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. 2018 Apr 30;41(4):271-281.
doi: 10.14348/molcells.2018.2091. Epub 2018 Apr 5.

IFIT1 Expression Patterns Induced by H9N2 Virus and Inactivated Viral Particle in Human Umbilical Vein Endothelial Cells and Bronchus Epithelial Cells

Bo Feng  1   2 , Qian Zhang  1   2 , Jianfang Wang  1 , Hong Dong  1 , Xiang Mu  1 , Ge Hu  1 , Tao Zhang  1
Affiliations

IFIT1 Expression Patterns Induced by H9N2 Virus and Inactivated Viral Particle in Human Umbilical Vein Endothelial Cells and Bronchus Epithelial Cells

Bo Feng et al. Mol Cells. .

Abstract

IFIT1 (also known as ISG56) is a member of the interferon-inducible protein with tetratricopeptide repeats (IFITs) family. IFITs are strongly induced by type I interferon (IFN), double-stranded RNA and virus infection. Here, we investigated IFIT1 expression in human umbilical vein endothelial cells (HUVECs) and in human bronchus epithelial cells (BEAS-2Bs) induced by the H9N2 virus and inactivated viral particle at different time points. We also investigated the effect of H9N2 virus and viral particle infection on IFN-α/β production, and assessed whether hemagglutinin or neuraminidase protein induced IFIT1 expression. Results showed that both H9N2 virus infection and viral particle inoculation induced the expression of IFIT1 at mRNA and protein levels in the two cell lines. Hemagglutinin or neuraminidase protein binding alone is not sufficient to induce IFIT1 expression. Surprisingly, the expression patterns of IFIT1 in response to H9N2 virus and viral particles in the two cell lines were opposite, and production kinetics of IFN-α/β also differed. An additional finding was that induction of IFIT1 in response to H9N2 virus infection or viral particle inoculation was more sensitive in HUVECs than in BEAS-2Bs. Our data offers new insight into the innate immune response of endothelial cells to H9N2 virus infection.

Keywords: H9N2 virus; IFIT1 expression pattern; IFN-α/β; endothelial cells.

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Figures

Fig. 1
Fig. 1. Replication of H9N2 virus in BEAS-2Bs
BEAS-2Bs grown on six-well plates were infected with H9N2 virus at MOIs. Culture supernatants were collected at a given time, and viral titers were determined by plaque-forming units. Values represent the means from three independent experiments plus standard deviations. (A) Replication of H9N2 virus at 24 h postinfection with different MOIs. (B) Replication of H9N2 virus at different time points after infection with MOI of 5 and 0.2, respectively.
Fig. 2
Fig. 2. IFIT1 expression induced by H9N2 virus infection and viral particle inoculation in BEAS-2Bs
BEAS-2Bs were infected with H9N2 virus (i.e., Virus) and inoculated with inactivated viral particle (i.e., Particle) at a MOI of 5, cells used for RT-PCR assay were collected at 6 h, 12 h and 24 h postinfection, cells used for western blot assay were collected at 12 h, 24 h and 36 h postinfection. Values represent the means from three independent experiments plus standard deviations. (A) Expression of IFIT1 at mRNA level. (B) Expression of IFIT1 at protein level. (C) The relative protein level of IFIT1 compared to β-actin. *means particle group and virus group compared with control group (*P < 0.05, **P < 0.01, ANOVE). # means particle group compared with virus group (#P < 0.05, ##P < 0.01, t-test).
Fig. 3
Fig. 3. IFIT1 expression induced by H9N2 virus infection and viral particle inoculation in HUVECs
HUVECs were infected with H9N2 virus (i.e., Virus) and inoculated with inactivated viral particle (i.e., Particle) at a MOI of 5, cells used for RT-PCR assay were collected at 6 h, 12 h and 24 h postinfection, cells used for western blot assay were collected at 12, 24 and 36 h postinfection. Values represent the means from three independent experiments plus standard deviations. (A) Expression of IFIT1 at mRNA level. (B) Expression of IFIT1 at protein level. (C) The relative protein level of IFIT1 compared to β-actin. *means particle group and virus group compared with control group (*P < 0.05, **P < 0.01, ANOVE). # means particle group compared with virus group (#P < 0.05, ##P < 0.01, t-test).
Fig. 4
Fig. 4. Evaluation of IFN-α/β production in HUVECs and BEAS-2Bs inoculated with H9N2 virus or viral particle
HUVECs and BEAS-2Bs were infected with H9N2 virus (i.e., Virus) and inoculated with inactivated viral particle (i.e., Particle) at a MOI of 5, and then supernatants were collected at 6 h, 12 h and 24 h postinfection. Levels of IFN-α/β were detected using human ELISA kits. Values represent the means from three independent experiments plus standard deviations. (A) IFN-α levels in BEAS-2Bs. (B) IFN-β levels in BEAS-2B cells. (C) IFN-α levels in HUVECs. (D) IFN-β levels in HUVECs. *means particle group and virus group compared with control group (*P < 0.05, **P < 0.01, ANOVE). # means particle group compared with virus group (#P < 0.05, ##P < 0.01, t-test).
Fig. 5
Fig. 5. IFIT1 mRNA levels induced by HA or NA protein in HUVECs and BEAS-2Bs
HUVECs and BEAS-2Bs were incubated with HA or NA at concentrations of 0.1 or 1 μg/ml. Cells used for RT-PCR analysis were collected at 6 h, 12 h and 24 h postinfection. (A) IFIT1 mRNA levels induced by HA in HUVECs. (B) IFIT1 mRNA levels induced by HA in BEAS-2Bs. (C) IFIT1 mRNA levels induced by NA in HUVECs. (d) IFIT1 mRNA levels induced by NA in BEAS-2Bs.
Fig. 6
Fig. 6. Localization of viral particles in HUVECs and BEAS-2Bs
BEAS-2Bs and HUVECs were inoculated with viral particles at a MOI of 5 for 24 h, then cells were double-stained with an anti-HA antibody (green) and 4′, 6-diamidino-2-phenylindole (DAPI, blue).
Fig. 7
Fig. 7. Antiviral response induced by viral particle inoculation in BEAS-2B2 and HUVECs
HUVECs and BEAS-2Bs cells were pretreated inactivated viral particle (i.e., Particle) at a MOI of 5 for 24 h, and then HUVECs and BEAS-2Bs were infected with H9N2 virus at a MOI of 5. At 24 h postinfection, supernatant in each group was collected, and viral titers were determined by plaque-forming units. Values represent the means from three independent experiments plus standard deviations. (A) Virus titers in HUVECs treated with viral particles. (B) Virus titers in BEAS-2Bs treated with viral particles. *means viral particle group compared with control group (*P < 0.05, t-test).
Fig. 8
Fig. 8. Overexpression of IFIT1 could not significantly reduced virus titers in HUVECs and BEAS-2Bs
HUVECs and BEAS-2Bs were transfected with control plasmid (Control) or IFIT1 CRISPR activation plasmid (IFIT1) for 36 h, then cells were infected with H9N2 virus at MOI of 5. Virus titer of each group was detected by plaque assay at 36 h postinfection. The overexpression of IFIT1 was detected by Western blot. (A) IFIT1 protein levels after transfected with plasmid in BEAS-2Bs and HUVECs. (B) Virus titers in BEAS-2Bs and HUVECs transfected with control plasmid or IFIT1 CRISPR activation plasmid.
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