Viperin Differentially Induces Interferon-Stimulated Genes in Distinct Cell Types

Jeong Jin Kim; Ku Sul Kim; John Eom; Jae Bong Lee; Jun-Young Seo

doi:10.4110/in.2018.19.e33

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Kim, Kim, Eom, Lee, and Seo: Viperin Differentially Induces Interferon-Stimulated Genes in Distinct Cell Types

Original Article

Immune Netw 2019;19(5):e33.

Published online: 4 October 2019

DOI: https://doi.org/10.4110/in.2018.19.e33

Viperin Differentially Induces Interferon-Stimulated Genes in Distinct Cell Types

Jeong Jin Kim, Ku Sul Kim, John Eom, Jae Bong Lee, Jun-Young Seo^*

Severance Biomedical Science Institute, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, Korea

^*Correspondence to Jun-Young Seo Severance Biomedical Science Institute, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea. E-mail: jyseo0724@yuhs.ac

Conflict of Interest The authors declare no potential conflicts of interest.

Received 29 October 2019 Revised 13 February 2019 Accepted 13 February 2019

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Viperin is an IFN-stimulated gene (ISG)-encoded protein that was identified in human primary macrophages treated with IFN-γ and in human primary fibroblasts infected with cytomegalovirus (CMV). This protein plays multiple roles in various cell types. It inhibits viral replication, mediates signaling pathways, and regulates cellular metabolism. Recent studies have shown that viperin inhibits IFN expression in macrophages, while it enhances TLR7 and TLR9-mediated IFN production in plasmacytoid dendritic cells, suggesting that viperin can play different roles in activation of the same pathway in different cell types. Viperin also controls induction of ISGs in macrophages. However, the effect of viperin on induction of ISGs in cell types other than macrophages is unknown. Here, we show that viperin differentially induces ISGs in 2 distinct cell types, macrophages and fibroblasts isolated from wild type and viperin knockout mice. Unlike in bone marrow-derived macrophages (BMDMs), viperin downregulates the expression levels of ISGs such as bone marrow stromal cell antigen-2, Isg15, Isg54, myxovirus resistance dynamin like GTPase 2, and guanylate binding protein 2 in murine embryonic fibroblasts (MEFs) treated with type I or II IFN. However, viperin upregulates expression of these ISGs in both BMDMs and MEFs stimulated with polyinosinic-polycytidylic acid or CpG DNA and infected with murine CMV. The efficiency of viral entry is inversely proportional to the expression levels of ISGs in both cell types. The data indicate that viperin differentially regulates induction of ISGs in a cell type-dependent manner, which might provide different innate immune responses in distinct cell types against infections.

Keywords: Viperin, Interferons, ISG, Macrophages, Fibroblasts, Cytomegalovirus

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Figure 1.

Viperin differentially regulates ISG expression in BMDMs and MEFs treated with type I IFN. (A-D) The effect of viperin on expression of ISGs in BMDMs and MEFs upon type I IFN treatment. The bone marrow cells isolated from WT and viperin KO C57BL/6 mice were differentiated into BMDMs in M-CSF conditioned media for 7 days. The BMDMs were washed and plated in media without M-CSF for 24 h (A, B). MEFs were isolated from these mice and immortalized (C, D). The cells were treated with or without type I IFN (1,000 U/ml) for 24 h. The mRNA expression levels of ISGs in the cells were measured by qRT-PCR and normalized to β-actin mRNA (A, C). Data are presented as mean ± SEM of triplicate samples and are representative of three individual experiments. Expression of viperin and ISG15 proteins in the cells was detected by immunoblot using anti-viperin (MaP.VIP) or anti-ISG15 antibody (B, D). GRP94 served as a protein-loading control. Quantitation of ISG15 protein level was normalized to GRP94. * p<0.05; ^** p<0.01; ^*** p<0.001.

Figure 2.

Viperin differently induces ISG expression in BMDMs and MEFs treated with type II IFN. (A-D) The effect of viperin on expression of ISGs in BMDMs and MEFs upon type II IFN treatment. WT and viperin KO BMDMs (A, B) or MEFs (C, D) were treated with or without IFN-γ (100 ng/ml) for 24 h. The mRNA expression levels of ISGs in the cells were measured by qRT-PCR and normalized to β-actin mRNA (A, C). Data are presented as mean±SEM of triplicate samples and are representative of three individual experiments. Expression of viperin and ISG15 proteins in the cells was detected by immunoblot using anti-viperin (MaP.VIP) or anti-ISG15 antibody (B, D). GRP94 served as a protein-loading control. Quantitation of ISG15 protein level was normalized to GRP94. * p<0.05; ^** p<0.01; ^*** p<0.001.

Figure 3.

Viperin enhances ISG expression in both BMDMs and MEFs transfected with poly(I:C) or CpG DNA. (A-F) The effect of viperin on expression of ISGs in BMDMs and MEFs upon poly(I:C) or CpG DNA treatment. WT and viperin KO BMDMs (A-C) or MEFs (D-F) were treated with lipofectamine 2000 (Lipo), or transfected with poly(I:C) (1 μg/ml) or CpG DNA (1 μg/ml) for 24 h. The mRNA expression levels of ISGs in the cells were measured by qRT-PCR and normalized to β-actin mRNA (A, D). Data are presented as mean±SEM of triplicate samples and are representative of three individual experiments. Expression of viperin and ISG15 proteins in the cells was detected by immunoblot using anti-viperin (MaP.VIP) or anti-ISG15 antibody (B, C, E, F). GRP94 served as a protein-loading control. Quantitation of ISG15 protein level was normalized to GRP94. * p<0.05; ^** p<0.01; ^*** p<0.001.

Figure 4.

Viperin differentially regulates ISG expression in BMDMs and MEFs treated with MCMV and/or type I IFN. (A, B) The effect of viperin on expression of ISGs in BMDMs and MEFs infected with MCMV in the absence or presence of type I IFN. WT and viperin KO BMDMs (A) or MEFs (B) were treated with or without type I IFN (1,000 U/ml) for 8 h and then infected with MCMV at an MOI of 0.2 for 24 h. The mRNA expression levels of ISGs in the cells were measured by qRT-PCR and normalized to β-actin mRNA. Data are presented as mean ± SEM of triplicate samples and are representative of three individual experiments. * p<0.05; ^** p<0.01; ^*** p<0.001.

Figure 5.

Viperin differently induces ISG expression in BMDMs and MEFs treated with MCMV and/or type II IFN. (A, B) The effect of viperin on expression of ISGs in BMDMs and MEFs infected with MCMV in the absence or presence of type II IFN. WT and viperin KO BMDMs (A) or MEFs (B) were treated with or without IFN-γ (100 ng/ml) for 8 h and then infected with MCMV at an MOI of 0.2 for 24 h. The mRNA expression levels of ISGs in the cells were measured by qRT-PCR and normalized to β-actin mRNA. Data are presented as mean±SEM of triplicate samples and are representative of three individual experiments. * p<0.05; ^** p<0.01; ^*** p<0.001.

Figure 6.

MCMV entry is inversely proportional to viperin-mediated ISG expression. (A-D) The efficiency of MCMV entry into BMDMs and MEFs infected with MCMV in the absence or presence of IFNs. WT and viperin KO BMDMs (A, B) or MEFs (C, D) were treated with or without type I IFN (1,000 U/ml) or IFN-γ (100 ng/ml) for 8 h and then infected with MCMV at an MOI of 2 for 24 h. The cells were stained with antibody specific to the MCMV protein IE1 (green) to identify infected cells. Nuclei were stained with DAPI (blue). A representative image from two individual experiments was shown (scale bar=100 μm) (A, C). The efficiency of MCMV entry into the cells was quantitated (B, D). The infected cells were counted in each image (n=10). The percentage of MCMV positive cells per total cells in each image was calculated. Data are presented as mean±SEM. * p<0.05; ^*** p<0.001.

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