells (8 × 105) were infected with PR8 at an MOI of 0.5 and subsequently treated with dimethyl sulfoxide (DMSO) or the indicated concentrations of TSA (in DMSO) for 24 h. (A) The culture medium and the cells were harvested separately, and the virion yield in the culture medium was measured by microplaque assay. The data presented are means ± the standard errors of the means of three independent experiments; the P value was calculated by using one-way ANOVA. (B) A549 cells were infected and treated with DMSO and TSA for 24 h, and the cell viability was determined by an MTT assay. The viability of DMSO-treated infected cells was considered 100% for comparison to TSA-treated infected cells. (C) Total cell lysates were prepared, and Acet H3, total H3, and NP were detected by WB as the markers of TSA potency, loading control, and infection, respectively. MW, molecular weight.
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FIG 6The inhibition of HDAC activity inhibits the phosphorylation of STAT1 and expression of ISGs. (A to E) A549 cells (8 × 105) were infected with PR8 at an MOI of 0.5 and subsequently treated with DMSO or the indicated concentrations of TSA (in DMSO) for 24 h. Total cell lysates were prepared, and phosphorylated STAT1 (pSTAT1, 91/84 kDa), along with total STAT1 (tSTAT1) as a loading control (A), and IFITM3 (15 kDa) (B), ISG15 (15 kDa) (C), or viperin (42 kDa) (D), along with PDI as loading control, were detected in uninfected (UNI) and infected (INF) cell lysates by WB. (E) In the same lysates, Acet H3, total H3, and NP were detected by WB as the markers of TSA potency, loading control, and infection, respectively. MW, molecular weight.
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FIG 7HDAC1 is involved in the expression of viperin in IAV-infected cells. (A and B) The knockdown of HDAC1 expression decreased viperin expression in infected cells. A549 cells (2 × 105) were transfected with nontargeting control (CT) siRNA or HDAC1-targeting (HD1) siRNA for 72 h. The cells were then infected with PR8 at an MOI of 0.5 for 24 h. (A) Total cell lysates were prepared, and HDAC1, viperin, PDI, and NP were detected in uninfected (UNI) and infected (INF) cell lysates by WB. (B) The viperin and PDI protein bands were quantified as Fig. 1C, and the amount of viperin was normalized to PDI. The normalized amount of viperin in CT siRNA-transfected INF cells was considered 100% for comparisons to HD1 siRNA-transfected INF cells. The data presented are means ± the standard errors of the means of three independent experiments; the P value was calculated using a t test. (C and D) The overexpression of HDAC1 increased viperin expression in infected cells. A549 cells (8 × 105) were transfected with empty plasmid pcDNA3 or pcDNA3 containing HDAC1 for 48 h. The cells were then infected with PR8 at an MOI of 0.5 for 24 h. (C) Total cell lysates were prepared, and HDAC1, viperin, PDI, and NP were detected in uninfected (UNI) and infected (INF) cell lysates by WB. Lanes 1 to 3 were combined with lanes 4 and 5 of the same blot to remove an unwanted lane from the middle. (D) The percent change in viperin level was calculated as described above. The data presented are means ± the standard errors of the means of three independent experiments; the P value was calculated using a t test. MW, molecular weight.
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