Plasminogen activator inhibitor-1 regulates Zika virus infection

Plasminogen activator inhibitor-1 regulates Zika virus infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Plasminogen activator inhibitor-1 regulates Zika virus infection Milos Stojanov, Yen-Chi Chiu, Amal Fahmi, Isabel Schultz-Pernice, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5866223/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Zika virus (ZIKV) infection can lead to severe congenital outcomes, yet the mechanisms governing its entry into host cells remain understood. ZIKV is a flavivirus known to exploit multiple cellular receptors and cofactors, particularly in neural cells, where infection can result in congenital Zika syndrome (CZS). Here we show that plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor involved in hemostasis, directly interacts with ZIKV particles and critically enhances viral replication in diverse cell types, including human neural progenitor cells and three-dimensional neural organoids. Our findings reveal that PAI-1 may contribute to ZIKV infection through distinct or complementary pathways, underscoring the virus’s versatile entry mechanisms. Inhibition of PAI-1 via tiplaxtinin (TPX) dramatically reduces viral load and impedes infectious particle release, demonstrating a dose-dependent effect that is especially potent in neural models relevant to CZS. These results highlight PAI-1 as an essential mediator of ZIKV pathogenesis and suggest that targeting PAI-1 function could represent a novel therapeutic avenue. Given the risk of future ZIKV outbreaks and the devastating impact of CZS, interventions aimed at PAI-1 may hold promise for reducing the global burden of ZIKV infection. Health sciences/Diseases/Infectious diseases/Viral infection Health sciences/Pathogenesis/Infection Zika virus orthoflavivirus virus entry pregnancy microcephaly congenital Zika syndrome ligand-based receptor capture LRC-TriCEPS neural organoids cerebral organoids plasminogen activator inhibitor-1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The Zika virus (ZIKV) outbreak of 2015-16 in the Americas and associated territories has been defined as a “perfect storm”, in which a neglected virus was introduced in a fully naïve population 1,2 . Recent estimates indicate that 132 million people were infected by the virus by 2018 3 . A member of the Orthoflavivirus genus, ZIKV was first described in 1947 in Uganda, Africa 4 , and is classified into African and Asian lineages 5 . In the last two decades, the Asian clade of ZIKV has spread through the Asian continent to the Pacific 6 , reaching South America, where it was identified in Bahia, Brazil, in March 2015 7 . The Brazilian outbreak extended throughout most of the country and led to more than one million confirmed cases by the end of the year. Surprisingly, an unexpected increase in cases of microcephaly was observed in the regions affected by the outbreak approximately six months later, leading to a confirmation of ZIKV as a causative agent of birth defects 8 . While the epidemic in the Americas was waning, it became clear that microcephaly cases were just the tip of the iceberg for in utero ZIKV infection sequelae. From a clinical perspective, resulting brain pathologies include microcephaly, calcifications, underdevelopment of the corpus callosum or cerebellum, ocular anomalies and reduced myelination 9 , leading to congenital ZIKV syndrome (CZS). As a result, infants from cohorts established during the 2015-16 outbreak have been observed to experience long-term disabilities, even in the absence of abnormal brain structures 10 . CZS was also observed in animal models, where even asymptomatic in utero infections have been shown to cause inflammation of fetal organs and long-term consequences 11,12 . The initial efforts after the 2015-16 outbreak, resulting in an unprecedented focus and funding support in the scientific community, did not lead to concrete development of prophylactic and therapeutic measures against ZIKV infections 13 . The main vaccine candidate developed to date, Zika virus purified inactivated vaccine (ZPIV), while showing good safety features, does not have an optimal immunogenicity profile 14,15 . Application of ZPIV where yellow fever and Japanese encephalitis virus vaccines are commonly administered, mainly South-East Asia, could therefore pose a challenge 14 . Thus, other preventive and/or therapeutic strategies are needed. Unique characteristics of ZIKV pathogenesis, such as vertical and sexual transmission, distinguish it from other orthoflaviviruses and underscore specific features in the virus’s biology, particularly its capacity to infect a broad range of cell types. A key step in the viral replication cycle is the attachment to the host cell, which involves the envelop (E) protein, whose structure is well conserved among orthoflaviviruses 16 . It binds cellular receptors and host factors and, upon attachment, drives the membrane fusion and the consequent entry of viral genetic material into the cell, where replication can take place. In addition to E protein, negatively charged lipids present in the viral membrane, such as phosphatidylserine (PS), were shown to participate in the entry process of orthoflaviviruses 17 . Despite considerable efforts, the precise mechanisms by which ZIKV enters host cells remain unclear. Similar to other orthoflaviviruses, ZIKV may interact with multiple factors during the entry process, as the interplay of multiple receptors may contribute to stronger binding, compared to an individual receptor 18 . Initially, the C-type lectin dendritic cell-specific intracellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) was reported to promote ZIKV entry in dendritic cells and immature macrophages in vitro 19 . Transmembrane PS receptors, including TIM (TIM1, TIM3, and TIM4) and TAM (TYRO3, AXL, and MER) family members, have been shown to contribute to the internalization of orthoflaviviruses 19,20 . In the case of ZIKV, several studies have specifically suggested that AXL, a transmembrane receptor tyrosine kinase, acts as an entry receptor and/or immune modulator for the virus in a cell type-specific manner 21 . However, other findings have suggested that TAM proteins are not the critical receptors for ZIKV infectivity 22 . Indeed, ZIKV challenge of TAM-knockout mice showed that viral replication was not affected in the spleen, placenta, vagina, and brain 22 . Additional studies carried out with ALX-knockout mice have also demonstrated that these proteins are not essential for ZIKV infectivity 23 . Another study reported that genetic ablation of AXL did not affect virus infectivity or ZIKV-induced cell death in neural progenitor cells (NPCs) and neural organoids 24 . While ZIKV appears to utilize well-established orthoflavivirus receptors for internalization across various infection models, its specific tropism and routes of transmission suggest the involvement of additional entry mechanisms. Using a ligand-based receptor capture method, we report here the identification of plasminogen activator inhibitor-1 (PAI-1), encoded by the SERPINE1 gene , as a novel cellular factor involved in ZIKV infection. PAI-1 belongs to the serine protease inhibitor family and plays a major role in the regulation of tissue hemostasis 25 . We utilized several cell lines to confirm the role of PAI-1 in ZIKV entry, including human brain glioblastoma cells (U-87 MG), and Vero cells, which are commonly used for viral propagation under laboratory conditions. Given the physiological relevance of the viral entry process, we focused our analyses on human neural progenitor cells (NPCs), which exhibit characteristics of neural cells enriched in the human fetal brain. To further validate our findings, we employed human neural organoids (hNOs), demonstrating the potential critical role of PAI-1 in ZIKV infection. These three-dimensional structures closely replicate the architecture and cellular diversity of the developing human brain, allowing us to investigate the potential critical role of PAI-1 in a more complex environment. Several factors suggest a potential rise in future orthoflavivirus outbreaks, including those caused by ZIKV 26 . Statistical models indicate that vector populations, comprising of A. aegypti , will increase their ecological niches due to globalization, urbanization, and climate change 27 . The devastating outcomes of CZS demand great concern, mainly in non-immune populations of the Northern hemisphere, and warrant further effort by the scientific community to find concrete solutions. While ZIKV prevalence has decreased since the 2015–2016 outbreak, the virus persists, with over 40,000 cases in the Americas in 2022, Brazil reporting the majority 28 . Therefore, the threat remains, alongside emerging evidence of ZIKV causing additional brain damage. The situation underscores the ongoing risk and the need for valid therapeutic alternatives. Results Identification of cell surface factors interacting with ZIKV Ligand-receptor capture technology LRC-TriCEPS was used to identify putative cellular factors involved in the internalization of ZIKV. The analysis was initially performed in U-87 MG and Vero cell lines with transferrin used as a control to validate the assay and remove background signal. A total of 374 membrane associated proteins were identified in the U-87 MG cell line, with 189 hits identified by more than two peptides (Supplementary table 1). On the other hand, 62 proteins were identified for the Vero cell line, of which 35 were recognized by more than two peptides (Supplementary table 2). Comparison with the transferrin control narrowed down the number of putative candidates to 11 for U-87 MG and 4 for Vero cells (Fig. 1 and Table 1 ). In both analyses, viral E protein (ZIKV_E) was successfully identified in infected cells (Fig. 1 , Supplementary tables 1 and 2). Table 1. Potential cellular factors identified in U-87 MG and Vero cell lines. Candidates common to the two cell lines are highlighted in grey. * Log 2 fold change relatively to the control assay. ** -Log 10 adjusted p-value. Three candidate proteins, PAI-1, fetuin A (AHSG), and haemoglobin subunit beta (HBB), were common to both cell lines (Table 1 ). Given PAI-1’s broad involvement in a range of diseases, its significance across multiple organ systems, and its known role in the pathogenesis of other viruses, we prioritized PAI-1 for further experimental investigation. PAI-1 binds to ZIKV and influences viral replication We sought to verify the direct interaction of PAI-1 with ZIKV using transmission electron microscopy (TEM). To test this, we incubated purified recombinant human PAI-1 protein with viral particles. The presence of PAI-1 on the surface of the virions was revealed by a PAI-1-specific antibody, coupled to protein A conjugated with nanogold particles (Fig. 2 ). Our results showed that the surface of virions was decorated with regularly interspaced pattern of gold particles, suggesting an interaction with a motif on the envelope protein (Fig. 2 B, left panel). In the untreated control, where PAI-1 was not added, no interaction of the virions with nanogold particles was observed (Fig. 2 B, right panel). We next sought to determine whether the addition of purified PAI-1 could enhance ZIKV infection. An inoculum consisting of ZIKV preincubated with PAI-1 was used to infect U-87 MG and Vero cell lines. In both cases, ZIKV RNA levels increased significantly compared to the negative control (no preincubation with PAI-1) (Supplementary Fig. 1). Specifically, we observed a two-fold increase of viral RNA in the U-87 MG cell line and a four-fold increase in the Vero cell line. We also observed a general trend of increased release of infectious viral titers in the supernatant, which, however, remained however below a one-log difference (Supplementary Fig. 1). Next, we overexpressed PAI-1 in the two cell lines using an expression plasmid encoding human SERPINE1 cDNA. Control experiments indicated that PAI-1 RNA levels, as measured by RT-qPCR, increased by 8-fold in U-87 MG cells and 4-fold in Vero cells (Supplementary Fig. 2A). The efficiency of ZIKV infection was compared in cells overexpressing PAI-1 and wild type cell lines, showing significantly increased viral RNA levels with higher PAI-1 expression (Fig. 3 A, top). In U-87 MG cells, we observed a 4-fold increase in viral RNA at 24 hours post infection (HPI) and a 7-fold increase at 48 HPI. In Vero cells, while a 3-fold increase in viral RNA was observed at 24 HPI, the difference was not significant at 48 HPI. As observed with the preincubation experiments, increased PAI-1 levels were accompanied by an increase in infectious viral titers in the supernatant compared to negative controls, remaining under a one-log difference, except for Vero cells at 48 HPI, similarly to viral RNA quantification (Fig. 3 A, bottom). To analyse the effects of PAI-1 depletion, we tested the ability of ZIKV to infect cell lines in which the SERPINE1 gene was knocked out (Fig. 3 B, Supplementary Fig. 2B). The absence of PAI-1 generally resulted in lower viral RNA loads and decreased release of viral particles into the supernatant. RNA synthesis was reduced by approximately half, except for U-87 MG cells at 24 HPI, where no significant difference was observed between wild type and PAI-1-depleted cells (Fig. 3 B, top). This difference was more prominent at 48 HPI, compared to 24 HPI. Infectious viral titers in the supernatant were approximately reduced by approximately one log at all timepoints tested compared to wild type background, except for U-87 MG cells at 24 HPI (Fig. 3 B, bottom). Several molecules inhibiting PAI-1 activity have been developed and, among them, tiplaxtinin (TPX) is the most studied inhibitor, whose efficacy has been showed in multiple models 29 . TPX is a small molecule that works by binding to the active site of PAI-1, stabilizing it in an inactive conformation. We tested the effect of TPX to potentially interfere with ZIKV replication, by treating cells with the drug for 24 hours before the infection and subsequently supplementing TPX in the cell culture medium. While in U-87 MG at 24 HPI a clear though non-significant effect was documented, TPX significantly reduced the viral RNA load at all other time points, in both cell lines, reaching significance except for in U-87 MG cells at 24 HPI (Fig. 3 C, top). The effect on viral replication was even more marked when supernatants were analysed, as we observed a reduction of infectious viral titers of at least 2 orders of magnitude at all time points tested (Fig. 3 C, bottom). Of note, infectious titers in the supernatant of U-87 MG cells treated with TPX at 48 HPI were reduced by five orders of magnitude. The effect of TPX on viral replication was dose-dependent, with increasing concentrations (5, 25, and 50 µM) resulting in a stronger inhibition of viral replication (Supplementary Fig. 3). Inhibition of PAI-1 blocks ZIKV infection in neural progenitor cells Infection of NPCs in the fetal brain represents a central component in ZIKV teratogenesis leading to CZS. To further evaluate the impact of PAI-1 on ZIKV replication, we used human iPSC-derived NPCs (Fig. 4 A). Successful iPSC to NPC transition was confirmed by the robust expression of Nestin, a marker for neural stem cells and the expected absence of NANOG, indicating the loss of pluripotency and commitment to a neuroectodermal lineage (Fig. 4 B). As expected, these cells were readily infected by ZIKV under our experimental conditions (Fig. 4 C). We therefore applied the LRC-TriCEPS method to the iPSC-derived NPCs (Fig. 4 D). Interestingly, a higher number of proteins potentially interacting with ZIKV was identified by using NPCs compared to U-87 MG and Vero cell lines (Table 1 ). A total of 438 membrane-associated or extracellular proteins were detected, of which 253 were identified by more than two peptides and were therefore used for statistical analysis (Supplementary table 3). After applying a two-fold enrichment cutoff and ensuring statistical significance, 19 candidates were selected (Table 2 ). We therefore confirmed that PAI-1 was also identified as a protein interacting with ZIKV (Fig. 4 D), confirming previous LRC-TriCEPS experiments with U-87 MG and Vero cell lines. Of note, AHSG (Fetuin A), previously identified in U-87MG and Vero cell lines, was also found as a molecule potentially interacting with ZIKV. This was not the case for HBB. Given the strong effect of TPX on ZIKV replication in U-87 MG and Vero cell lines, we repeated this analysis using NPCs. Treatment with 50 µM TPX completely abolished viral replication compared to positive controls (control and DMSO-treated cells) (Fig. 4 E). Table 2. Potential cellular factors identified in NPCs. Candidates common to the previous analysis (Table 1 ) are highlighted in grey. *Log 2 fold change relatively to the control assay. **-Log 10 adjusted p-value. PAI-1 inhibition blocks ZIKV infection in human neural organoids To investigate the impact of PAI-1 on ZIKV infection in a model that mimics the developing human brain, we utilized ESC-derived hNOs. This system closely replicates key aspects of human brain formation and has been previously used to model microcephaly in vitro 30 . To highlight the contribution of PAI-1 to ZIKV pathogenesis, we investigated the impact of TPX treatment on ZIKV-challenged hNOs. Macroscopically, infection by ZIKV and different treatment conditions, including presence or absence of DMSO, TPX and PAI-1, did not have any apparent effect on the organoids, as compared to the controls, after 4 days of incubation (Fig. 5 A). In agreement, no significant difference was detected in organoid surface area measurements, despite ZIKV-infected, untreated organoids showing the smallest size increase (0.5 mm 2 ) between day 0 post-infection and day 4 post-infection, compared to all other conditions (0.6 mm 2 – 1.1 mm 2 ) (Fig. 5 A, Supplementary Fig. 4). As observed in cell lines and NPCs, increasing concentrations of TPX had a profound effect on viral replication in human brain organoids (Fig. 5 B and C). Flow cytometry analysis of organoids dissociated into single cells confirmed the critical impact of TPX treatment on viral infection. At 4 days post-infection the percentage of ZIKV E-protein harbouring cells was documented to be significantly reduced compared to untreated, infected organoids (Fig. 5 B). While drug concentrations of 25 µM reduced viral titers in the supernatant by 2 logs, 50 µM completely abolished the release of viral particles from the organoids. These observations were additionally confirmed by confocal microscopy analysis of stained, sectioned organoids (Fig. 5 D). As previously reported 31,32 , a ventricle-dominated hNO tissue structure, composed of densely packed, sex determining region Y-box 2 (SOX2) positive NPCs, was observed in mock-treated organoids after 15 days of culture (Fig. 5 D). No morphological abnormalities and no viral particles could be identified through immunofluorescence staining in organoids treated with 25 µM of TPX compared to mock-treated cultures, indicating the important reduction in viral loads upon TPX treatment. However, while no viral particles could be detected in organoids cultured in medium supplemented with 50 µM of TPX, a reduction in ventricle size and alterations in tissue and outer border morphology were observed, indicating possible toxic effects induced by high TPX concentrations on exposed cultures. Interestingly, while no significant impact of PAI-1 treatment on viral loads has been observed (Fig. 5 C), incubation with PAI-1 induced a massive but localized invasion of ZIKV in a small portion of the organoids (Fig. 5 D). Discussion We identified PAI-1 as a novel host factor that significantly influences ZIKV infection. PAI-1 belongs to the serine protease inhibitor family and plays a major role in the regulation of hemostasis 33 . It is a plasma-circulating protein with a short half-life whose primary function is to specifically inhibit plasminogen activators. These activators normally convert plasminogen into its active form, plasmin, through proteolytic processes, thereby increasing fibrinolytic activity 25 . Beyond its primary function in the coagulation system, PAI-1 exhibits pleiotropic effects, impacting various physiological and pathological processes across different systems in the body including cell migration 34,35 , vascularization 36 , metabolic processes 37,38 , inflammation 39 , cancer progression 40 .and viral infection 41 . We demonstrated that PAI-1 physically interacts with ZIKV in multiple cell types, including U-87 MG glioblastoma cells, Vero cells and NPCs. Direct interaction was further confirmed by TEM, where PAI-1 was shown to bind to the surface of ZIKV particles. Our functional assays revealed that increasing PAI-1 levels, either through overexpression or by addition of recombinant human PAI-1, enhanced ZIKV replication and viral particle production in U-87 MG and Vero cell lines. Conversely, deletion of the SERPINE1 gene, which encodes PAI-1, led to a reduction in viral RNA levels and decreased infectious viral titers in the supernatant of U-87 MG and Vero cell lines. Most notably, inhibition of PAI-1’s enzymatic activity using TPX resulted in a significant, dose-dependent decrease of ZIKV replication across all models tested. In NPCs and hNOs, TPX treatment completely abolished viral replication at higher concentrations, underscoring the potentially crucial role of PAI-1 in the ZIKV life cycle. The enhancement of ZIKV infection by PAI-1 in U-87 MG and Vero cells could be attributed to several potential mechanisms. PAI-1 might act as an attachment factor, increasing viral binding affinity to host cells. Alternatively, it could modulate cellular pathways that favour viral entry or replication. Given that PAI-1 is known to influence cell migration, adhesion, and extracellular matrix remodeling 42 , its interaction with ZIKV might alter the cellular environment to be more permissive to infection. These mechanisms may however be cell type specific since incubation with recombinant PAI-1 did not influence viral infection in NPCs and hNOs. Previous research on ZIKV entry mechanisms has focused on AXL as the main receptor in multiple cell types 43,44 . However, the exact pathways facilitating ZIKV entry into neural cells remain incompletely understood. Our study introduces PAI-1 as a novel player in this process, offering new insights into ZIKV-host interactions. Furthermore, our experiments primarily focused on the early stages of infection. The observation that TPX treatment reduced viral titers even when administered post-infection suggests that PAI-1 may also influence later stages of the viral life cycle, such as assembly or release. Interestingly, PAI-1 has been linked with the infectious process of other viruses. It has been proposed that domain III of the Dengue virus’ (DENV) E protein is able to activate the expression of PAI-1 during the early phases of infection and is linked with hemorrhage 41 . Furthermore, the maturation of influenza A virus (IAV) was shown to be inhibited by PAI-1 in the extracellular environment due to the blockage of IAV glycoprotein cleavage 45 . As a result, the infectivity of progeny IAV was observed to be attenuated 45 . PAI-1 mRNA levels and protein expression were documented to be downregulated in Hepatitis C virus-infected cells and patients 46 . Interestingly, PAI-1 levels were observed to increase in pregnant women after the 20th week of pregnancy 47 . Its production was localized within the placenta 48 and may be a hypothetic link with the materno-fetal transmission of ZIKV. Despite these advancements, our study has limitations. The precise molecular mechanisms by which PAI-1 enhances ZIKV infection are yet to be fully elucidated. Additionally, while TPX effectively inhibited ZIKV replication, it has only been used in preclinical models and it is not an FDA approved drug 49,50 . In conclusion, our study identifies PAI-1 as a critical host factor in ZIKV infection, particularly in neural cells relevant to CZS. Targeting PAI-1 or its interaction with ZIKV presents a promising therapeutic avenue. Given the ongoing risk of ZIKV outbreaks and the severe consequences of congenital infections, the challenge to develop an effective vaccine due to cross-antibody effects with other vaccines/viruses, developing strategies to inhibit PAI-1 function could contribute to preventing or mitigating the impact of ZIKV. Future studies should aim to uncover the detailed mechanisms of PAI-1-mediated enhancement of ZIKV infection and evaluate the potential of PAI-1 inhibitors in animal models and clinical settings. Materials and methods Ethics statement In accordance with Articles 13 and 14 of the Federal Act on Research on Embryonic Stem Cells, and Article 20 of the Ordinance on Research on Embryonic Stem Cells (ESCs), the use of the human H1 ESC line was approved by the Cantonal Ethics Committee of Bern, Switzerland, under authorization number R-FP-S-2-0023-0000. Cell lines and viruses Cell lines and viral strains used in this study are depicted in Table 3 . Human brain glioblastoma (U-87 MG) cells and Cercopithecus aethiops kidney epithelial (Vero) cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, 61965026, Gibco, Basel, Switzerland) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS, F7524, Sigma-Aldrich, Buchs, Switzerland). Deletion of SERPINE1 gene in U-87 MG and Vero cell lines was performed by Ubigene Biosciences (Guangzhou, China). Table 3 Cell lines and virus strain used in this study Origin Details Cell lines U-87 MG Human brain glioblastoma Growth medium: DMEM + 10% FBS Vero Cercopithecus aethiops kidney epithelial cells Growth medium: DMEM + 10% FBS NPC iPS-derived neural progenitor cells Obtained by differentiating PB12 human induced pluripotent stem cells Viral strains PRVABC-59 ZIKV, Asian-lineage strain Pandemic strain isolated from a viremic patient in Puerto Rico in 2015 NPCs differentiation followed a previously described protocol 51 . Briefly, human iPSC colonies (PB12 line 52 ) were initially cultured in N2B27 medium consisting of 50% DMEM/F-12 GlutaMAX (Thermo Fisher Scientific), 50% Neurobasal medium (Thermo Fisher Scientific), 2% B27 supplement (without vitamin A, Thermo Fisher Scientific), 1% N2 supplement (Thermo Fisher Scientific), and 50 µM β-mercaptoethanol (Thermo Fisher Scientific). Human iPSCs were first seeded and cultured for 6 hours in a low-adhesion culture plate in N2-B27 medium supplemented with SB431542 (20 µM, Tocris), LDN-193189 (0.1 µM, Sigma-Aldrich), and Y-27632 (10 µM, 72304, STEMCELL Technologies). The resulting aggregates were transferred to poly-L-ornithine and laminin-coated dishes. Media were changed daily from division 0 (DIV0) to DIV20. Y-27632 was removed at DIV1, XAV-939 (1 µM, 3748, Tocris) was added until DIV9. Between DIV5 and DIV9, SB431542 was removed and FGF2 (10 ng/mL, Peprotech) and cyclopamine (1 µM, Sigma-Aldrich) were added. From DIV10 to DIV17, LDN-193189 and XAV-939 were removed and CHIR99021 (0.4 µM, STE04-0004-02, Ozyme) was added. At DIV17, NPCs were enzymatically dissociated using accutase (11599686, Thermo Fisher Scientific) and cryopreserved in Cryostor cell cryopreservation medium (100–1061, STEMCELL Technologies) for storage in liquid nitrogen vapor at -150°C. ZIKV infection in cell lines and NPCs U-87 MG cells and Vero cells were seeded into a 24-well plate (approximately 6x10 4 cells per well) one day before infection with ZIKV. NPCs were prepared by coating 24-well plates with poly-L-ornithine (17 µg/ml, Sigma-Aldrich) and laminin (2.4 µg/ml, Gibco). Following coating, NPCs were seeded at approximately 2x10⁵ cells per well in N2B27 medium supplemented with 10 µM Y-27632 (ROCK inhibitor, STEMCELL Technologies) and 20 ng/ml FGF (PreproTech). ZIKV infection was carried out with a multiplicity of infection (MOI) of 1. After an incubation period of 30 minutes, ZIKV inoculum was removed and replaced with fresh medium. Tiplaxtinin (PZ0295, Sigma-Aldrich), a well-known PAI-1 small-molecule inhibitor 53 , was used after dissolution in dimethyl sulfoxide (DMSO, A3672, PanReac AppliChem, Darmstadt, Germany). Recombinant human PAI-1 (PAI-1, 528205, Sigma-Aldrich), was dissolved in a solution comprising of 150 mM NaCl, 50 mM sodium phosphate buffer, and 1 mM EDTA at pH 6.6. One hour prior to infection, ZIKV and 1 µg/ml PAI-1 were mixed in an appropriate amount of medium and incubated at 37°C, shaking at 200 rounds per minute (rpm). Following incubation, the mixture was added to the cells and incubated for 30 minutes. Subsequently, cells were washed once with phosphate-buffered saline (PBS, Gibco), before adding fresh cell growth medium to the cultures. Neural stem cells were fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences) and 4% sucrose (Sigma-Aldrich) in phosphate-buffered saline (PBS) for 10 minutes, followed by three washes with PBS at room temperature. Subsequently, the samples were subjected to an overnight incubation at 4°C with primary antibodies diluted in a PBS blocking solution containing 2% BSA (Sigma-Aldrich) and 0.2% Triton X-100 (Sigma-Aldrich) to facilitate membrane permeabilization. The primary antibodies used were: NESTIN (MAB5326, Millipore), NANOG (4903, Cell Signaling), Concanavalin A (Vector Laboratories Ltd), and ZIKV (HB-112, ATCC). Following three washes with PBS, cells were incubated for one hour at room temperature with species-specific secondary antibodies conjugated to Alexa Fluor 488 and 555 (Invitrogen) and DAPI (Calbiochem) in a PBS solution containing 2% BSA and 0.1% Triton X-100. Quantification of viral RNA Extraction of RNA from lysed cells was performed with the NucleoSpin RNA II kit (740955.50, Macherey-Nagel AG, Düren, Germany). Reverse transcription was carried out with random hexamers (100026484, Invitrogen, Carlsbad, CA, US) and SuperScript II Reverse Transcriptase (18064014, Invitrogen). The quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed using the iTaq Universal Probes Supermix (1725131, Bio-Rad, Basel, Switzerland). Relative quantification was performed with the Delta-Delta Ct method 54 using GAPDH as the housekeeping gene. The primers and TaqMan probes used for qRT-PCR were purchased from Microsynth AG (Baglach, Switzerland) and are listed in Table 4 . Table 4 Primers and probes for used for qRT-PCR analyses. Target Primer/probe Sequence ZIKV 55 ZIKV_fw 5’-CGYTGCCCAACACAAGG-3’ ZIKV_rev 5’-CCACYAAYGTTCTTTTGCABACAT-3’ ZIKV_probe 5’-6FAM-AGCCTACCTTGAYAAGCARTCAGACACYCAA-BHQ1-3’ GAPDH 56 GAPDH_fw 5'-GAAGGTGAAGGTCGGAGTCAAC-3' GAPDH_rev 5'-CAGAGTTAAAAGCAGCCCTGGT-3' GAPDH_probe 5'-6JOE-TTTGGTCGTATTGGGCGCCT-BHQ1-3' PAI-1 57 PAI-1_fw PAI-1_rev PAI-1_probe 5’-GGCTGACTTCACGAGTCTTTCA-3’ 5’-TTCACTTTCTGCAGCGCCT-3’ 5’-6FAM-ACCAAGAGCCTCTCCACGTCGCG-BHQ1-3’ 6-FAM: 6-carboxyfluorescein; 6-JOE: 6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein; BHQ-1: black hole quencher-1. Ligand-receptor capture assay Identification of proteins potentially interacting with ZIKV was performed using the ligand-based receptor capture (LRC) technology (Dualsystems Biotech AG, Schlieren, Switzerland). For each cell line, 2x10 7 cells were seed into T175 cell culture flask (355000, BD Switzerland, Basel, Switzerland) and used for the assay. For each replicate, 0.5 µl of the TriCEPS molecule were incubated with ZIKV strain PRVABC-59 for 90 minutes at 37°C. In parallel, cells were mildly oxidized on ice using 1.5 mM NaIO 4 in PBS (pH 6.5) for 15 minutes. Oxidized cells were incubated with ZIKV-TriCEPS mixture (MOI of 4) for 30 minutes at 37°C. Cells were collected by scraping and lysed immediately. Following cell lysis, target proteins were purified using solid phase chromatography and processed to remove unspecific interactions as previously described 58 . Proteins were then reduced, alkylated and digested with trypsin. The tryptic peptides were collected for liquid chromatography-mass spectrometry/mass spectrometry. To allow for statistical analysis, the experiment was performed in biochemical triplicates. Samples were analysed on a Thermo Orbitrap Elite spectrometer fitted with an electrospray ion source. Tryptic peptides were measured in data dependent acquisition mode (TOP20) in a 120-minute gradient using a 15 cm C18 packed column. The Progenesis software was used for raw file alignment and feature detection, Comet search engine was used for spectra identification and the Trans proteomic pipeline was used for statistical validation of putative identifications and protein inference. Upon protein inference, relative quantification of control and ligand samples was performed based on ion extracted intensity and differential protein abundance was tested using a statistical ANOVA model followed by multiple testing correction. This model assumes that the measurement error follows Gaussian distribution and views individual features as replicates of a protein's abundance and explicitly accounts for this redundancy. It tests each protein for differential abundance in all pairwise comparisons of ligand and control samples and reports the p-values. Next, p-values are adjusted for multiple comparisons to control the experiment-wide false discovery rate (FDR). The adjusted p-value (q-value) obtained for every protein is plotted against the magnitude of the fold enrichment between the two experimental conditions. In the present study, the annotated human proteome database from Uniprot was used for the analysis of the human cell lines. The proteome database of Cercopithecus aethiops from Uniprot was used for the analysis of the samples from the Vero cell line. The sequence of the genome polyprotein (POLG, Q32ZE1) of ZIKV was included in the analysis. The results for the human cell lines were filtered for membrane associated proteins. Quantification of infectious virus Cell supernatants were collected at 24 and 48 HPI and stored at − 80°C until analysis. The day before titration, Vero cells were seeded into 96-well plates (CLS351172, Corning, Root, Switzerland) at a density of 2x10 4 /well. Serial 10-fold dilutions of cell supernatants were prepared in Vero cell growth medium, inoculated on Vero cells and incubated at 37°C with 5% CO 2 for 72 hours. Subsequently, cells were fixed by 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, US) for 20 minutes. Following fixation, cells were washed three times with 0.1% saponin in PBS solution, and incubated for 40 minutes with anti-orthoflavivirus group antigen antibody 4G2 (clone D1-4G2-4-15, ATCC, HB-112) at 37°C. Cells were subsequently washed twice with 0.1% saponin after which samples were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (G-21040, Thermo Fischer Scientific) for 40 minutes at 37°C. Following three more washes with 0.1% saponin solution, staining was performed with the 3-amino-9-ethylcarbazole substrate (Sigma-Aldrich). Infectious virus titers were calculated and expressed as 50% tissue culture infective dose per ml (TCID 50 /ml) using the Reed and Muench method 59 . Overexpression of PAI-1 protein in cell lines A commercially available plasmid (HG10296-NF, Sino Biological Inc., Beijing, China) was used for the overexpression of PAI-1. U-87 MG cells and Vero cells were cultured in 6-well plates (140675, Thermo Fisher Scientific) to form a monolayer by the day of transfection. Transfection was performed using Lipofectamine 3000 transfection reagent (L3000008, Invitrogen) following the manufacturer’s instructions. The cells were briefly washed with PBS before the transfection mixture was added. After incubation at 37°C in a 5% CO₂ atmosphere for 1 hour in U-87 MG cells and 6 hours in Vero cells, the transfection mixture was removed, and fresh growth medium was added. Beginning 48 hours after transfection, U-87 MG cells were treated with 100 µg/ml and Vero cells with 400 µg/ml of hygromycin B (30-240-CR, Corning). Antibiotic selection was continued for two weeks to ensure the propagation of successfully transfected cells. Immunogold labelling PAI-1 and ZIKV PRVABC-59 were mixed at a 1:1 ratio and incubated at 37°C for 1 hour. Then, 4 µl of the mixture were loaded onto a carbon film Cu grid (CF200-Cu, Electron Microscopy Sciences), which had been previously glow-discharged for 30 seconds. After 1 minute, the samples were transferred to Whatman filter paper (WHA1001325, Whatman, Massachusetts, US). The blocking buffer containing 5% bovine serum albumin (Gibco) in PBS was applied to the grid for 10 minutes. Subsequently, PAI-1 was detected using a primary antibody (G-21040, Thermo Fisher Scientific) at a 10-fold dilution in 1% blocking buffer for 1 hour. The grid was then washed three times with PBS and incubated with 10-nm immunogold-conjugated protein A, diluted 25x in HB buffer, to detect the primary antibody. After a 1-hour incubation, the grid was washed three times with PBS, then fixed with 2% PFA for 10 minutes and washed once with ddH 2 O. Finally, the sample was negatively stained with 2% uranyl acetate for 1 minute. Images were captured using a Philips CM100 at 80 kV with a TVIPS F416 camera (4kx4k, Thermo Fisher Scientific). Generation of human neural organoids hNOs were generated starting from H1 embryonic stem cells (ESCs) as previously described 32 . Briefly, on day 0, H1 ESCs were dissociated through a 4-minute incubation with accutase (Sigma-Aldrich), followed by repeated pipetting to obtain a single-cell suspension. Cells were subsequently counted and pelleted through room-temperature (RT) centrifugation at 200 g for 5 minutes. Cells were then resuspended in an appropriate volume of RT Formation Medium (STEMCELL Technologies, Zürich, Switzerland) previously supplemented with 50 µM Rho-associated protein kinase (ROCK) inhibitor Y-27632 (STEMCELL Technologies) and the obtained single cell suspension distributed in ultra-low attachment 96-well plates (Corning) at a density of 10,000 cells and 100 µl per well. Cells were incubated at 37°C, 5% CO 2 and 100 µl of medium without Y-27632 were added to each well in two-day intervals. On the same day (day 0), remaining cells were used for stemness quality control by flow cytometry as described below (Flow cytometry analysis of human neural organoids). After 5 days, cultures confirmed to have passed the stemness check were inspected for embryoid body formation and further subjected to visual quality control before being transferred into 24-well ultra-low attachment plates (Corning) prepared with 500 µl of pre-warmed Induction Medium (STEMCELL Technologies) using 200 µl ART wide bore filtered pipette tips (Thermo Fisher Scientific). The cultures were maintained at 37°C, 5% CO 2 and neuroepithelium induction was observed in the following two days. On day 7 of culture, embryoid bodies were singularly embedded into 30 µl Matrigel (Corning) droplets and transferred into 6-well ultra-low attachment plates (Corning) containing 3 ml of pre-warmed home-made Expansion Medium, prepared according to the previously published protocol by Lancaster and colleagues 31 . The expanding hNOs were incubated at 37°C with 5% CO 2 for 3 more days. On day 10 of culture, the Expansion Medium was removed completely and replaced with 3.5 ml of RT home-made Maturation Medium, prepared using the previously cited protocol 31 . During the whole maturation period and until the end of each experiment, organoids were cultured on an orbital shaker, gently moving at 65 rpm, at 37°C with 5% CO 2 . Maturation Medium was replaced at regular intervals of 2 to 3 days. Fifteen-days old organoids were subjected once more to visual quality control before being used for infection experiments. Human neural organoid infection with ZIKV hNOs were challenged with ZIKV after a total culture time of 15 days. One day before infection, organoid cell numbers were quantified through flow cytometry as described below (Flow cytometry analysis of human neural organoids). Obtained mean cell counts were used for MOI calculation. Infections were performed based on the previously described procedure 32 . The required volume ZIKV stock was added to RT Maturation Medium or Maturation Medium complemented with PAI-1 as previously described (ZIKV infection in cell lines), vortexed to obtain a homogeneous suspension and distributed on hNOs to obtain a final MOI of 0.1 TCID 50 /cell and 2 ml per well. Mock-spiked Maturation Medium was prepared and distributed analogously. Organoids were incubated at 37°C with 5% CO 2 in the virus-spiked Maturation Medium for 2 hours, constantly shaking at 65 rpm. The medium was subsequently completely removed, the cultures washed three times with 3 ml of RT sterile Dulbecco's Phosphate Buffered Saline (DPBS, Gibco) and 3.5 ml of virus-free RT Maturation Medium were added to each well. Organoids were placed back on the orbital shaker, shaking at 65 rpm at 37°C with 5% CO 2 . Supernatant and tissue samples were taken at regular intervals, processed directly or stored at -80°C, and used for subsequent analysis as described below. Biosafety level 2 procedures were strictly adhered to during each protocol step. Flow cytometry analysis of human neural organoids For quality control of H1-cells on day 0 of organoid generation, stemness analysis through double staining with markers SSEA-5 (STEMCELL Technologies, #60063AZ, 1:50) and TRA-1-60 (STEMCELL Technologies, #60064PE, 1:50) was performed, as previously described 32 . For this, antibodies were diluted in BD CellWASH (BD Switzerland). A sample of H1-cells were pelleted by centrifugation at 280 g for 8 minutes at 4°C. The supernatant was aspirated, the cells resuspended by vortexing in the previously prepared antibody solution and incubated for 15 minutes on ice, protected from light. Following incubation, cells were washed by addition of 1 ml of BD CellWASH and spun down once more at 280 g, 8 minutes at 4°C. The obtained supernatant was aspirated, and the cells resuspended in 100 µl of BD CellWASH by vortexing. Flow cytometry acquisition was performed on a FACS Canto II (BD Switzerland) in combination with the DIVA software and results analyzed using FlowJo (TreeStar). The minimum acceptable cut-off value used was 80% double-positive cells. Prior to virus infection and for accurate MOI calculation, cell numbers of single, representative organoids were analyzed and calculated through flow cytometry analysis. For this purpose, single organoids were dissociated by 10-minute incubation at 37°C, 5% CO 2 in 500 µl of accutase. A single-cell suspension was obtained through repeated pipetting. 100 µl of the obtained suspension were transferred to a flow cytometry tube (Falcon) and washed by adding 2.5 ml of DPBS before pelleting the cells by centrifugation at 350 g for 10 minutes at 4°C. Supernatant was subsequently carefully removed through aspiration and cells resuspended in 300 µl of CellWASH (BD Switzerland). CountBright Absolute Counting Beads for flow cytometry (Invitrogen) were allowed to reach RT, vortexed for 30 seconds and 50 µl of the bead solution was rapidly transferred to the resuspended cells. The mixture was thoroughly vortexed before proceeding to flow cytometry acquisition and data analysis as previously described. Cell counts were calculated according to CountBright Absolute Counting Beads for flow cytometry user manual. Flow cytometry analysis was additionally used to quantify the percentage of infected cells in organoids. As previously described, hNOs were dissociated by incubating single organoids in 500 µl of accutase for 10 minutes at 37°C, 5% CO 2 . Cultures were dissociated into single cells by repeated pipetting and accutase diluted by addition of 2 ml of DPBS into each tube. A volume of 0.5 ml of cell solution, corresponding to approximately 10 6 cells, was transferred to a new flow cytometry tube and transferred to biocontainment centrifuge buckets, for pelleting at 350 x g for 7 minutes at 4°C. To allow discrimination between live and dead cells, LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen, L34966) was diluted 1:1000 in DPBS and vortexed. The supernatant of each cell pellet was removed through aspiration and cells were resuspended in 200 µl of the previously prepared LIVE/DEAD Fixable Aqua Dead Cell Stain solution. Cells were resuspended by vortexing and incubated 30 minutes on ice, protected from light. Following incubation, cells were washed by adding 1 ml of DPBS to each tube and subsequently pelleted by centrifugation in biocontainment buckets at 380 g for 5 minutes at 4°C. The supernatant was removed from each cell pellet through aspiration and the cells fixed using 200 µl of undiluted Fixation/Permeabilization Concentrate (Invitrogen) per tube. Cells were resuspended by vortexing and left to incubate on ice for 30 minutes. In the meantime, 10x Perm/Wash Buffer (BD Switzerland) was diluted 1:10 in distilled water. Following incubation, cells were washed by adding 1 ml of the previously prepared Perm/Wash solution to each tube. Cells were pelleted by centrifugation at 380 g for 5 minutes at 4°C. Following primary antibody solutions were prepared by diluting antibodies in Perm/Wash solution at given concentrations: home-made orthoflavivirus envelope protein antibody (4G2, 1:10) and cleaved caspase-3 (Cell Signaling Technology, #9661, 1:1000). After centrifugation, supernatants were removed by aspiration and cells were resuspended in 100 µl of primary antibody solution, followed by vortexing and a 15-minute incubation on ice. One ml of perm-wash solution was subsequently added to each tube and cells spun down by centrifuging at 380 g for 5 minutes at 4°C. Secondary antibody solutions were prepared as previously described, using the following antibodies at given concentrations: anti-mouse AF488 (Invitrogen, A21131, 1:500) and anti-rabbit AF647 (Invitrogen, A21245, 1:500). Supernatants were subsequently aspirated, and cells resuspended in 100 µl of secondary antibody solution, vortexed and incubated for 15 minutes on ice. Following secondary antibody staining, cells were washed once more with 1 ml of Perm/Wash solution and pelleted at 380 g for 5 minutes at 4°C. Supernatants were once more removed and the cells resuspended in 80 µl of Perm/Wash by vortexing. Unstained and live/dead staining controls were prepared for each analysis. To allow clear live and dead cell separation, live/dead control-cells were heated at 75°C for 5 minutes shaking at 300 rpm, prior to staining. Biosafety level 2 procedures were strictly adhered to during each protocol step. Human neural organoids staining Whole ZIKV-challenged and mock-treated organoids were collected at regular intervals during ZIKV-infection experiments for cryopreservation and subsequent immunofluorescent analysis. An adapted version of the procedure for “Cryogenic Tissue Processing and Section Immunofluorescence of Cerebral Organoids” published by STEMCELL Technologies was followed, as previously described 32 . Briefly, harvested organoids were washed twice with sterile DPBS, pre-fixed for 10 minutes in 2% formalin solution and subsequently preserved in 4% formalin solution (Sigma-Aldrich). For cryoprotection, fixed organoids were equilibrated in 30% sucrose (Sigma-Aldrich) in DPBS solution overnight at 4°C. Complete equilibration was confirmed by organoids sinking to the tubes’ bottom. The tissue was then transferred to a pre-heated 7.5% gelatin (Sigma-Aldrich), 10% sucrose in DMEM/F-12 (Gibco) solution and left to incubate at 37°C for 1 hour, to allow organoid encapsulation. Finally, hNOs were transferred together with a sufficient amount of warm gelatin-solution into a Cryomold (Biosystems Switzerland AG, Muttenz, Switzerland), and after initial polymerization at RT deep-frozen by gently placing the Cryomold onto the surface of a previously prepared dry-ice and 100% ethanol slurry. Frozen Cryomolds were stored at -80°C until further processing. For immunofluorescence staining, frozen organoid-bearing gelatin blocks were processed into 18 µm thick slices using a Leica CM1950 cryostat. Sections were collected on SuperFrost Plus Adhesion slides (Epredia, Dreieich, Germany) and stored at -20°C until the day of staining. For further processing, slides were left to thaw at RT for 30 minutes, before being transferred to a pre-warmed 0.1% Tween-20 (Sigma-Aldrich) in DPBS solution (0.1% T-DPBS) and incubated at 37°C for 10 minutes to allow gelatine removal. Areas of interested were subsequently encircled with ReadyProbes Hyrophobic Barrier Pap Pen (Thermo Fisher Scientific) and unspecific staining prevented by 1 hour RT incubation with a 5% donkey serum (Abcam, Paris, France) or 5% goat serum (Sigma-Aldrich) in 0.1% T-DPBS solution. Primary antibodies were diluted in 0.1% T-DPBS supplemented with 5% bovine serum albumin (Sigma-Aldrich) at pre-established concentrations as follows: SOX2 (Invitrogen, 14-9811-82, 1:200) and home-made orthoflavivirus envelope protein antibody (4G2, 1:3). Blocking solutions were removed by gently tapping slides on clean tissue, 100 µl of primary antibody solution were pipetted on top of each area of interest and left to incubate on tissue slices overnight at 4°C in a humidified chamber. On the following day, the solution was removed and the sections washed in Coplin jars filled with 0.1% T-DPBS three times for 5 minutes each. The following secondary antibodies were diluted in 0.1% T-DPBS at given concentrations: anti-mouse AF488 (Invitrogen, A21131, 1:200), anti-rat AF647 (Invitrogen, A21247, 1:500). For nuclear staining DAPI (Sigma-Aldrich, 1:1000) was added to the solution. The obtained antibody mixtures were pipetted on the selected areas and left to incubate for 2 hours at RT. Slides were subsequently washed three more times as previously described and mounted in EMS Shield Mount with DABCO (Electron Microscopy Sciences). 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Ligand-based receptor identification on living cells and tissues using TRICEPS. Nat Protoc 8 , 1321–1336 (2013). REED, L. J. & MUENCH, H. A SIMPLE METHOD OF ESTIMATING FIFTY PER CENT ENDPOINTS12. American Journal of Epidemiology 27 , 493–497 (1938). R Core Team. R: A language and environment for statistical ## computing. R Foundation for Statistical Computing, Vienna, Austria. (2021). Additional Declarations There is NO Competing Interest. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5866223","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":415267938,"identity":"adc9c0b7-fc64-4843-a62a-6291ba242c0a","order_by":0,"name":"Milos Stojanov","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYDACZuYGKOsAiLBhMABRjA3YVUO0QGQloFrSiNDCANcCBocJa9FtZ2x8+IOhro6f8fCzBx/bzsubS+QYMPzcgVuL2WHGZmMehsMSkg3HzA1ntt023Dkjx4Cx9wxeLW3SQH9IGBw4YCbNc+Z2gsGNtARmxjb8WiSBDgNqOf4NqOUccVokeBiYgVrOAG2pOADUknyAkBagXwwOS85sOFMmOaMi2XDDmccHDvbi03L+8MGHPyrq+Pkljm+T+GBgJ29wPLHxwU88WiAAFBcSBxD8AzjUoQH+BuLUjYJRMApGwcgDACC6Unxjf4xGAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-5166-5841","institution":"University Hospital of Lausanne","correspondingAuthor":true,"prefix":"","firstName":"Milos","middleName":"","lastName":"Stojanov","suffix":""},{"id":415267939,"identity":"59f80e76-34f0-4f67-83dd-22a418c1472e","order_by":1,"name":"Yen-Chi Chiu","email":"","orcid":"","institution":"University Hospital of Lausanne","correspondingAuthor":false,"prefix":"","firstName":"Yen-Chi","middleName":"","lastName":"Chiu","suffix":""},{"id":415267940,"identity":"4c7f5114-ada0-45d2-9b9d-587a4f3481d0","order_by":2,"name":"Amal Fahmi","email":"","orcid":"","institution":"University of Bern","correspondingAuthor":false,"prefix":"","firstName":"Amal","middleName":"","lastName":"Fahmi","suffix":""},{"id":415267941,"identity":"28be43f3-1741-4355-8d6a-db5e8d8c9135","order_by":3,"name":"Isabel Schultz-Pernice","email":"","orcid":"https://orcid.org/0000-0003-3841-501X","institution":"University of Bern","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"","lastName":"Schultz-Pernice","suffix":""},{"id":415267942,"identity":"b7e6d024-8def-48ac-a515-612c79389f16","order_by":4,"name":"Teodora David","email":"","orcid":"","institution":"University of Bern","correspondingAuthor":false,"prefix":"","firstName":"Teodora","middleName":"","lastName":"David","suffix":""},{"id":415267943,"identity":"7359f8ca-0958-4596-a4b4-28e8b5b77943","order_by":5,"name":"Margot Jarrige","email":"","orcid":"","institution":"Université Paris-Saclay","correspondingAuthor":false,"prefix":"","firstName":"Margot","middleName":"","lastName":"Jarrige","suffix":""},{"id":415267944,"identity":"3c18743a-aac2-46a3-974f-83762a6c729b","order_by":6,"name":"Donya El Akrouti","email":"","orcid":"","institution":"Université Paris-Saclay","correspondingAuthor":false,"prefix":"","firstName":"Donya","middleName":"El","lastName":"Akrouti","suffix":""},{"id":415267945,"identity":"cfb61f9d-6939-4b41-ac6c-85eaa34ae7bf","order_by":7,"name":"Nicole Déglon","email":"","orcid":"https://orcid.org/0000-0003-4475-9476","institution":"University Hospital of Lausanne (CHUV)","correspondingAuthor":false,"prefix":"","firstName":"Nicole","middleName":"","lastName":"Déglon","suffix":""},{"id":415267946,"identity":"b373deb7-253c-42ff-972b-f77152bd4ac8","order_by":8,"name":"Anselme Perrier","email":"","orcid":"","institution":"Université Paris-Saclay","correspondingAuthor":false,"prefix":"","firstName":"Anselme","middleName":"","lastName":"Perrier","suffix":""},{"id":415267947,"identity":"fd573d31-d05a-422a-aa13-709b7011ac5f","order_by":9,"name":"Marco Alves","email":"","orcid":"https://orcid.org/0000-0002-8495-6098","institution":"University of Bern","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Alves","suffix":""},{"id":415267948,"identity":"d349567f-de4b-425f-93e6-4352c61ee7b8","order_by":10,"name":"David Baud","email":"","orcid":"","institution":"University Hospital of Lausanne","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Baud","suffix":""}],"badges":[],"createdAt":"2025-01-20 13:20:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5866223/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5866223/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78877071,"identity":"2b4d1efc-6ec3-4d4a-9a2a-5434ed4daa66","added_by":"auto","created_at":"2025-03-20 07:35:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":58129,"visible":true,"origin":"","legend":"\u003cp\u003eVolcano plots depicting the results of LRC-TriCEPS in U-87 MG (left panel) and Vero (right panel) cell lines. The X-axis shows the fold change compared to the control reaction (transferrin), with a four-fold enrichment considered significant. The Y-axis displays the statistical significance (ANOVA model) of the obtained peptides. Common candidates are highlighted in red. ZIKV E protein is shown in green, while transferrin receptor, expected to be enriched in the control experiments, is shown in pink.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5866223/v1/fd8434945778e5a0d639688f.png"},{"id":78877822,"identity":"bb08d0ec-cba2-48bc-b7dd-5f08237a6b01","added_by":"auto","created_at":"2025-03-20 07:43:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":371696,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction between PAI-1 and ZIKV. A. Schematic representation of the immunogold assay. B. Representative TEM micrographs of ZIKV in the presence (left) and absence (right) of recombinant human PAI-1 protein. Scale bars, 50 nm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5866223/v1/60bb06d8931c7bf255216427.png"},{"id":78877068,"identity":"a03417f9-4cce-49d7-9fb0-98339995b078","added_by":"auto","created_at":"2025-03-20 07:35:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90384,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of PAI-1 expression levels and activity on ZIKV replication in U-87 MG and Vero cell lines, showing effects on viral RNA replication (top panels) and infectious viral titers in the supernatant (lower panels). A. Impact of PAI-1 overexpression at 24 and 48 HPI. B. Effect of \u003cem\u003eSERPINE1\u003c/em\u003e gene deletion on viral replication at 24 and 48 HPI. C. Effect of TPX on viral replication at 24 and 48 HPI. Viral RNA quantification was normalized to GAPDH, and data represent the averages of triplicate experiments ± SD. Statistical analysis was performed using a two-way ANOVA, with significance levels * p\u0026lt;0.05; ** p\u0026lt;0.01; *** p\u0026lt;0.001. Viral titers, quantified by TCID\u003csub\u003e50\u003c/sub\u003e method, were calculated by pooling 6 replicates in the same analysis.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5866223/v1/91ff14252cbcd8a7f56b6ca0.png"},{"id":78877070,"identity":"998778e1-821b-49b4-a909-d416b5835938","added_by":"auto","created_at":"2025-03-20 07:35:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":256245,"visible":true,"origin":"","legend":"\u003cp\u003ePAI-1 and ZIKV infection in neural progenitor cells. A. Representative bright field image of rosette-like structures commonly observed in \u003cem\u003ein vitro\u003c/em\u003e cultures of NPCs. B. Characterisation of NPCs for the expression of Nestin (green), NANOG (red) and DAPI staining (blue). C. Susceptibility of NPCs to ZIKV. Top panel show mock-infected cells, while the bottom panel show cells infected wth ZIKV. Cells were stained with rhodamine-Concanavalin A (ConA, red) and ZIKV using the 4G2 antibody (green). D. Volcano plot depicting the results of LRC-TriCEPS in NPCs. The X-axis shows the fold change compared to the control reaction (transferrin), with a four-fold enrichment considered significant. The Y-axis displays the statistical significance (ANOVA model) of the obtained peptides. PAI-1 is highlighted in red, while ZIKV E protein in green. Transferrin receptor (control experiment) is shown in pink. E. Effect of TPX and purified PAI-1 on ZIKV replication in NPCs compared to positive controls (control and DMSO) at 24 and 48 HPI. Scale bars, 100 μm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5866223/v1/dce4df1df4cd73e31ada17f6.png"},{"id":78877073,"identity":"62dc0c6f-5e06-4fb9-a0af-3bbfed5dc78e","added_by":"auto","created_at":"2025-03-20 07:35:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":863055,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of TPX on ZIKV infection in human neural organoids. A. Representative micrographs of hNOs at day 0 and day 4 post-infection under different treatment conditions. Scale bars, 1 mm. B. Expression of viral E protein within the organoids in the presence of varying TPX concentrations as determined by flow cytometry. Dashed line depicts the detection limit. Statistical analysis was performed using a nonparametric Welch's t-test, with significance levels *** p\u0026lt;0.001. C. Effect TPX treatment on the release of viral particles from hNOs, quantified by TCID\u003csub\u003e50\u003c/sub\u003e assay at day 1, 2, 3, and 4 post-infection. D. Confocal microscopy analysis of neural organoids sections. The effect of different conditions on ZIKV replication is shown, with images of entire organoids in the left panels and magnified areas, indicated by white squares, in the right panels. Cells were stained with DAPI (blue) and SOX2 (pink), while ZIKV presence was detected by staining for the viral E protein (yellow). Scale bars represent 300 μm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5866223/v1/973fa37840c0f3a72cb110f8.png"},{"id":78877966,"identity":"584768f7-6177-4029-931e-52b65779f340","added_by":"auto","created_at":"2025-03-20 07:51:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2621391,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5866223/v1/2407be4d-5d73-41a7-9076-a780b2e016b8.pdf"},{"id":78877072,"identity":"d091e639-f117-4959-a317-5518966b60cc","added_by":"auto","created_at":"2025-03-20 07:35:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":500081,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5866223/v1/2d8599d5e3da1ef06acf1cfe.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Plasminogen activator inhibitor-1 regulates Zika virus infection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Zika virus (ZIKV) outbreak of 2015-16 in the Americas and associated territories has been defined as a \u0026ldquo;perfect storm\u0026rdquo;, in which a neglected virus was introduced in a fully na\u0026iuml;ve population\u003csup\u003e1,2\u003c/sup\u003e. Recent estimates indicate that 132\u0026nbsp;million people were infected by the virus by 2018\u003csup\u003e3\u003c/sup\u003e. A member of the \u003cem\u003eOrthoflavivirus\u003c/em\u003e genus, ZIKV was first described in 1947 in Uganda, Africa\u003csup\u003e4\u003c/sup\u003e, and is classified into African and Asian lineages\u003csup\u003e5\u003c/sup\u003e. In the last two decades, the Asian clade of ZIKV has spread through the Asian continent to the Pacific\u003csup\u003e6\u003c/sup\u003e, reaching South America, where it was identified in Bahia, Brazil, in March 2015\u003csup\u003e7\u003c/sup\u003e. The Brazilian outbreak extended throughout most of the country and led to more than one million confirmed cases by the end of the year. Surprisingly, an unexpected increase in cases of microcephaly was observed in the regions affected by the outbreak approximately six months later, leading to a confirmation of ZIKV as a causative agent of birth defects\u003csup\u003e8\u003c/sup\u003e. While the epidemic in the Americas was waning, it became clear that microcephaly cases were just the tip of the iceberg for \u003cem\u003ein utero\u003c/em\u003e ZIKV infection sequelae. From a clinical perspective, resulting brain pathologies include microcephaly, calcifications, underdevelopment of the corpus callosum or cerebellum, ocular anomalies and reduced myelination\u003csup\u003e9\u003c/sup\u003e, leading to congenital ZIKV syndrome (CZS). As a result, infants from cohorts established during the 2015-16 outbreak have been observed to experience long-term disabilities, even in the absence of abnormal brain structures\u003csup\u003e10\u003c/sup\u003e. CZS was also observed in animal models, where even asymptomatic \u003cem\u003ein utero\u003c/em\u003e infections have been shown to cause inflammation of fetal organs and long-term consequences\u003csup\u003e11,12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe initial efforts after the 2015-16 outbreak, resulting in an unprecedented focus and funding support in the scientific community, did not lead to concrete development of prophylactic and therapeutic measures against ZIKV infections\u003csup\u003e13\u003c/sup\u003e. The main vaccine candidate developed to date, Zika virus purified inactivated vaccine (ZPIV), while showing good safety features, does not have an optimal immunogenicity profile\u003csup\u003e14,15\u003c/sup\u003e. Application of ZPIV where yellow fever and Japanese encephalitis virus vaccines are commonly administered, mainly South-East Asia, could therefore pose a challenge\u003csup\u003e14\u003c/sup\u003e. Thus, other preventive and/or therapeutic strategies are needed.\u003c/p\u003e \u003cp\u003eUnique characteristics of ZIKV pathogenesis, such as vertical and sexual transmission, distinguish it from other orthoflaviviruses and underscore specific features in the virus\u0026rsquo;s biology, particularly its capacity to infect a broad range of cell types. A key step in the viral replication cycle is the attachment to the host cell, which involves the envelop (E) protein, whose structure is well conserved among orthoflaviviruses\u003csup\u003e16\u003c/sup\u003e. It binds cellular receptors and host factors and, upon attachment, drives the membrane fusion and the consequent entry of viral genetic material into the cell, where replication can take place. In addition to E protein, negatively charged lipids present in the viral membrane, such as phosphatidylserine (PS), were shown to participate in the entry process of orthoflaviviruses\u003csup\u003e17\u003c/sup\u003e. Despite considerable efforts, the precise mechanisms by which ZIKV enters host cells remain unclear. Similar to other orthoflaviviruses, ZIKV may interact with multiple factors during the entry process, as the interplay of multiple receptors may contribute to stronger binding, compared to an individual receptor\u003csup\u003e18\u003c/sup\u003e. Initially, the C-type lectin dendritic cell-specific intracellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) was reported to promote ZIKV entry in dendritic cells and immature macrophages \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e19\u003c/sup\u003e. Transmembrane PS receptors, including TIM (TIM1, TIM3, and TIM4) and TAM (TYRO3, AXL, and MER) family members, have been shown to contribute to the internalization of orthoflaviviruses\u003csup\u003e19,20\u003c/sup\u003e. In the case of ZIKV, several studies have specifically suggested that AXL, a transmembrane receptor tyrosine kinase, acts as an entry receptor and/or immune modulator for the virus in a cell type-specific manner\u003csup\u003e21\u003c/sup\u003e. However, other findings have suggested that TAM proteins are not the critical receptors for ZIKV infectivity\u003csup\u003e22\u003c/sup\u003e. Indeed, ZIKV challenge of TAM-knockout mice showed that viral replication was not affected in the spleen, placenta, vagina, and brain\u003csup\u003e22\u003c/sup\u003e. Additional studies carried out with ALX-knockout mice have also demonstrated that these proteins are not essential for ZIKV infectivity\u003csup\u003e23\u003c/sup\u003e. Another study reported that genetic ablation of AXL did not affect virus infectivity or ZIKV-induced cell death in neural progenitor cells (NPCs) and neural organoids\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile ZIKV appears to utilize well-established orthoflavivirus receptors for internalization across various infection models, its specific tropism and routes of transmission suggest the involvement of additional entry mechanisms. Using a ligand-based receptor capture method, we report here the identification of plasminogen activator inhibitor-1 (PAI-1), encoded by the \u003cem\u003eSERPINE1 gene\u003c/em\u003e, as a novel cellular factor involved in ZIKV infection. PAI-1 belongs to the serine protease inhibitor family and plays a major role in the regulation of tissue hemostasis\u003csup\u003e25\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe utilized several cell lines to confirm the role of PAI-1 in ZIKV entry, including human brain glioblastoma cells (U-87 MG), and Vero cells, which are commonly used for viral propagation under laboratory conditions. Given the physiological relevance of the viral entry process, we focused our analyses on human neural progenitor cells (NPCs), which exhibit characteristics of neural cells enriched in the human fetal brain. To further validate our findings, we employed human neural organoids (hNOs), demonstrating the potential critical role of PAI-1 in ZIKV infection. These three-dimensional structures closely replicate the architecture and cellular diversity of the developing human brain, allowing us to investigate the potential critical role of PAI-1 in a more complex environment.\u003c/p\u003e \u003cp\u003eSeveral factors suggest a potential rise in future orthoflavivirus outbreaks, including those caused by ZIKV\u003csup\u003e26\u003c/sup\u003e. Statistical models indicate that vector populations, comprising of \u003cem\u003eA. aegypti\u003c/em\u003e, will increase their ecological niches due to globalization, urbanization, and climate change\u003csup\u003e27\u003c/sup\u003e. The devastating outcomes of CZS demand great concern, mainly in non-immune populations of the Northern hemisphere, and warrant further effort by the scientific community to find concrete solutions. While ZIKV prevalence has decreased since the 2015\u0026ndash;2016 outbreak, the virus persists, with over 40,000 cases in the Americas in 2022, Brazil reporting the majority\u003csup\u003e28\u003c/sup\u003e. Therefore, the threat remains, alongside emerging evidence of ZIKV causing additional brain damage. The situation underscores the ongoing risk and the need for valid therapeutic alternatives.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eIdentification of cell surface factors interacting with ZIKV\u003c/h2\u003e\n \u003cp\u003eLigand-receptor capture technology LRC-TriCEPS was used to identify putative cellular factors involved in the internalization of ZIKV. The analysis was initially performed in U-87 MG and Vero cell lines with transferrin used as a control to validate the assay and remove background signal. A total of 374 membrane associated proteins were identified in the U-87 MG cell line, with 189 hits identified by more than two peptides (Supplementary table 1). On the other hand, 62 proteins were identified for the Vero cell line, of which 35 were recognized by more than two peptides (Supplementary table 2). Comparison with the transferrin control narrowed down the number of putative candidates to 11 for U-87 MG and 4 for Vero cells (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In both analyses, viral E protein (ZIKV_E) was successfully identified in infected cells (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary tables 1 and 2).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Potential cellular factors identified in U-87 MG and Vero cell lines.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eCandidates common to the two cell lines are highlighted in grey. * Log\u003csub\u003e2\u003c/sub\u003e fold change relatively to the control assay. ** -Log\u003csub\u003e10\u003c/sub\u003e adjusted p-value.\u003c/p\u003e\n \u003cp\u003eThree candidate proteins, PAI-1, fetuin A (AHSG), and haemoglobin subunit beta (HBB), were common to both cell lines (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Given PAI-1\u0026rsquo;s broad involvement in a range of diseases, its significance across multiple organ systems, and its known role in the pathogenesis of other viruses, we prioritized PAI-1 for further experimental investigation.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003ePAI-1 binds to ZIKV and influences viral replication\u003c/h3\u003e\n\u003cp\u003eWe sought to verify the direct interaction of PAI-1 with ZIKV using transmission electron microscopy (TEM). To test this, we incubated purified recombinant human PAI-1 protein with viral particles. The presence of PAI-1 on the surface of the virions was revealed by a PAI-1-specific antibody, coupled to protein A conjugated with nanogold particles (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Our results showed that the surface of virions was decorated with regularly interspaced pattern of gold particles, suggesting an interaction with a motif on the envelope protein (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, left panel). In the untreated control, where PAI-1 was not added, no interaction of the virions with nanogold particles was observed (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, right panel).\u003c/p\u003e\n\u003cp\u003eWe next sought to determine whether the addition of purified PAI-1 could enhance ZIKV infection. An inoculum consisting of ZIKV preincubated with PAI-1 was used to infect U-87 MG and Vero cell lines. In both cases, ZIKV RNA levels increased significantly compared to the negative control (no preincubation with PAI-1) (Supplementary Fig.\u0026nbsp;1). Specifically, we observed a two-fold increase of viral RNA in the U-87 MG cell line and a four-fold increase in the Vero cell line. We also observed a general trend of increased release of infectious viral titers in the supernatant, which, however, remained however below a one-log difference (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eNext, we overexpressed PAI-1 in the two cell lines using an expression plasmid encoding human \u003cem\u003eSERPINE1\u003c/em\u003e cDNA. Control experiments indicated that PAI-1 RNA levels, as measured by RT-qPCR, increased by 8-fold in U-87 MG cells and 4-fold in Vero cells (Supplementary Fig. 2A). The efficiency of ZIKV infection was compared in cells overexpressing PAI-1 and wild type cell lines, showing significantly increased viral RNA levels with higher PAI-1 expression (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, top). In U-87 MG cells, we observed a 4-fold increase in viral RNA at 24 hours post infection (HPI) and a 7-fold increase at 48 HPI. In Vero cells, while a 3-fold increase in viral RNA was observed at 24 HPI, the difference was not significant at 48 HPI. As observed with the preincubation experiments, increased PAI-1 levels were accompanied by an increase in infectious viral titers in the supernatant compared to negative controls, remaining under a one-log difference, except for Vero cells at 48 HPI, similarly to viral RNA quantification (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, bottom).\u003c/p\u003e\n\u003cp\u003eTo analyse the effects of PAI-1 depletion, we tested the ability of ZIKV to infect cell lines in which the \u003cem\u003eSERPINE1\u003c/em\u003e gene was knocked out (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, Supplementary Fig. 2B). The absence of PAI-1 generally resulted in lower viral RNA loads and decreased release of viral particles into the supernatant. RNA synthesis was reduced by approximately half, except for U-87 MG cells at 24 HPI, where no significant difference was observed between wild type and PAI-1-depleted cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, top). This difference was more prominent at 48 HPI, compared to 24 HPI. Infectious viral titers in the supernatant were approximately reduced by approximately one log at all timepoints tested compared to wild type background, except for U-87 MG cells at 24 HPI (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, bottom).\u003c/p\u003e\n\u003cp\u003eSeveral molecules inhibiting PAI-1 activity have been developed and, among them, tiplaxtinin (TPX) is the most studied inhibitor, whose efficacy has been showed in multiple models\u003csup\u003e29\u003c/sup\u003e. TPX is a small molecule that works by binding to the active site of PAI-1, stabilizing it in an inactive conformation. We tested the effect of TPX to potentially interfere with ZIKV replication, by treating cells with the drug for 24 hours before the infection and subsequently supplementing TPX in the cell culture medium. While in U-87 MG at 24 HPI a clear though non-significant effect was documented, TPX significantly reduced the viral RNA load at all other time points, in both cell lines, reaching significance except for in U-87 MG cells at 24 HPI (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, top). The effect on viral replication was even more marked when supernatants were analysed, as we observed a reduction of infectious viral titers of at least 2 orders of magnitude at all time points tested (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, bottom). Of note, infectious titers in the supernatant of U-87 MG cells treated with TPX at 48 HPI were reduced by five orders of magnitude. The effect of TPX on viral replication was dose-dependent, with increasing concentrations (5, 25, and 50 \u0026micro;M) resulting in a stronger inhibition of viral replication (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e\n\u003ch3\u003eInhibition of PAI-1 blocks ZIKV infection in neural progenitor cells\u003c/h3\u003e\n\u003cp\u003eInfection of NPCs in the fetal brain represents a central component in ZIKV teratogenesis leading to CZS. To further evaluate the impact of PAI-1 on ZIKV replication, we used human iPSC-derived NPCs (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Successful iPSC to NPC transition was confirmed by the robust expression of Nestin, a marker for neural stem cells and the expected absence of NANOG, indicating the loss of pluripotency and commitment to a neuroectodermal lineage (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). As expected, these cells were readily infected by ZIKV under our experimental conditions (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). We therefore applied the LRC-TriCEPS method to the iPSC-derived NPCs (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). Interestingly, a higher number of proteins potentially interacting with ZIKV was identified by using NPCs compared to U-87 MG and Vero cell lines (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). A total of 438 membrane-associated or extracellular proteins were detected, of which 253 were identified by more than two peptides and were therefore used for statistical analysis (Supplementary table 3). After applying a two-fold enrichment cutoff and ensuring statistical significance, 19 candidates were selected (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). We therefore confirmed that PAI-1 was also identified as a protein interacting with ZIKV (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD), confirming previous LRC-TriCEPS experiments with U-87 MG and Vero cell lines. Of note, AHSG (Fetuin A), previously identified in U-87MG and Vero cell lines, was also found as a molecule potentially interacting with ZIKV. This was not the case for HBB.\u003c/p\u003e\n\u003cp\u003eGiven the strong effect of TPX on ZIKV replication in U-87 MG and Vero cell lines, we repeated this analysis using NPCs. Treatment with 50 \u0026micro;M TPX completely abolished viral replication compared to positive controls (control and DMSO-treated cells) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Potential cellular factors identified in NPCs.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eCandidates common to the previous analysis (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) are highlighted in grey. *Log\u003csub\u003e2\u003c/sub\u003e fold change relatively to the control assay. **-Log\u003csub\u003e10\u003c/sub\u003e adjusted p-value.\u003c/p\u003e\n\u003ch3\u003ePAI-1 inhibition blocks ZIKV infection in human neural organoids\u003c/h3\u003e\n\u003cp\u003eTo investigate the impact of PAI-1 on ZIKV infection in a model that mimics the developing human brain, we utilized ESC-derived hNOs. This system closely replicates key aspects of human brain formation and has been previously used to model microcephaly \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e30\u003c/sup\u003e. To highlight the contribution of PAI-1 to ZIKV pathogenesis, we investigated the impact of TPX treatment on ZIKV-challenged hNOs. Macroscopically, infection by ZIKV and different treatment conditions, including presence or absence of DMSO, TPX and PAI-1, did not have any apparent effect on the organoids, as compared to the controls, after 4 days of incubation (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). In agreement, no significant difference was detected in organoid surface area measurements, despite ZIKV-infected, untreated organoids showing the smallest size increase (0.5 mm\u003csup\u003e2\u003c/sup\u003e) between day 0 post-infection and day 4 post-infection, compared to all other conditions (0.6 mm\u003csup\u003e2\u003c/sup\u003e \u0026ndash; 1.1 mm\u003csup\u003e2\u003c/sup\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e\n\u003cp\u003eAs observed in cell lines and NPCs, increasing concentrations of TPX had a profound effect on viral replication in human brain organoids (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB and C). Flow cytometry analysis of organoids dissociated into single cells confirmed the critical impact of TPX treatment on viral infection. At 4 days post-infection the percentage of ZIKV E-protein harbouring cells was documented to be significantly reduced compared to untreated, infected organoids (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). While drug concentrations of 25 \u0026micro;M reduced viral titers in the supernatant by 2 logs, 50 \u0026micro;M completely abolished the release of viral particles from the organoids. These observations were additionally confirmed by confocal microscopy analysis of stained, sectioned organoids (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). As previously reported\u003csup\u003e31,32\u003c/sup\u003e, a ventricle-dominated hNO tissue structure, composed of densely packed, sex determining region Y-box 2 (SOX2) positive NPCs, was observed in mock-treated organoids after 15 days of culture (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). No morphological abnormalities and no viral particles could be identified through immunofluorescence staining in organoids treated with 25 \u0026micro;M of TPX compared to mock-treated cultures, indicating the important reduction in viral loads upon TPX treatment. However, while no viral particles could be detected in organoids cultured in medium supplemented with 50 \u0026micro;M of TPX, a reduction in ventricle size and alterations in tissue and outer border morphology were observed, indicating possible toxic effects induced by high TPX concentrations on exposed cultures. Interestingly, while no significant impact of PAI-1 treatment on viral loads has been observed (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC), incubation with PAI-1 induced a massive but localized invasion of ZIKV in a small portion of the organoids (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe identified PAI-1 as a novel host factor that significantly influences ZIKV infection. PAI-1 belongs to the serine protease inhibitor family and plays a major role in the regulation of hemostasis\u003csup\u003e33\u003c/sup\u003e. It is a plasma-circulating protein with a short half-life whose primary function is to specifically inhibit plasminogen activators. These activators normally convert plasminogen into its active form, plasmin, through proteolytic processes, thereby increasing fibrinolytic activity\u003csup\u003e25\u003c/sup\u003e. Beyond its primary function in the coagulation system, PAI-1 exhibits pleiotropic effects, impacting various physiological and pathological processes across different systems in the body including cell migration\u003csup\u003e34,35\u003c/sup\u003e, vascularization\u003csup\u003e36\u003c/sup\u003e, metabolic processes\u003csup\u003e37,38\u003c/sup\u003e, inflammation\u003csup\u003e39\u003c/sup\u003e, cancer progression\u003csup\u003e40\u003c/sup\u003e .and viral infection\u003csup\u003e41\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe demonstrated that PAI-1 physically interacts with ZIKV in multiple cell types, including U-87 MG glioblastoma cells, Vero cells and NPCs. Direct interaction was further confirmed by TEM, where PAI-1 was shown to bind to the surface of ZIKV particles. Our functional assays revealed that increasing PAI-1 levels, either through overexpression or by addition of recombinant human PAI-1, enhanced ZIKV replication and viral particle production in U-87 MG and Vero cell lines. Conversely, deletion of the \u003cem\u003eSERPINE1\u003c/em\u003e gene, which encodes PAI-1, led to a reduction in viral RNA levels and decreased infectious viral titers in the supernatant of U-87 MG and Vero cell lines. Most notably, inhibition of PAI-1\u0026rsquo;s enzymatic activity using TPX resulted in a significant, dose-dependent decrease of ZIKV replication across all models tested. In NPCs and hNOs, TPX treatment completely abolished viral replication at higher concentrations, underscoring the potentially crucial role of PAI-1 in the ZIKV life cycle.\u003c/p\u003e \u003cp\u003eThe enhancement of ZIKV infection by PAI-1 in U-87 MG and Vero cells could be attributed to several potential mechanisms. PAI-1 might act as an attachment factor, increasing viral binding affinity to host cells. Alternatively, it could modulate cellular pathways that favour viral entry or replication. Given that PAI-1 is known to influence cell migration, adhesion, and extracellular matrix remodeling\u003csup\u003e42\u003c/sup\u003e, its interaction with ZIKV might alter the cellular environment to be more permissive to infection. These mechanisms may however be cell type specific since incubation with recombinant PAI-1 did not influence viral infection in NPCs and hNOs.\u003c/p\u003e \u003cp\u003ePrevious research on ZIKV entry mechanisms has focused on AXL as the main receptor in multiple cell types\u003csup\u003e43,44\u003c/sup\u003e. However, the exact pathways facilitating ZIKV entry into neural cells remain incompletely understood. Our study introduces PAI-1 as a novel player in this process, offering new insights into ZIKV-host interactions. Furthermore, our experiments primarily focused on the early stages of infection. The observation that TPX treatment reduced viral titers even when administered post-infection suggests that PAI-1 may also influence later stages of the viral life cycle, such as assembly or release. Interestingly, PAI-1 has been linked with the infectious process of other viruses. It has been proposed that domain III of the Dengue virus\u0026rsquo; (DENV) E protein is able to activate the expression of PAI-1 during the early phases of infection and is linked with hemorrhage\u003csup\u003e41\u003c/sup\u003e. Furthermore, the maturation of influenza A virus (IAV) was shown to be inhibited by PAI-1 in the extracellular environment due to the blockage of IAV glycoprotein cleavage\u003csup\u003e45\u003c/sup\u003e. As a result, the infectivity of progeny IAV was observed to be attenuated\u003csup\u003e45\u003c/sup\u003e. PAI-1 mRNA levels and protein expression were documented to be downregulated in Hepatitis C virus-infected cells and patients\u003csup\u003e46\u003c/sup\u003e. Interestingly, PAI-1 levels were observed to increase in pregnant women after the 20th week of pregnancy\u003csup\u003e47\u003c/sup\u003e. Its production was localized within the placenta\u003csup\u003e48\u003c/sup\u003e and may be a hypothetic link with the materno-fetal transmission of ZIKV.\u003c/p\u003e \u003cp\u003eDespite these advancements, our study has limitations. The precise molecular mechanisms by which PAI-1 enhances ZIKV infection are yet to be fully elucidated. Additionally, while TPX effectively inhibited ZIKV replication, it has only been used in preclinical models and it is not an FDA approved drug\u003csup\u003e49,50\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, our study identifies PAI-1 as a critical host factor in ZIKV infection, particularly in neural cells relevant to CZS. Targeting PAI-1 or its interaction with ZIKV presents a promising therapeutic avenue. Given the ongoing risk of ZIKV outbreaks and the severe consequences of congenital infections, the challenge to develop an effective vaccine due to cross-antibody effects with other vaccines/viruses, developing strategies to inhibit PAI-1 function could contribute to preventing or mitigating the impact of ZIKV. Future studies should aim to uncover the detailed mechanisms of PAI-1-mediated enhancement of ZIKV infection and evaluate the potential of PAI-1 inhibitors in animal models and clinical settings.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eIn accordance with Articles 13 and 14 of the Federal Act on Research on Embryonic Stem Cells, and Article 20 of the Ordinance on Research on Embryonic Stem Cells (ESCs), the use of the human H1 ESC line was approved by the Cantonal Ethics Committee of Bern, Switzerland, under authorization number R-FP-S-2-0023-0000.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell lines and viruses\u003c/h3\u003e\n\u003cp\u003eCell lines and viral strains used in this study are depicted in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Human brain glioblastoma (U-87 MG) cells and \u003cem\u003eCercopithecus aethiops\u003c/em\u003e kidney epithelial (Vero) cells were grown in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, 61965026, Gibco, Basel, Switzerland) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS, F7524, Sigma-Aldrich, Buchs, Switzerland). Deletion of \u003cem\u003eSERPINE1\u003c/em\u003e gene in U-87 MG and Vero cell lines was performed by Ubigene Biosciences (Guangzhou, China).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCell lines and virus strain used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOrigin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDetails\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCell lines\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eU-87 MG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman brain glioblastoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrowth medium: DMEM\u0026thinsp;+\u0026thinsp;10% FBS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCercopithecus aethiops\u003c/em\u003e kidney epithelial cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrowth medium: DMEM\u0026thinsp;+\u0026thinsp;10% FBS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eiPS-derived neural progenitor cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eObtained by differentiating PB12 human induced pluripotent stem cells\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eViral strains\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePRVABC-59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZIKV, Asian-lineage strain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePandemic strain isolated from a viremic patient in Puerto Rico in 2015\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNPCs differentiation followed a previously described protocol\u003csup\u003e51\u003c/sup\u003e. Briefly, human iPSC colonies (PB12 line\u003csup\u003e52\u003c/sup\u003e) were initially cultured in N2B27 medium consisting of 50% DMEM/F-12 GlutaMAX (Thermo Fisher Scientific), 50% Neurobasal medium (Thermo Fisher Scientific), 2% B27 supplement (without vitamin A, Thermo Fisher Scientific), 1% N2 supplement (Thermo Fisher Scientific), and 50 \u0026micro;M β-mercaptoethanol (Thermo Fisher Scientific). Human iPSCs were first seeded and cultured for 6 hours in a low-adhesion culture plate in N2-B27 medium supplemented with SB431542 (20 \u0026micro;M, Tocris), LDN-193189 (0.1 \u0026micro;M, Sigma-Aldrich), and Y-27632 (10 \u0026micro;M, 72304, STEMCELL Technologies). The resulting aggregates were transferred to poly-L-ornithine and laminin-coated dishes. Media were changed daily from division 0 (DIV0) to DIV20. Y-27632 was removed at DIV1, XAV-939 (1 \u0026micro;M, 3748, Tocris) was added until DIV9. Between DIV5 and DIV9, SB431542 was removed and FGF2 (10 ng/mL, Peprotech) and cyclopamine (1 \u0026micro;M, Sigma-Aldrich) were added. From DIV10 to DIV17, LDN-193189 and XAV-939 were removed and CHIR99021 (0.4 \u0026micro;M, STE04-0004-02, Ozyme) was added. At DIV17, NPCs were enzymatically dissociated using accutase (11599686, Thermo Fisher Scientific) and cryopreserved in Cryostor cell cryopreservation medium (100\u0026ndash;1061, STEMCELL Technologies) for storage in liquid nitrogen vapor at -150\u0026deg;C.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eZIKV infection in cell lines and NPCs\u003c/h2\u003e \u003cp\u003eU-87 MG cells and Vero cells were seeded into a 24-well plate (approximately 6x10\u003csup\u003e4\u003c/sup\u003e cells per well) one day before infection with ZIKV. NPCs were prepared by coating 24-well plates with poly-L-ornithine (17 \u0026micro;g/ml, Sigma-Aldrich) and laminin (2.4 \u0026micro;g/ml, Gibco). Following coating, NPCs were seeded at approximately 2x10⁵ cells per well in N2B27 medium supplemented with 10 \u0026micro;M Y-27632 (ROCK inhibitor, STEMCELL Technologies) and 20 ng/ml FGF (PreproTech). ZIKV infection was carried out with a multiplicity of infection (MOI) of 1. After an incubation period of 30 minutes, ZIKV inoculum was removed and replaced with fresh medium. Tiplaxtinin (PZ0295, Sigma-Aldrich), a well-known PAI-1 small-molecule inhibitor\u003csup\u003e53\u003c/sup\u003e, was used after dissolution in dimethyl sulfoxide (DMSO, A3672, PanReac AppliChem, Darmstadt, Germany).\u003c/p\u003e \u003cp\u003eRecombinant human PAI-1 (PAI-1, 528205, Sigma-Aldrich), was dissolved in a solution comprising of 150 mM NaCl, 50 mM sodium phosphate buffer, and 1 mM EDTA at pH 6.6. One hour prior to infection, ZIKV and 1 \u0026micro;g/ml PAI-1 were mixed in an appropriate amount of medium and incubated at 37\u0026deg;C, shaking at 200 rounds per minute (rpm). Following incubation, the mixture was added to the cells and incubated for 30 minutes. Subsequently, cells were washed once with phosphate-buffered saline (PBS, Gibco), before adding fresh cell growth medium to the cultures.\u003c/p\u003e \u003cp\u003eNeural stem cells were fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences) and 4% sucrose (Sigma-Aldrich) in phosphate-buffered saline (PBS) for 10 minutes, followed by three washes with PBS at room temperature. Subsequently, the samples were subjected to an overnight incubation at 4\u0026deg;C with primary antibodies diluted in a PBS blocking solution containing 2% BSA (Sigma-Aldrich) and 0.2% Triton X-100 (Sigma-Aldrich) to facilitate membrane permeabilization. The primary antibodies used were: NESTIN (MAB5326, Millipore), NANOG (4903, Cell Signaling), Concanavalin A (Vector Laboratories Ltd), and ZIKV (HB-112, ATCC). Following three washes with PBS, cells were incubated for one hour at room temperature with species-specific secondary antibodies conjugated to Alexa Fluor 488 and 555 (Invitrogen) and DAPI (Calbiochem) in a PBS solution containing 2% BSA and 0.1% Triton X-100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of viral RNA\u003c/h2\u003e \u003cp\u003eExtraction of RNA from lysed cells was performed with the NucleoSpin RNA II kit (740955.50, Macherey-Nagel AG, D\u0026uuml;ren, Germany). Reverse transcription was carried out with random hexamers (100026484, Invitrogen, Carlsbad, CA, US) and SuperScript II Reverse Transcriptase (18064014, Invitrogen). The quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed using the iTaq Universal Probes Supermix (1725131, Bio-Rad, Basel, Switzerland). Relative quantification was performed with the Delta-Delta Ct method\u003csup\u003e54\u003c/sup\u003e using GAPDH as the housekeeping gene. The primers and TaqMan probes used for qRT-PCR were purchased from Microsynth AG (Baglach, Switzerland) and are listed in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers and probes for used for qRT-PCR analyses.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer/probe\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eZIKV\u003csup\u003e55\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZIKV_fw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CGYTGCCCAACACAAGG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZIKV_rev\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CCACYAAYGTTCTTTTGCABACAT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZIKV_probe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-6FAM-AGCCTACCTTGAYAAGCARTCAGACACYCAA-BHQ1-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eGAPDH\u003csup\u003e56\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGAPDH_fw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-GAAGGTGAAGGTCGGAGTCAAC-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGAPDH_rev\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-CAGAGTTAAAAGCAGCCCTGGT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGAPDH_probe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-6JOE-TTTGGTCGTATTGGGCGCCT-BHQ1-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAI-1\u003csup\u003e57\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePAI-1_fw\u003c/p\u003e \u003cp\u003ePAI-1_rev\u003c/p\u003e \u003cp\u003ePAI-1_probe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GGCTGACTTCACGAGTCTTTCA-3\u0026rsquo;\u003c/p\u003e \u003cp\u003e5\u0026rsquo;-TTCACTTTCTGCAGCGCCT-3\u0026rsquo;\u003c/p\u003e \u003cp\u003e5\u0026rsquo;-6FAM-ACCAAGAGCCTCTCCACGTCGCG-BHQ1-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e6-FAM: 6-carboxyfluorescein; 6-JOE: 6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein; BHQ-1: black hole quencher-1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLigand-receptor capture assay\u003c/h2\u003e \u003cp\u003eIdentification of proteins potentially interacting with ZIKV was performed using the ligand-based receptor capture (LRC) technology (Dualsystems Biotech AG, Schlieren, Switzerland). For each cell line, 2x10\u003csup\u003e7\u003c/sup\u003e cells were seed into T175 cell culture flask (355000, BD Switzerland, Basel, Switzerland) and used for the assay. For each replicate, 0.5 \u0026micro;l of the TriCEPS molecule were incubated with ZIKV strain PRVABC-59 for 90 minutes at 37\u0026deg;C. In parallel, cells were mildly oxidized on ice using 1.5 mM NaIO\u003csub\u003e4\u003c/sub\u003e in PBS (pH 6.5) for 15 minutes. Oxidized cells were incubated with ZIKV-TriCEPS mixture (MOI of 4) for 30 minutes at 37\u0026deg;C. Cells were collected by scraping and lysed immediately.\u003c/p\u003e \u003cp\u003eFollowing cell lysis, target proteins were purified using solid phase chromatography and processed to remove unspecific interactions as previously described\u003csup\u003e58\u003c/sup\u003e. Proteins were then reduced, alkylated and digested with trypsin. The tryptic peptides were collected for liquid chromatography-mass spectrometry/mass spectrometry. To allow for statistical analysis, the experiment was performed in biochemical triplicates.\u003c/p\u003e \u003cp\u003eSamples were analysed on a Thermo Orbitrap Elite spectrometer fitted with an electrospray ion source. Tryptic peptides were measured in data dependent acquisition mode (TOP20) in a 120-minute gradient using a 15 cm C18 packed column. The Progenesis software was used for raw file alignment and feature detection, Comet search engine was used for spectra identification and the Trans proteomic pipeline was used for statistical validation of putative identifications and protein inference. Upon protein inference, relative quantification of control and ligand samples was performed based on ion extracted intensity and differential protein abundance was tested using a statistical ANOVA model followed by multiple testing correction. This model assumes that the measurement error follows Gaussian distribution and views individual features as replicates of a protein's abundance and explicitly accounts for this redundancy. It tests each protein for differential abundance in all pairwise comparisons of ligand and control samples and reports the p-values. Next, p-values are adjusted for multiple comparisons to control the experiment-wide false discovery rate (FDR). The adjusted p-value (q-value) obtained for every protein is plotted against the magnitude of the fold enrichment between the two experimental conditions. In the present study, the annotated human proteome database from Uniprot was used for the analysis of the human cell lines. The proteome database of \u003cem\u003eCercopithecus aethiops\u003c/em\u003e from Uniprot was used for the analysis of the samples from the Vero cell line. The sequence of the genome polyprotein (POLG, Q32ZE1) of ZIKV was included in the analysis. The results for the human cell lines were filtered for membrane associated proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of infectious virus\u003c/h2\u003e \u003cp\u003eCell supernatants were collected at 24 and 48 HPI and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis. The day before titration, Vero cells were seeded into 96-well plates (CLS351172, Corning, Root, Switzerland) at a density of 2x10\u003csup\u003e4\u003c/sup\u003e/well. Serial 10-fold dilutions of cell supernatants were prepared in Vero cell growth medium, inoculated on Vero cells and incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 72 hours. Subsequently, cells were fixed by 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, US) for 20 minutes. Following fixation, cells were washed three times with 0.1% saponin in PBS solution, and incubated for 40 minutes with anti-orthoflavivirus group antigen antibody 4G2 (clone D1-4G2-4-15, ATCC, HB-112) at 37\u0026deg;C. Cells were subsequently washed twice with 0.1% saponin after which samples were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (G-21040, Thermo Fischer Scientific) for 40 minutes at 37\u0026deg;C. Following three more washes with 0.1% saponin solution, staining was performed with the 3-amino-9-ethylcarbazole substrate (Sigma-Aldrich). Infectious virus titers were calculated and expressed as 50% tissue culture infective dose per ml (TCID\u003csub\u003e50\u003c/sub\u003e/ml) using the Reed and Muench method\u003csup\u003e59\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOverexpression of PAI-1 protein in cell lines\u003c/h2\u003e \u003cp\u003eA commercially available plasmid (HG10296-NF, Sino Biological Inc., Beijing, China) was used for the overexpression of PAI-1. U-87 MG cells and Vero cells were cultured in 6-well plates (140675, Thermo Fisher Scientific) to form a monolayer by the day of transfection. Transfection was performed using Lipofectamine 3000 transfection reagent (L3000008, Invitrogen) following the manufacturer\u0026rsquo;s instructions. The cells were briefly washed with PBS before the transfection mixture was added. After incubation at 37\u0026deg;C in a 5% CO₂ atmosphere for 1 hour in U-87 MG cells and 6 hours in Vero cells, the transfection mixture was removed, and fresh growth medium was added. Beginning 48 hours after transfection, U-87 MG cells were treated with 100 \u0026micro;g/ml and Vero cells with 400 \u0026micro;g/ml of hygromycin B (30-240-CR, Corning). Antibiotic selection was continued for two weeks to ensure the propagation of successfully transfected cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunogold labelling\u003c/h2\u003e \u003cp\u003ePAI-1 and ZIKV PRVABC-59 were mixed at a 1:1 ratio and incubated at 37\u0026deg;C for 1 hour. Then, 4 \u0026micro;l of the mixture were loaded onto a carbon film Cu grid (CF200-Cu, Electron Microscopy Sciences), which had been previously glow-discharged for 30 seconds. After 1 minute, the samples were transferred to Whatman filter paper (WHA1001325, Whatman, Massachusetts, US). The blocking buffer containing 5% bovine serum albumin (Gibco) in PBS was applied to the grid for 10 minutes. Subsequently, PAI-1 was detected using a primary antibody (G-21040, Thermo Fisher Scientific) at a 10-fold dilution in 1% blocking buffer for 1 hour. The grid was then washed three times with PBS and incubated with 10-nm immunogold-conjugated protein A, diluted 25x in HB buffer, to detect the primary antibody. After a 1-hour incubation, the grid was washed three times with PBS, then fixed with 2% PFA for 10 minutes and washed once with ddH\u003csub\u003e2\u003c/sub\u003eO. Finally, the sample was negatively stained with 2% uranyl acetate for 1 minute. Images were captured using a Philips CM100 at 80 kV with a TVIPS F416 camera (4kx4k, Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of human neural organoids\u003c/h2\u003e \u003cp\u003ehNOs were generated starting from H1 embryonic stem cells (ESCs) as previously described\u003csup\u003e32\u003c/sup\u003e. Briefly, on day 0, H1 ESCs were dissociated through a 4-minute incubation with accutase (Sigma-Aldrich), followed by repeated pipetting to obtain a single-cell suspension. Cells were subsequently counted and pelleted through room-temperature (RT) centrifugation at 200 g for 5 minutes. Cells were then resuspended in an appropriate volume of RT Formation Medium (STEMCELL Technologies, Z\u0026uuml;rich, Switzerland) previously supplemented with 50 \u0026micro;M Rho-associated protein kinase (ROCK) inhibitor Y-27632 (STEMCELL Technologies) and the obtained single cell suspension distributed in ultra-low attachment 96-well plates (Corning) at a density of 10,000 cells and 100 \u0026micro;l per well. Cells were incubated at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e and 100 \u0026micro;l of medium without Y-27632 were added to each well in two-day intervals. On the same day (day 0), remaining cells were used for stemness quality control by flow cytometry as described below (Flow cytometry analysis of human neural organoids). After 5 days, cultures confirmed to have passed the stemness check were inspected for embryoid body formation and further subjected to visual quality control before being transferred into 24-well ultra-low attachment plates (Corning) prepared with 500 \u0026micro;l of pre-warmed Induction Medium (STEMCELL Technologies) using 200 \u0026micro;l ART wide bore filtered pipette tips (Thermo Fisher Scientific). The cultures were maintained at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e and neuroepithelium induction was observed in the following two days. On day 7 of culture, embryoid bodies were singularly embedded into 30 \u0026micro;l Matrigel (Corning) droplets and transferred into 6-well ultra-low attachment plates (Corning) containing 3 ml of pre-warmed home-made Expansion Medium, prepared according to the previously published protocol by Lancaster and colleagues\u003csup\u003e31\u003c/sup\u003e. The expanding hNOs were incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 3 more days. On day 10 of culture, the Expansion Medium was removed completely and replaced with 3.5 ml of RT home-made Maturation Medium, prepared using the previously cited protocol\u003csup\u003e31\u003c/sup\u003e. During the whole maturation period and until the end of each experiment, organoids were cultured on an orbital shaker, gently moving at 65 rpm, at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Maturation Medium was replaced at regular intervals of 2 to 3 days. Fifteen-days old organoids were subjected once more to visual quality control before being used for infection experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHuman neural organoid infection with ZIKV\u003c/h2\u003e \u003cp\u003ehNOs were challenged with ZIKV after a total culture time of 15 days. One day before infection, organoid cell numbers were quantified through flow cytometry as described below (Flow cytometry analysis of human neural organoids). Obtained mean cell counts were used for MOI calculation. Infections were performed based on the previously described procedure\u003csup\u003e32\u003c/sup\u003e. The required volume ZIKV stock was added to RT Maturation Medium or Maturation Medium complemented with PAI-1 as previously described (ZIKV infection in cell lines), vortexed to obtain a homogeneous suspension and distributed on hNOs to obtain a final MOI of 0.1 TCID\u003csub\u003e50\u003c/sub\u003e/cell and 2 ml per well. Mock-spiked Maturation Medium was prepared and distributed analogously. Organoids were incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in the virus-spiked Maturation Medium for 2 hours, constantly shaking at 65 rpm. The medium was subsequently completely removed, the cultures washed three times with 3 ml of RT sterile Dulbecco's Phosphate Buffered Saline (DPBS, Gibco) and 3.5 ml of virus-free RT Maturation Medium were added to each well. Organoids were placed back on the orbital shaker, shaking at 65 rpm at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Supernatant and tissue samples were taken at regular intervals, processed directly or stored at -80\u0026deg;C, and used for subsequent analysis as described below. Biosafety level 2 procedures were strictly adhered to during each protocol step.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis of human neural organoids\u003c/h2\u003e \u003cp\u003eFor quality control of H1-cells on day 0 of organoid generation, stemness analysis through double staining with markers SSEA-5 (STEMCELL Technologies, #60063AZ, 1:50) and TRA-1-60 (STEMCELL Technologies, #60064PE, 1:50) was performed, as previously described\u003csup\u003e32\u003c/sup\u003e. For this, antibodies were diluted in BD CellWASH (BD Switzerland). A sample of H1-cells were pelleted by centrifugation at 280 g for 8 minutes at 4\u0026deg;C. The supernatant was aspirated, the cells resuspended by vortexing in the previously prepared antibody solution and incubated for 15 minutes on ice, protected from light. Following incubation, cells were washed by addition of 1 ml of BD CellWASH and spun down once more at 280 g, 8 minutes at 4\u0026deg;C. The obtained supernatant was aspirated, and the cells resuspended in 100 \u0026micro;l of BD CellWASH by vortexing. Flow cytometry acquisition was performed on a FACS Canto II (BD Switzerland) in combination with the DIVA software and results analyzed using FlowJo (TreeStar). The minimum acceptable cut-off value used was 80% double-positive cells.\u003c/p\u003e \u003cp\u003ePrior to virus infection and for accurate MOI calculation, cell numbers of single, representative organoids were analyzed and calculated through flow cytometry analysis. For this purpose, single organoids were dissociated by 10-minute incubation at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e in 500 \u0026micro;l of accutase. A single-cell suspension was obtained through repeated pipetting. 100 \u0026micro;l of the obtained suspension were transferred to a flow cytometry tube (Falcon) and washed by adding 2.5 ml of DPBS before pelleting the cells by centrifugation at 350 g for 10 minutes at 4\u0026deg;C. Supernatant was subsequently carefully removed through aspiration and cells resuspended in 300 \u0026micro;l of CellWASH (BD Switzerland). CountBright Absolute Counting Beads for flow cytometry (Invitrogen) were allowed to reach RT, vortexed for 30 seconds and 50 \u0026micro;l of the bead solution was rapidly transferred to the resuspended cells. The mixture was thoroughly vortexed before proceeding to flow cytometry acquisition and data analysis as previously described. Cell counts were calculated according to CountBright Absolute Counting Beads for flow cytometry user manual.\u003c/p\u003e \u003cp\u003eFlow cytometry analysis was additionally used to quantify the percentage of infected cells in organoids. As previously described, hNOs were dissociated by incubating single organoids in 500 \u0026micro;l of accutase for 10 minutes at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. Cultures were dissociated into single cells by repeated pipetting and accutase diluted by addition of 2 ml of DPBS into each tube. A volume of 0.5 ml of cell solution, corresponding to approximately 10\u003csup\u003e6\u003c/sup\u003e cells, was transferred to a new flow cytometry tube and transferred to biocontainment centrifuge buckets, for pelleting at 350 x g for 7 minutes at 4\u0026deg;C. To allow discrimination between live and dead cells, LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen, L34966) was diluted 1:1000 in DPBS and vortexed. The supernatant of each cell pellet was removed through aspiration and cells were resuspended in 200 \u0026micro;l of the previously prepared LIVE/DEAD Fixable Aqua Dead Cell Stain solution. Cells were resuspended by vortexing and incubated 30 minutes on ice, protected from light. Following incubation, cells were washed by adding 1 ml of DPBS to each tube and subsequently pelleted by centrifugation in biocontainment buckets at 380 g for 5 minutes at 4\u0026deg;C. The supernatant was removed from each cell pellet through aspiration and the cells fixed using 200 \u0026micro;l of undiluted Fixation/Permeabilization Concentrate (Invitrogen) per tube. Cells were resuspended by vortexing and left to incubate on ice for 30 minutes. In the meantime, 10x Perm/Wash Buffer (BD Switzerland) was diluted 1:10 in distilled water. Following incubation, cells were washed by adding 1 ml of the previously prepared Perm/Wash solution to each tube. Cells were pelleted by centrifugation at 380 g for 5 minutes at 4\u0026deg;C. Following primary antibody solutions were prepared by diluting antibodies in Perm/Wash solution at given concentrations: home-made orthoflavivirus envelope protein antibody (4G2, 1:10) and cleaved caspase-3 (Cell Signaling Technology, #9661, 1:1000). After centrifugation, supernatants were removed by aspiration and cells were resuspended in 100 \u0026micro;l of primary antibody solution, followed by vortexing and a 15-minute incubation on ice. One ml of perm-wash solution was subsequently added to each tube and cells spun down by centrifuging at 380 g for 5 minutes at 4\u0026deg;C. Secondary antibody solutions were prepared as previously described, using the following antibodies at given concentrations: anti-mouse AF488 (Invitrogen, A21131, 1:500) and anti-rabbit AF647 (Invitrogen, A21245, 1:500). Supernatants were subsequently aspirated, and cells resuspended in 100 \u0026micro;l of secondary antibody solution, vortexed and incubated for 15 minutes on ice. Following secondary antibody staining, cells were washed once more with 1 ml of Perm/Wash solution and pelleted at 380 g for 5 minutes at 4\u0026deg;C. Supernatants were once more removed and the cells resuspended in 80 \u0026micro;l of Perm/Wash by vortexing. Unstained and live/dead staining controls were prepared for each analysis. To allow clear live and dead cell separation, live/dead control-cells were heated at 75\u0026deg;C for 5 minutes shaking at 300 rpm, prior to staining. Biosafety level 2 procedures were strictly adhered to during each protocol step.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eHuman neural organoids staining\u003c/h2\u003e \u003cp\u003eWhole ZIKV-challenged and mock-treated organoids were collected at regular intervals during ZIKV-infection experiments for cryopreservation and subsequent immunofluorescent analysis. An adapted version of the procedure for \u0026ldquo;Cryogenic Tissue Processing and Section Immunofluorescence of Cerebral Organoids\u0026rdquo; published by STEMCELL Technologies was followed, as previously described\u003csup\u003e32\u003c/sup\u003e. Briefly, harvested organoids were washed twice with sterile DPBS, pre-fixed for 10 minutes in 2% formalin solution and subsequently preserved in 4% formalin solution (Sigma-Aldrich). For cryoprotection, fixed organoids were equilibrated in 30% sucrose (Sigma-Aldrich) in DPBS solution overnight at 4\u0026deg;C. Complete equilibration was confirmed by organoids sinking to the tubes\u0026rsquo; bottom. The tissue was then transferred to a pre-heated 7.5% gelatin (Sigma-Aldrich), 10% sucrose in DMEM/F-12 (Gibco) solution and left to incubate at 37\u0026deg;C for 1 hour, to allow organoid encapsulation. Finally, hNOs were transferred together with a sufficient amount of warm gelatin-solution into a Cryomold (Biosystems Switzerland AG, Muttenz, Switzerland), and after initial polymerization at RT deep-frozen by gently placing the Cryomold onto the surface of a previously prepared dry-ice and 100% ethanol slurry. Frozen Cryomolds were stored at -80\u0026deg;C until further processing. For immunofluorescence staining, frozen organoid-bearing gelatin blocks were processed into 18 \u0026micro;m thick slices using a Leica CM1950 cryostat. Sections were collected on SuperFrost Plus Adhesion slides (Epredia, Dreieich, Germany) and stored at -20\u0026deg;C until the day of staining. For further processing, slides were left to thaw at RT for 30 minutes, before being transferred to a pre-warmed 0.1% Tween-20 (Sigma-Aldrich) in DPBS solution (0.1% T-DPBS) and incubated at 37\u0026deg;C for 10 minutes to allow gelatine removal. Areas of interested were subsequently encircled with ReadyProbes Hyrophobic Barrier Pap Pen (Thermo Fisher Scientific) and unspecific staining prevented by 1 hour RT incubation with a 5% donkey serum (Abcam, Paris, France) or 5% goat serum (Sigma-Aldrich) in 0.1% T-DPBS solution. Primary antibodies were diluted in 0.1% T-DPBS supplemented with 5% bovine serum albumin (Sigma-Aldrich) at pre-established concentrations as follows: SOX2 (Invitrogen, 14-9811-82, 1:200) and home-made orthoflavivirus envelope protein antibody (4G2, 1:3). Blocking solutions were removed by gently tapping slides on clean tissue, 100 \u0026micro;l of primary antibody solution were pipetted on top of each area of interest and left to incubate on tissue slices overnight at 4\u0026deg;C in a humidified chamber. On the following day, the solution was removed and the sections washed in Coplin jars filled with 0.1% T-DPBS three times for 5 minutes each. The following secondary antibodies were diluted in 0.1% T-DPBS at given concentrations: anti-mouse AF488 (Invitrogen, A21131, 1:200), anti-rat AF647 (Invitrogen, A21247, 1:500). For nuclear staining DAPI (Sigma-Aldrich, 1:1000) was added to the solution. The obtained antibody mixtures were pipetted on the selected areas and left to incubate for 2 hours at RT. Slides were subsequently washed three more times as previously described and mounted in EMS Shield Mount with DABCO (Electron Microscopy Sciences). Confocal imaging was conducted with a Carl Zeiss LSM710 confocal microscope at the Microscopy imaging center (MIC) of the University of Bern, Switzerland. Images were analyzed using Imaris (Oxford Instruments).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe GraphPad Prism 5 software (GraphPad software, La Jolla, CA USA) and R software\u003csup\u003e60\u003c/sup\u003e were used for statistical analysis. Statistical significance: * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Musso, D., Ko, A. I. \u0026amp; Baud, D. 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(2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Zika virus, orthoflavivirus, virus entry, pregnancy, microcephaly, congenital Zika syndrome, ligand-based receptor capture, LRC-TriCEPS, neural organoids, cerebral organoids, plasminogen activator inhibitor-1","lastPublishedDoi":"10.21203/rs.3.rs-5866223/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5866223/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eZika virus (ZIKV) infection can lead to severe congenital outcomes, yet the mechanisms governing its entry into host cells remain understood. ZIKV is a flavivirus known to exploit multiple cellular receptors and cofactors, particularly in neural cells, where infection can result in congenital Zika syndrome (CZS). Here we show that plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor involved in hemostasis, directly interacts with ZIKV particles and critically enhances viral replication in diverse cell types, including human neural progenitor cells and three-dimensional neural organoids. Our findings reveal that PAI-1 may contribute to ZIKV infection through distinct or complementary pathways, underscoring the virus\u0026rsquo;s versatile entry mechanisms. Inhibition of PAI-1 via tiplaxtinin (TPX) dramatically reduces viral load and impedes infectious particle release, demonstrating a dose-dependent effect that is especially potent in neural models relevant to CZS. These results highlight PAI-1 as an essential mediator of ZIKV pathogenesis and suggest that targeting PAI-1 function could represent a novel therapeutic avenue. Given the risk of future ZIKV outbreaks and the devastating impact of CZS, interventions aimed at PAI-1 may hold promise for reducing the global burden of ZIKV infection.\u003c/p\u003e","manuscriptTitle":"Plasminogen activator inhibitor-1 regulates Zika virus infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-20 07:35:39","doi":"10.21203/rs.3.rs-5866223/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f2da6764-f8ee-4321-9a42-032947c103ca","owner":[],"postedDate":"March 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":44281471,"name":"Health sciences/Diseases/Infectious diseases/Viral infection"},{"id":44281472,"name":"Health sciences/Pathogenesis/Infection"}],"tags":[],"updatedAt":"2025-03-20T07:35:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-20 07:35:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5866223","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5866223","identity":"rs-5866223","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}