Post-Transcriptional Induction of the Antiviral Host Factor GILT/IFI30 by Interferon Gamma
<p>IFN-γ induces GILT protein expression but not its mRNA in HeLa cells. (<b>A</b>) HeLa cells were treated with 0.2 μg/mL of IFN-γ for indicated time period. GILT and actin proteins were analyzed using western blotting. (<b>B</b>) Fluorescent intensities of GILT, FAT10, IDO1, and IFI6 mRNA in both IFN-γ (0.2 μg/mL)-treated and untreated cells were measured with microarray (Kubo et al., 2022) [<a href="#B12-ijms-25-09663" class="html-bibr">12</a>]. Fold inductions by IFN-γ are also indicated. (<b>C</b>) The copy numbers of GAPDH, GILT, and IFI6 mRNAs were quantified using ddPCR. Normalized copy numbers are presented with error bars indicating standard deviations (<span class="html-italic">n</span> = 3). Significance in difference between specified groups is denoted by the <span class="html-italic">p</span>-value from Student’s t-test. (<b>D</b>) Copy numbers of GILT and GAPDH mRNAs in the cytoplasm and nuclei were measured using ddPCR, and the ratios of normalized copy numbers of GILT cDNAs in the cytoplasmic and nuclear fractions to the total copy numbers of GILT cDNA are indicated (<span class="html-italic">n</span> = 3). Error bars show standard deviations.</p> "> Figure 2
<p>IFN-γ induces GILT protein expression but not its mRNA in TE671 cells. (<b>A</b>) TE671 cells, JEG3 cells, and PBMCs were treated with 0.2 μg/mL of IFN-γ for 3 days, and cell lysates and total RNA samples were extracted. GILT and actin proteins were analyzed using western blotting using their antibodies. (<b>B</b>) Phosphorylated and total STAT1 proteins were analyzed with western blotting, using their specific antibodies. (<b>C</b>) The copy numbers of GAPDH, GILT, and IFI6 mRNAs in TE671 and JEG3 cells were quantified using ddPCR. Normalized copy numbers are presented with error bars indicating standard deviations (<span class="html-italic">n</span> = 3). Significance in difference between specified groups is denoted by the <span class="html-italic">p</span>-value from Student’s t-test.</p> "> Figure 3
<p>GILT promoter is not activated by IFN-γ. (<b>A</b>) The promoter/enhancer region of the GILT gene was amplified with PCR, and its nucleotide sequence is indicated. Bold and underlined letters show putative GAS and CAT sequences, respectively. (<b>B</b>) HeLa cells were transfected with expression plasmids for nano luciferase (NanoLuc) under the control of the GILT promoter/enhancer and for firefly luciferase (FLuc) under the control of the GAS sequence from the LMP2 gene and treated with IFN-γ. Cell lysates were prepared from the treated cells 3 days after the treatment. NanoLuc and FLuc activities of the cell lysates were measured (<span class="html-italic">n</span> = 3). Luciferase activity of the untreated cells is always set to 1. Relative luciferase activities of the IFN-γ-treated cells to those of untreated cells are indicated. Error bars show standard deviations. The <span class="html-italic">p</span> value between the FLuc activities in untreated and IFN-γ-treated cells is indicated.</p> "> Figure 4
<p>Stability of GILT protein is not changed by IFN-γ. (<b>A</b>) The translation inhibitor cycloheximide (100 μM final concentration) was added to HeLa cells transduced with an MLV vector expressing GILT and culture for indicated time periods. Cell lysates from the treated cells were analyzed with western blotting using anti-GILT or anti-actin antibody. (<b>B</b>) The intensities of the mature GILT protein detected in the western blotting analysis were measured. The GILT intensities in the untreated GILT-expressing HeLa cells are always set to 1. Relative intensities to the untreated cells are indicated (<span class="html-italic">n</span> = 3). Error bars indicate standard deviations.</p> "> Figure 5
<p>Impact of the mTOR inhibitor rapamycin on GILT protein expression, GILT promoter activity. (<b>A</b>) HeLa cells were treated with DMSO, rapamycin, and/or IFN-γ for 3 days. Cell lysates from the treated cells were analyzed with western blotting using anti-GILT and anti-actin antibodies. (<b>B</b>) HeLa cells were treated with DMSO or rapamycin for 3 days because rapamycin was dissolved with DMSO. Cell numbers were counted (<span class="html-italic">n</span> = 3). Error bars indicate standard deviations. (<b>C</b>) HeLa cells were transfected with the expression plasmids for NanoLuc and FLuc under the control of the GILT promoter and GAS, respectively. The transfected cells were treated with DMSO, rapamycin, and/or IFN-γ for 3 days as indicated. NanoLuc and FLuc activities were measured (<span class="html-italic">n</span> = 3). Luciferase activities of the DMSO-treated cells are always set to 1. Relative luciferase activities to those of the DMSO-treated cells are indicated. Error bars indicate standard deviations. The <span class="html-italic">p</span> values of Student’s t-test and ANOVA are shown. (<b>D</b>) The copy numbers of GAPDH, GILT, and IFI6 mRNA were measured using ddPCR. Normalized copy numbers of GILT and IFI6 are indicated (<span class="html-italic">n</span> = 3). Error bars show standard deviations. The <span class="html-italic">p</span>-values of ANOVA and Student’s t-test are indicated.</p> "> Figure 6
<p>Untranslated regions of the GILT mRNA inhibit GILT protein expression. (<b>A</b>) An expression plasmid containing full-length GILT mRNA was obtained (Full GILT). A DNA fragment containing the 5′ UTR and GILT protein-coding region was amplified and ligated into pcDNA3.1 (5′UTR-GILT). A DNA fragment containing the GILT protein-coding region and 3′ UTR was amplified and ligated to pcDNA3.1 (GILT-3′UTR). (<b>B</b>) HeLa cells were transfected with the Renilla luciferase expression plasmid together with the Full GILT, 5′UTR-GILT, or GILT-3′UTR expression plasmid and then were treated with IFN-γ for 24 h. Cell lysates from the treated cells were analyzed with western blotting using anti-GILT and anti-actin antibodies. (<b>C</b>) Renilla luciferase activities of the cell lysates were measaured (<span class="html-italic">n</span> = 3). Error bars show standard deviations.</p> "> Figure 7
<p>3′ untranslated region of the GILT mRNA inhibits luciferase protein expression but not in the presence of IFN-γ. (<b>A</b>) The 5′ and 3′ UTRs were fused to the 5′ and 3′ ends of the RLuc-coding region, respectively (CMV-5′UTR-RLuc and CMV-RLuc-3′UTR). (<b>B</b>) These expression plasmids were transfected into HeLa cells. RLuc activities were measured. Luminescence levels are indicated with standard deviations (<span class="html-italic">n</span> = 3). (<b>C</b>) The 3′ half region of the full-length GILT cDNA was linked to the 3′ end of the RLuc-coding region (CMV-RLuc-3′GILT). The 5′ half region of the GILT cDNA was fused to the 5′ end of the RLuc (CMV-5′GILT-RLuc). The resulting DNA fragments were ligated to pcDNA3.1. (<b>D</b>) HeLa cells were transfected with CMV-RLuc, CMV-RLuc-3′GILT, or CMV-5′GILT-RLuc expression plasmid and then were treated with IFN-γ for 2 days. RLuc activities of the treated cells were measured. (<b>E</b>) HeLa cells were transfected with CMV-RLuc, CMV-RLuc-3′GILT, or CMV-5′GILT-RLuc expression plasmid and then were treated with IFN-γ and/or rapamycin for 2 days. Relative luciferase activities to the CMV-RLuc-transfected cells in the absence and presence of IFN-γ are indicated (<span class="html-italic">n</span> = 3). Error bars show standard deviations. The <span class="html-italic">p</span> values of the Student’s t-test and ANOVA are demonstrated.</p> "> Figure 8
<p>STAT1 phosphorylation is required for the initiation of GILT translation by IFN-γ. (<b>A</b>) TE671 cells were treated with IFN-γ and/or FLU, and cell lysates were prepared. Phosphorylated STAT1, total STAT1, and GILT proteins were analyzed using western blotting. (<b>B</b>) The copy numbers of GAPDH, GILT, and IFI6 mRNA were measured using ddPCR. Normalized copy numbers of GILT and IFI6 are indicated (<span class="html-italic">n</span> = 3). Error bars show standard deviations. The <span class="html-italic">p</span> values of the Student’s t-test and ANOVA are demonstrated.</p> "> Figure 9
<p>Mechanism of GILT protein expression in the absence and presence of IFN-γ.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Post-Transcriptional Elevation of GILT Protein by IFN-γ in HeLa Cells
2.2. Post-Transcriptional Elevation of GILT Protein by IFN-γ in Other Cell Types
2.3. Absence of Activation of GILT Transcription by IFN-γ
2.4. GILT Protein Stability and Its Relationship with IFN-γ
2.5. Impact of Rapamycin on GILT Protein Induction by IFN-γ
2.6. Untranslated Region of GILT mRNA Inhibits Its Translation in the Absence of IFN-γ
2.7. STAT1 Phosphorylation Is Necessary for IFN-γ-Mediated GILT Protein Induction
3. Discussion
4. Materials and Methods
4.1. Plasmids
4.2. Cells
4.3. Peripheral Blood Mononuclear Cells
4.4. Amphotropic Murine Leukemia Virus Vector
4.5. Western Blotting
4.6. Luciferase Activity
4.7. Protein Stability
4.8. Droplet Digital PCR
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Maric, M.; Arunachalam, B.; Phan, U.T.; Dong, C.; Garrett, W.S.; Cannon, K.S.; Alfonso, C.; Karlsson, L.; Flavell, R.A.; Cresswell, P. Defective antigen processing in GILT-free mice. Science 2002, 294, 1361–1365. [Google Scholar] [CrossRef]
- Singh, R.; Cresswell, P. Defective cross-presentation of viral antigens in GILT-free mice. Science 2010, 328, 1394–1398. [Google Scholar] [CrossRef] [PubMed]
- Ewanchuk, B.W.; Yates, R.M. The phagosome and redox control of antigen processing. Free. Radic. Biol. Med. 2018, 125, 53–61. [Google Scholar] [CrossRef] [PubMed]
- Rausch, M.P.; Hastings, K.T. Diverse cellular and organismal functions of the lysosomal thiol reductase. Mol. Immunol. 2015, 68, 124–128. [Google Scholar] [CrossRef] [PubMed]
- Izumida, M.; Hayashi, H.; Smith, C.; Ishibashi, F.; Suga, K.; Kubo, Y. Antivirus activity, but not thiolreductase activity, is conserved in interferon-gamma-inducible GILT protein in arthropod. Mol. Immunol. 2021, 140, 240–249. [Google Scholar] [CrossRef]
- Kubo, Y.; Izumida, M.; Yashima, Y.; Yoshii-Kamiyama, H.; Tanaka, Y.; Yasui, K.; Hayashi, H.; Mastuyama, T. Gamma-interferon-inducible, lysosome/endosome-localized thiolreductase, GILT, has anti-retroviral activity and its expression is counteracted by HIV-1. Oncotarget 2016, 7, 71255–71273. [Google Scholar] [CrossRef]
- Schleicher, T.R.; Yang, J.; Freudzon, M.; Rembisz, A.; Craft, S.; Hamilton, M.; Graham, M.; Mlambo, G.; Tripathi, A.K.; Li, Y.; et al. A mosquito salivary gland protein partially inhibits Plasmodium sporozoite traversal and transmission. Nature Commum. 2018, 9, 2908. [Google Scholar] [CrossRef]
- Kongton, K.; McCall, K.; Phongdara, A. Identification of gamma-interferon-inducible lysosomal thiol reductase (GILT) homoloques in the fruit fly Drosophila melanogaster. Dev. Comp. Immunol. 2014, 44, 389–396. [Google Scholar] [CrossRef]
- Thipwong, J.; Saelim, H.; Panrat, T.; Phongdara, A. Penaeus monodon GILT enzyme restyricts WSSV infectivity by reducing disulfide bonds in WSSV proteins. Dis. Aquat. Organ. 2019, 135, 59–70. [Google Scholar] [CrossRef]
- Chen, D.; Hou, Z.; Jiang, D.; Zheng, M.; Li, G.; Zhang, Y.; Li, R.; Lin, H.; Chang, J.; Zeng, H.; et al. GILT restricts the cellular entry mediated by the envelope glycoproteins of SARS-CoV, Ebola virus, and Lassa fever virus. Emerg. Microbes Infect. 2019, 8, 1511–1523. [Google Scholar] [CrossRef]
- Darnell, J.E., Jr.; Kerr, I.M.; Stark, G.R. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994, 264, 1415. [Google Scholar] [CrossRef]
- Kubo, Y.; Yasui, K.; Izumida, M.; Hayashi, H.; Matsuyama, T. IDO1, FAT10, IFI6, and GILT are involved in the antiretroviral activity of γ-interferon and IDO1 restricts retrovirus infection by autophagy enhancement. Cells 2022, 11, 2240. [Google Scholar] [CrossRef] [PubMed]
- Inouye, S.; Watanabe, K.; Nakamura, H.; Shimomura, O. Secretional luciferase of the luminous shrimp Oplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone luciferase. FEBS Lett. 2000, 481, 19–25. [Google Scholar] [CrossRef]
- Chatterjee-Kishore, M.; Wright, K.L.; Ting, J.P.; Stark, G.R. How Stat1 mediates constitutive gene expression: A complex of unphosphorylated Stat1 and IRF1 supports transcription of the LMP2 gene. EMBO J. 2000, 19, 4111–4122. [Google Scholar] [CrossRef]
- Shi, G.; Ozog, S.; Torbett, B.E.; Compton, A.A. mTOR inhibitors lower an intrinsic barrier to virus infection mediated by IFITM3. Proc. Natl. Acad. Sci. USA 2018, 115, E10069–E10078. [Google Scholar] [CrossRef]
- Frank, D.A.; Mahajan, S.; Ritz, J. Fludarabine-induced immunosuppression is associated with inhibition of STAT1 signaling. Nature Med. 1999, 5, 444–447. [Google Scholar] [CrossRef] [PubMed]
- Buetow, K.H.; Meador, L.R.; Menon, H.; Lu, Y.K.; Brill, J.; Cui, H.; Roe, D.J.; DiCaudo, D.J.; Hastings, K.T. High GILT expression and an active and intact MHC class II antigen presentation pathway are associated with improved survival in melanoma. J. Immunol. 2019, 203, 2577–2587. [Google Scholar] [CrossRef] [PubMed]
- Nguyen, J.; Bernert, R.; In, K.; Kang, P.; Sebastiao, N.; Hu, C.; Hastings, K.T. Gamma-interferon-inducible lysosomal thiol reductase is upregulated in human melanoma. Melanoma Res. 2016, 26, 125–137. [Google Scholar] [CrossRef]
- Phipps-Yonas, H.; Cui, H.; Sebastiao, N.; Brunhoeber, P.S.; Haddock, E.; Deymier, M.J.; Klapper, W.; Lybarger, L.; Roe, D.J.; Hastings, K.T. Low GILT expression is associated with poor patient survival in diffuse large B-cell lymphoma. Front. Immunol. 2013, 4, 425. [Google Scholar] [CrossRef]
- Jiang, W.; Zheng, F.; Yao, T.; Gong, F.; Zheng, W.; Yao, N. IFI30 as a prognostic biomarker and correlation with immune infiltrates in glioma. Ann. Transl. Med. 2021, 9, 1686. [Google Scholar] [CrossRef]
- Liu, X.; Song, C.; Yang, S.; Ji, Q.; Chen, F.; Li, W. IFI30 expression is an independent unfavourable prognostic factor in glioma. J. Cell. Mol. Med. 2020, 24, 12433–12443. [Google Scholar] [CrossRef] [PubMed]
- Zhu, C.; Chen, X.; Guan, G.; Zou, C.; Guo, Q.; Cheng, P.; Cheng, W.; Wu, A. IFI30 is a novel immune-related target with predicting value of prognosis and treatment response in glioblastoma. Onco. Targets Ther. 2020, 13, 1129–1143. [Google Scholar] [CrossRef] [PubMed]
- Ye, C.; Zhou, W.; Wang, F.; Yin, G.; Zhang, X.; Kong, L.; Gao, Z.; Feng, M.; Zhou, C.; Sun, D.; et al. Profnostic value of gamma-interferon-inducible lysosomal thiol reductase expression in female patients diagnosed with breast cancer. Int. J. Cancer 2020, 150, 705–717. [Google Scholar] [CrossRef] [PubMed]
- Fan, Y.; Wang, X.; Li, Y. IFI30 expression predicts patient prognosis in breast cancer and dictates breast cancer cells proliferation via regulating autophagy. Int. J. Med. Sci. 2021, 18, 3342–3352. [Google Scholar] [CrossRef] [PubMed]
- Stefanovic, M.; Zivotic, I.; Stojkovic, L.; Dincic, E.; Stankovic, A.; Zivkovic, M. The association of genetic variants IL2RA rs2104286, IFI30 rs11554159, and IKZF3 rs12946510 with multiple sclerosis onset and severity in patients from Serbia. J. Neuroimmunol. 2020, 347, 577346. [Google Scholar] [CrossRef]
- Rausch, M.P.; Irvine, K.R.; Antony, P.A.; Restifo, N.P.; Cresswell, P.; Hastings, K.T. GILT accelerates autoimmunity to the melanoma antigen tyrosinase-related protein 1. J. Immunol. 2010, 185, 2828–2835. [Google Scholar] [CrossRef]
- Maric, M.; Barjaktarevic, I.; Bogunovic, B.; Stojakovic, M.; Maric, C.; Vukmanovic, S. Developmental up-regulation of IFN-gamma-inducible lysosomal thiol reductase expression leads to reduced T cell sensitivity and cell severe autoimmunity. J. Immunol. 2009, 182, 746–750. [Google Scholar] [CrossRef]
- O’Donnell, P.W.; Haque, A.; Klemsz, M.J.; Kaplan, M.H.; Blum, J.S. Induction of the antigen-processing enzyme IFN-γ-inducible lysosomal thiol reductase in melanoma cells is STAT1-dependent but CIITA-independent. J. Immunol. 2004, 173, 731–735. [Google Scholar] [CrossRef]
- Srinivasan, P.; Maric, M. Signal transducer and activator of transcription 1 negatively regulates constitutive gamma interferon-inducible lysosomal thiol reductase expression. Immunology 2010, 132, 209–216. [Google Scholar] [CrossRef]
- Burrows, G.G.; Meza-Romero, R.; Huan, J.; Sinha, S.; Mooney, J.L.; Vandenbark, A.A.; Offner, H. GILT required for RTL550-CYS-MOG to treat experimental sutoimmune encephalomyelitis. Metab. Brain Dis. 2012, 27, 143–149. [Google Scholar] [CrossRef]
- Maenaka, A.; Iwasaki, K.; Ota, A.; Miwa, Y.; Ohashi, W.; Horimi, K.; Matsuoka, Y.; Ohnishi, M.; Uchida, K.; Kobayashi, T. Interferon-γ-induced HLA class II expression on endothelial cells is decreased by inhibition of mTOR and HMG-CoA reductase. FEBS Open Bio. 2020, 10, 927–936. [Google Scholar] [CrossRef]
- Kroczynska, B.; Mehrotra, S.; Arslan, A.D.; Kaur, S.; Platanias, L.C. Regulation of interferon-dependent mRNA translation of target genes. J. Interferon Cytokine Res. 2014, 34, 289–296. [Google Scholar] [CrossRef]
- Kaur, S.; Lal, L.; Sassano, A.; Majchrzak-Kita, B.; Srikanth, M.; Baker, D.P.; Petroulakis, E.; Hay, N.; Sonenberg, N.; Fish, E.N.; et al. Regulatory effects of mammalian target of rapamycin-activated pathways in thpe I and II interferon signaling. J. Biol. Chem. 2007, 282, 1757–1768. [Google Scholar] [CrossRef]
- Kaur, S.; Sassano, A.; Dolniak, B.; Joshi, S.; Majchrzak-Kita, B.; Baker, D.P.; Hay, N.; Fish, E.N.; Platanias, L.C. Role of the Akt pathway in mRNA translation of interferon-stimulated genes. Proc. Natl. Acad. Sci. USA 2008, 105, 4808–4813. [Google Scholar] [CrossRef] [PubMed]
- Naldini, L.; Blomer, U.; Gallay, P.; Ory, D.; Mulligan, R.; Gage, F.H.; Verma, I.M.; Trono, D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996, 272, 263–267. [Google Scholar] [CrossRef] [PubMed]
- Kubo, Y.; Tominaga, C.; Yoshii, H.; Kamiyama, H.; Mitani, C.; Amanuma, H.; Yamamoto, N. Characterization of R peptide of murine leukemia virus envelope glycoproteins in syncytium formation and entry. Arch. Virol. 2007, 152, 2169–2182. [Google Scholar] [CrossRef] [PubMed]
- Chang, L.J.; Urlacher, V.; Iwakuma, T.; Cui, Y.; Zucali, J. Efficacy and safety analysis of a recombinant human immunodeficiency virus type 1 derived vector system. Gene Ther. 1999, 6, 715–728. [Google Scholar] [CrossRef]
- Meregildo-Rodriguez, E.D.; Asmat-Rubio, M.G.; Vasquez-Tirado, G.A. Droplet digital PCR vs. quantitative real time-PCR for diagnosis of pulmonary and extrapulmonary tuberculosis: Systematic review and meta-analysis. Front. Med. 2023, 10, 1248842. [Google Scholar] [CrossRef]
Primers | Nucleotide Sequences |
---|---|
5′ UTR sense | CTGCAGTCGCCACACCTTGC |
GILT sense | ATGACTTCGAAAGTTTATGAT |
GILT antisense | CACTTGAAGCAAACACTCCTG |
5′ UTR-RLuc sense | CTGCAGTCGCCACACCTTTGCCCCTGCTG (5′ UTR of GILT) CG ATGACTTCGAAAGTTTATGAT (RLuc) |
RLuc antisense | TTATTGTTCATTTTTGAGAACTCGC |
3′ UTR sense | TGGCCGGTGAGCTGCGGAGAG |
3′ UTR antisense | GCTTATTAAACTAGTTTTACTTTAGC |
GAPDH sense | CCATGCCATCACTGCCACCC |
GAPDH antisense | GCCAGTGAGCTTCCCGTTCAG |
GILT 532 antisense | CTACAGGCATAGTGGCAGACT |
IFI6 sense | GCGCGCGGCGCCACCATGCGG |
IFI6 antisense | TGGCTACTCCTCATCCTCCTC |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Nakamura, T.; Izumida, M.; Hans, M.B.; Suzuki, S.; Takahashi, K.; Hayashi, H.; Ariyoshi, K.; Kubo, Y. Post-Transcriptional Induction of the Antiviral Host Factor GILT/IFI30 by Interferon Gamma. Int. J. Mol. Sci. 2024, 25, 9663. https://doi.org/10.3390/ijms25179663
Nakamura T, Izumida M, Hans MB, Suzuki S, Takahashi K, Hayashi H, Ariyoshi K, Kubo Y. Post-Transcriptional Induction of the Antiviral Host Factor GILT/IFI30 by Interferon Gamma. International Journal of Molecular Sciences. 2024; 25(17):9663. https://doi.org/10.3390/ijms25179663
Chicago/Turabian StyleNakamura, Taisuke, Mai Izumida, Manya Bakatumana Hans, Shuichi Suzuki, Kensuke Takahashi, Hideki Hayashi, Koya Ariyoshi, and Yoshinao Kubo. 2024. "Post-Transcriptional Induction of the Antiviral Host Factor GILT/IFI30 by Interferon Gamma" International Journal of Molecular Sciences 25, no. 17: 9663. https://doi.org/10.3390/ijms25179663