Hippo Signaling Pathway Has a Critical Role in Zika Virus Replication and in the Pathogenesis of Neuroinflammation
2020; Elsevier BV; Volume: 190; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2019.12.005
ISSN1525-2191
AutoresGustavo Garcia, Sayan Paul, Sara Beshara, V. Krishnan Ramanujan, Arunachalam Ramaiah, Karin Nielsen‐Saines, Melody M. H. Li, Samuel W. French, Kouki Morizono, Ashok Kumar, Vaithilingaraja Arumugaswami,
Tópico(s)interferon and immune responses
ResumoZika virus (ZIKV) is a reemerging human pathogen that causes congenital abnormalities, including microcephaly and eye disease. The cellular/molecular basis of ZIKV and host interactions inducing ocular and neuronal pathogenesis are unclear. Herein, we noted that the Hippo/Salvador-Warts-Hippo signaling pathway, which controls organ size through progenitor cell proliferation and differentiation, is dysregulated after ZIKV infection. In human fetal retinal pigment epithelial cells, there is an early induction of transcriptional coactivator, Yes-associated protein (YAP), which is later degraded with a corresponding activation of the TANK binding kinase 1/interferon regulatory factor 3 type I interferon pathway. YAP/transcriptional co-activator with a PDZ-binding domain (TAZ) silencing results in reduced ZIKV replication, indicating a direct role of Hippo pathway in regulating ZIKV infection. Using an in vivo Ifnar1−/− knockout mouse model, ZIKV infection was found to reduce YAP/TAZ protein levels while increasing phosphorylated YAP Ser127 in the retina and brain. Hippo pathway is activated in major cellular components of the blood-brain barrier, including endothelial cells and astrocytes. In addition, this result suggests AMP-activated protein kinase signaling pathway's role in regulating YAP/TAZ in ZIKV-infected cells. These data demonstrate that ZIKV infection might initiate a cross talk among AMP-activated protein kinase–Hippo–TBK1 pathways, which could regulate antiviral and energy stress responses during oculoneuronal inflammation. Zika virus (ZIKV) is a reemerging human pathogen that causes congenital abnormalities, including microcephaly and eye disease. The cellular/molecular basis of ZIKV and host interactions inducing ocular and neuronal pathogenesis are unclear. Herein, we noted that the Hippo/Salvador-Warts-Hippo signaling pathway, which controls organ size through progenitor cell proliferation and differentiation, is dysregulated after ZIKV infection. In human fetal retinal pigment epithelial cells, there is an early induction of transcriptional coactivator, Yes-associated protein (YAP), which is later degraded with a corresponding activation of the TANK binding kinase 1/interferon regulatory factor 3 type I interferon pathway. YAP/transcriptional co-activator with a PDZ-binding domain (TAZ) silencing results in reduced ZIKV replication, indicating a direct role of Hippo pathway in regulating ZIKV infection. Using an in vivo Ifnar1−/− knockout mouse model, ZIKV infection was found to reduce YAP/TAZ protein levels while increasing phosphorylated YAP Ser127 in the retina and brain. Hippo pathway is activated in major cellular components of the blood-brain barrier, including endothelial cells and astrocytes. In addition, this result suggests AMP-activated protein kinase signaling pathway's role in regulating YAP/TAZ in ZIKV-infected cells. These data demonstrate that ZIKV infection might initiate a cross talk among AMP-activated protein kinase–Hippo–TBK1 pathways, which could regulate antiviral and energy stress responses during oculoneuronal inflammation. Zika virus (ZIKV) is a mosquito-borne positive-sense RNA virus that was first isolated in 1947 in Uganda, Africa. Since then, there have been numerous outbreaks throughout Africa and Asia, and, most recently, the pandemic in the Americas and Caribbean islands.1Duffy M.R. Chen T.-H. Hancock W.T. Powers A.M. Kool J.L. Lanciotti R.S. Pretrick M. Marfel M. Holzbauer S. Dubray C. Guillaumot L. Griggs A. Bel M. Lambert A.J. Laven J. Kosoy O. Panella A. Biggerstaff B.J. Fischer M. Hayes E.B. 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Hippo signalling governs cytosolic nucleic acid sensing through YAP/TAZ-mediated TBK1 blockade.Nat Cell Biol. 2017; 19: 362-374Crossref PubMed Scopus (123) Google Scholar resulting in antiviral response by stimulating the production of type I and III interferons. In this study, in vitro and in vivo models were used to characterize how ZIKV modulates the Hippo signaling pathway. We found that the level of YAP/TAZ changes during ZIKV infection because of Hippo pathway activation. shRNA knockdown showed that YAP/TAZ plays a direct role in ZIKV replication. Lastly, investigating the interplay between the host cell and ZIKV in mouse model revealed that the activation of Hippo pathway targets BBB and immune and inflammatory cells, contributing to energy stress, antiviral response, and neuroinflammation. The Institutional Animal Care Use Committee of the University of California, Los Angeles (UCLA) and Cedars-Sinai Medical Center have provided approval of the study. 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Integrated analysis of DNA methylation and RNA transcriptome during in vitro differentiation of human pluripotent stem cells into retinal pigment epithelial cells.PLoS One. 2014; 9: e91416Crossref PubMed Scopus (18) Google Scholar Fetal retinas (approximately 20 weeks old) were used for isolating RPEs at the laboratory of Dr. Fan using a previously reported protocol.44Liao J.L. Yu J. Huang K. Hu J. Diemer T. Ma Z. Dvash T. Yang X.J. Travis G.H. Williams D.S. Bok D. Fan G. Molecular signature of primary retinal pigment epithelium and stem-cell-derived RPE cells.Hum Mol Genet. 2010; 19: 4229-4238Crossref PubMed Scopus (151) Google Scholar,45Liu Z. Jiang R. Yuan S. Wang N. Feng Y. Hu G. Zhu X. Huang K. Ma J. Xu G. Liu Q. Xue Z. Fan G. Integrated analysis of DNA methylation and RNA transcriptome during in vitro differentiation of human pluripotent stem cells into retinal pigment epithelial cells.PLoS One. 2014; 9: e91416Crossref PubMed Scopus (18) Google Scholar The cells were cultured in minimal essential medium-α modification medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, 1% N2 supplement, 1% glutamine, 125 mg taurine, 1% nonessential amino acids, 10 μg hydrocortisone, and 6.5 ng triiodothyronine. Cells of early passage (4 to 8) were used. The human TIM-1 gene was cloned into the lentiviral vector plasmid, pLenti CMV Puro DEST (Addgene, Cambridge, MA).46Campeau E. Ruhl V.E. Rodier F. Smith C.L. Rahmberg B.L. Fuss J.O. Campisi J. Yaswen P. Cooper P.K. Kaufman P.D. A versatile viral system for expression and depletion of proteins in mammalian cells.PLoS One. 2009; 4: e6529Crossref PubMed Scopus (588) Google Scholar The TIM-1–expressing lentiviral vector was produced by calcium phosphate transfection method.47Morizono K. Chen I.S. Role of phosphatidylserine receptors in enveloped virus infection.J Virol. 2014; 88: 4275-4290Crossref PubMed Scopus (120) Google Scholar TIM–HEK-1 cells were generated by transducing HEK293 cells (ATCC, Manassas, VA) with lentiviral vectors expressing TIM-1, followed by culture in Iscove’s modified Dulbecco’s medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal calf serum (Sigma-Aldrich) and 1 μg/mL puromycin (Life Technologies, Camarillo, CA). Human glioblastoma cell line U-87 MG and Vero cell line were purchased from ATCC. The cells were incubated at 37°C, supplemented with 5% CO2, and subcultured when 90% confluence was reached at approximately every second to third day using 0.05% trypsin plus 0.53 mmol/L EDTA (Corning, Corning, NY). Use of these established cell lines for the study was approved by the Institutional Biosafety Committee of Cedars-Sinai Medical Center and UCLA. HEK–TIM-1 cells (2.5 × 104 cells/well) were added in a 48-well plate. After 24 hours, the pLKO.1-puro shRNA targeting YAP/TAZ (5′-CCGGCCCAGTTAAATGTTCACCAATCTCGAGATTGGTGAACATTTAACTGGGTTTTTG-3′) or pLKO.1-puro Non-Targeting shRNA Control plasmid (Sigma-Aldrich) was transfected using Lipofectamine 2000 (Invitrogen, Waltham, MA). At 48 hours after transfection, ZIKV with a multiplicity of infection of 1.0 was added. At 24 hours after infection, cell culture supernatant and cell protein lysates were collected for virus titer and YAP/TAZ protein knockdown efficiency assessments. Asian genotype Zika virus strain, PRVABC59 (https://www.ncbi.nlm.nih.gov; GenBank accession number KU501215), was used in this study. PRVABC59 was acquired from the CDC (Atlanta, GA). Viral stocks were generated in Vero cells. Viral titer was measured by plaque assay, as previously described.48Contreras D. Arumugaswami V. Zika virus infectious cell culture system and the in vitro prophylactic effect of interferons.J Vis Exp. 2016; 114: 54767Google Scholar For heat inactivation, ZIKV was subjected to heat treatment for 1 hour at 70°C. For infection studies, RPE, U-87 MG, and HEK–TIM-1 cells were plated in a 24-well plate (2.5 × 104 cells/well). After 24 hours, ZIKV inoculum (live or heat inactivated), with a multiplicity of infection of 1
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