The activation of the adaptor protein STING depends on its interactions with the phospholipid PI4P.

Activation of the endoplasmic reticulum (ER)-resident adaptor protein STING, a component of a cytosolic DNA-sensing pathway, induces the transcription of genes encoding type I interferons (IFNs) and other proinflammatory factors. Because STING is activated at the Golgi apparatus, control of the localization and activation of STING is important in stimulating antiviral and antitumor immune responses. Through a genome-wide CRISPR interference screen, we found that STING activation required the Golgi-resident protein ACBD3, which promotes the generation of phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network, as well as other PI4P-associated proteins. Appropriate localization and activation of STING at the Golgi apparatus required ACBD3 and the PI4P-generating kinase PI4KB. In contrast, STING activation was enhanced when the lipid-shuttling protein OSBP, which removes PI4P from the Golgi apparatus, was inhibited by the US Food and Drug Administration-approved antifungal itraconazole. The increase in the abundance of STING-activating phospholipids at the trans-Golgi network resulted in the increased production of IFN-ß and other cytokines in THP-1 cells. Furthermore, a mutant STING that could not bind to PI4P failed to traffic from the ER to the Golgi apparatus in response to a STING agonist, whereas forced relocalization of STING to PI4P-enriched areas elicited STING activation in the absence of stimulation with a STING agonist. Thus, PI4P is critical for STING activation, and manipulating PI4P abundance may therapeutically modulate STING-dependent immune responses.

MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via protein kinase C.

Glycosylated mucin proteins contribute to the essential barrier function of the intestinal epithelium. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in human intestinal epithelial monolayers, MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TEER buildup in ΔMUC13 cells could be prevented by addition of MLCK, ROCK or protein kinase C (PKC) inhibitors. The levels of TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not affect TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC. The responsible PKC member might be PKCδ (or PRKCD) based on elevated protein levels in the absence of full-length MUC13. Our results demonstrate for the first time that a mucin protein can negatively regulate TJ function and stimulate intestinal barrier permeability.

Glycosylated extracellular mucin domains protect against SARS-CoV-2 infection at the respiratory surface.

Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of terminal mucin glycans sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry.

The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy.

Naked viruses can escape host cells before the induction of lysis via release in extracellular vesicles (EVs). These nanosized EVs cloak the secreted virus particles in a host-derived membrane, which alters virus-host interactions that affect infection efficiency and antiviral immunity. Currently, little is known about the viral and host factors regulating this form of virus release. Here, we assessed the role of the encephalomyocarditis virus (EMCV) Leader protein, a 'viral security protein' that subverts the host antiviral response. EV release upon infection with wildtype virus or a Leader-deficient mutant was characterized at the single particle level using high-resolution flow cytometry. Inactivation of the Leader abolished EV induction during infection and strongly reduced EV-enclosed virus release. We demonstrate that the Leader promotes the release of virions within EVs by stimulating a secretory arm of autophagy. This newly discovered role of the EMCV Leader adds to the variety of mechanisms via which this protein affects virus-host interactions. Moreover, these data provide first evidence for a crucial role of a non-structural viral protein in the non-lytic release of picornaviruses via packaging in EVs.

The power of imaging to understand extracellular vesicle biology in vivo.

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.

Retinyl esters form lipid droplets independently of triacylglycerol and seipin.

Lipid droplets store neutral lipids, primarily triacylglycerol and steryl esters. Seipin plays a role in lipid droplet biogenesis and is thought to determine the site of lipid droplet biogenesis and the size of newly formed lipid droplets. Here we show a seipin-independent pathway of lipid droplet biogenesis. In silico and in vitro experiments reveal that retinyl esters have the intrinsic propensity to sequester and nucleate in lipid bilayers. Production of retinyl esters in mammalian and yeast cells that do not normally produce retinyl esters causes the formation of lipid droplets, even in a yeast strain that produces only retinyl esters and no other neutral lipids. Seipin does not determine the size or biogenesis site of lipid droplets composed of only retinyl esters or steryl esters. These findings indicate that the role of seipin in lipid droplet biogenesis depends on the type of neutral lipid stored in forming droplets.

Tissue Microarrays to Visualize Influenza D Attachment to Host Receptors in the Respiratory Tract of Farm Animals.

The trimeric hemagglutinin-esterase fusion protein (HEF) of influenza D virus (IDV) binds 9-O-acetylated sialic acid receptors, which are expressed in various host species. While cattle are the main reservoir for IDV, the viral genome has also been detected in domestic pigs. In addition, antibodies against IDV have been detected in other farm animals such as sheep, goats, and horses, and even in farmers working with IDV positive animals. Viruses belonging to various IDV clades circulate, but little is known about their differences in host and tissue tropism. Here we used recombinantly produced HEF proteins (HEF S57A) from the major clades D/Oklahoma (D/OK) and D/Oklahoma/660 (D/660) to study their host and tissue tropism and receptor interactions. To this end, we developed tissue microarrays (TMA) composed of respiratory tissues from various farm animals including cattle, domestic pigs, sheep, goats, and horses. Protein histochemical staining of farm animal respiratory tissue-microarrays with HEF proteins showed that cattle have receptors present over the entire respiratory tract while receptors are only present in the nasal and pharyngeal epithelium of pigs, sheep, goats, and horses. No differences in tropism for tissues and animals were observed between clades, while hemagglutination assays showed that D/OK has a 2-fold higher binding affinity than D/660 for receptors on red blood cells. The removal of O-acetylation from receptors via saponification treatment confirmed that receptor-binding of both clades was dependent on O-acetylated sialic acids.

The Transmembrane Mucin MUC1 Facilitates ß1-Integrin-Mediated Bacterial Invasion.

At the intestinal host-microbe interface, the transmembrane mucin MUC1 can function as a physical barrier as well as a receptor for bacteria. MUC1 also influences epithelial cell morphology and receptor function. Various bacterial pathogens can exploit integrins to infect eukaryotic cells. It is yet unclear whether MUC1 influences the interaction of bacteria with integrins. We used Escherichia coli expressing the invasin (inv) protein of Yersinia pseudotuberculosis (E. coli inv) to assess the effects of MUC1 on ß1 integrin (ITGB1)-mediated bacterial invasion. Our results show that expression of full-length MUC1 does not yield a physical barrier but slightly enhances E. coli inv uptake. Enzymatic removal of the MUC1 extracellular domain (ED) using a secreted protease of C1 esterase inhibitor (StcE) of pathogenic Escherichia coli had no additional effect on E. coli inv invasion. In contrast, expression of a truncated MUC1 that lacks the cytoplasmic tail (CT) reduced bacterial entry substantially. Substitution of tyrosine residues in the MUC1 CT also reduced bacterial uptake, while deletion of the C-terminal half of the cytoplasmic tail only had a minor effect, pointing to a regulatory role of tyrosine phosphorylation and the N-terminal region of the MUC1 CT in integrin-mediated uptake process. Unexpectedly, StcE removal of the ED in MUC1-ΔCT cells reversed the block in bacterial invasion. Together, these findings indicate that MUC1 can facilitate ß1-integrin-mediated bacterial invasion by a concerted action of the large glycosylated extracellular domain and the membrane-juxtaposed cytoplasmic tail region.IMPORTANCE Bacteria can exploit membrane receptor integrins for cellular invasion, either by direct binding of bacterial adhesins or utilizing extracellular matrix components. MUC1 is a large transmembrane glycoprotein expressed by most epithelial cells that can have direct defensive or receptor functions at the host-microbe interface and is involved in facilitating integrin clustering. We investigated the role of epithelial MUC1 on ß1 integrin-mediated bacterial invasion. We discovered that MUC1 does not act as a barrier but facilitates bacterial entry through ß1 integrins. This process involves a concerted action of the MUC1 O-glycosylated extracellular domain and cytoplasmic tail. Our findings add a new dimension to the complexity of bacterial invasion mechanisms and provide novel insights into the distinct functions of MUC1 domains at the host-microbe interface.

Excessive E2F Transcription in Single Cancer Cells Precludes Transient Cell-Cycle Exit after DNA Damage.

E2F transcription factors control the expression of cell-cycle genes. Cancers often demonstrate enhanced E2F target gene expression, which can be explained by increased percentages of replicating cells. However, we demonstrate in human cancer biopsy specimens that individual neoplastic cells display abnormally high levels of E2F-dependent transcription. To mimic this situation, we delete the atypical E2F repressors (E2F7/8) or overexpress the E2F3 activator in untransformed cells. Cells with elevated E2F activity during S/G2 phase fail to exit the cell cycle after DNA damage and undergo mitosis. In contrast, wild-type cells complete S phase and then exit the cell cycle by activating the APC/CCdh1 via repression of the E2F target Emi1. Many arrested wild-type cells eventually inactivate APC/CCdh1 to execute a second round of DNA replication and mitosis, thereby becoming tetraploid. Cells with elevated E2F transcription fail to exit the cell cycle after DNA damage, which potentially causes genomic instability, promotes malignant progression, and reduces drug sensitivity.

Dendritic cells release exosomes together with phagocytosed pathogen; potential implications for the role of exosomes in antigen presentation.

Dendritic cells (DC) have the unique capacity to activate naïve T cells by presenting T cell receptor specific peptides from exogenously acquired antigens bound to Major Histocompatibility Complex (MHC) molecules. MHC molecules are displayed on the DC plasma membrane as well as on extracellular vesicles (EV) that are released by DC, and both have antigen-presenting capacities. However, the physiological role of antigen presentation by EV is still unclear. We here demonstrate that the release of small EV by activated DC is strongly stimulated by phagocytic events. We show that, concomitant with the enhanced release of EV, a significant proportion of phagocytosed bacteria was expulsed back into the medium. High-resolution fluorescence microscopic images revealed that bacteria in phagosomes were surrounded by EV marker-proteins. Moreover, expulsed bacteria were often found associated with clustered HLA II and CD63. Together, these observations suggest that exosomes may be formed by the inward budding into phagosomes, whereupon they are secreted together with the phagosomal content. These findings may have important implications for selective loading of peptides derived from phagocytosed pathogens onto exosome associated HLA molecules, and have important implications for vaccine design.

Bystander T-Cells Support Clonal T-Cell Activation by Controlling the Release of Dendritic Cell-Derived Immune-Stimulatory Extracellular Vesicles.

Extracellular vesicles (EV) that are released by immune cells are studied intensively for their functions in immune regulation and are scrutinized for their potential in human immunotherapy, for example against cancer. In our search for signals that stimulate the release of functional EV by dendritic cells we observed that LPS-activated human monocyte-derived dendritic cells (moDC) changed their morphological characteristics upon contact with non-cognate activated bystander T-cells, while non-activated bystander T-cells had no effect. Exposure to activated bystander T-cells also stimulated the release of EV-associated proteins by moDC, particularly CD63, and ICAM-1, although the extent of stimulation varied between individual donors. Stimulation of moDC with activated bystander T-cells also increased the release of EV-associated miR155, which is a known central modulator of T-cell responses. Functionally, we observed that EV from moDC that were licensed by activated bystander T-cells exhibited a capacity for antigen-specific T-cell activation. Taken together, these results suggest that non-cognatei interactions between DC and bystander T-cells modulates third party antigen-specific T-cell responses via EV.

MUC1 is a receptor for the Salmonella SiiE adhesin that enables apical invasion into enterocytes.

The cellular invasion machinery of the enteric pathogen Salmonella consists of a type III secretion system (T3SS) with injectable virulence factors that induce uptake by macropinocytosis. Salmonella invasion at the apical surface of intestinal epithelial cells is inefficient, presumably because of a glycosylated barrier formed by transmembrane mucins that prevents T3SS contact with host cells. We observed that Salmonella is capable of apical invasion of intestinal epithelial cells that express the transmembrane mucin MUC1. Knockout of MUC1 in HT29-MTX cells or removal of MUC1 sialic acids by neuraminidase treatment reduced Salmonella apical invasion but did not affect lateral invasion that is not hampered by a defensive barrier. A Salmonella deletion strain lacking the SiiE giant adhesin was unable to invade intestinal epithelial cells through MUC1. SiiE-positive Salmonella closely associated with the MUC1 layer at the apical surface, but invaded Salmonella were negative for the adhesin. Our findings uncover that the transmembrane mucin MUC1 is required for Salmonella SiiE-mediated entry of enterocytes via the apical route.

Inhibition and Eradication of Pseudomonas aeruginosa Biofilms by Host Defence Peptides.

P. aeruginosa is a notorious biofilm producer that causes a wide variety of acute and chronic infections. In this study the in vitro anti-biofilm activity of 13 Host Defence Peptides from different species was tested against P. aeruginosa biofilms. Most HDPs were able to prevent biofilm attachment, due to their antimicrobial effect on planktonic bacteria in the starting inoculum. Activity of HDPs against pre-formed biofilms was also observed, although mainly at short incubation times. Several HDPs were able to kill bacteria in the biofilm (colony counting of biofilm associated bacteria) but only CRAMP eradicated the whole biofilm (crystal violet staining). These results were quantitatively confirmed by confocal microscopy studies using a live/dead stain of the biofilms. Furthermore, for chicken CATH-2 (one of the more potent HDPs) it was shown that the peptide could indeed penetrate the biofilm structures and kill bacteria within the biofilm. These studies highlight the potency but also the limitations of HDPs as new potential anti-biofilm agents.

Lysosome-mediated degradation of a distinct pool of lipid droplets during hepatic stellate cell activation.

Activation of hepatic stellate cells (HSCs) is a critical step in the development of liver fibrosis. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerols (TAGs), cholesteryl esters, and retinyl esters (REs). We previously provided evidence for the presence of two distinct LD pools, a preexisting and a dynamic LD pool. Here we investigate the mechanisms of neutral lipid metabolism in the preexisting LD pool. To investigate the involvement of lysosomal degradation of neutral lipids, we studied the effect of lalistat, a specific lysosomal acid lipase (LAL/Lipa) inhibitor on LD degradation in HSCs during activation in vitro The LAL inhibitor increased the levels of TAG, cholesteryl ester, and RE in both rat and mouse HSCs. Lalistat was less potent in inhibiting the degradation of newly synthesized TAG species as compared with a more general lipase inhibitor orlistat. Lalistat also induced the presence of RE-containing LDs in an acidic compartment. However, targeted deletion of the Lipa gene in mice decreased the liver levels of RE, most likely as the result of a gradual disappearance of HSCs in livers of Lipa-/- mice. Lalistat partially inhibited the induction of activation marker α-smooth muscle actin (α-SMA) in rat and mouse HSCs. Our data suggest that LAL/Lipa is involved in the degradation of a specific preexisting pool of LDs and that inhibition of this pathway attenuates HSC activation.

Stearoyl-CoA desaturase activity in bovine cumulus cells protects the oocyte against saturated fatty acid stress.

Metabolic rich and poor conditions are both characterized by elevated free fatty acid levels and have been associated with impaired female fertility. In particular, saturated free fatty acids have a dose-dependent negative impact on oocyte developmental competence, while monounsaturated free fatty acids appear less harmful. Cumulus cells seem to protect the oocyte against free fatty acids, and the aim of this study was to determine the mechanism behind this protection In particular, the role of the enzyme stearoyl-CoA desaturase (SCD) that converts saturated into monounsaturated fatty acids was investigated. SCD gene and protein were abundantly expressed in cumulus cells, but expression was low in oocytes. The level of SCD protein expression in cumulus cells did not change when COCs were exposed to saturated stearic acid during maturation. SCD inhibition in the presence of stearic acid significantly reduced the developmental competence of oocytes and increased the incidence of apoptosis in cumulus cells. The esterified oleic/stearic acid ratio of the neutral lipid fraction in cumulus cells decreased in the presence of SCD inhibitors when COCs were exposed to saturated free fatty acids during maturation, indicating the SCD-specific conversion of saturated fatty acids under noninhibiting conditions. The observation that cumulus cells can desaturate the potentially toxic stearic acid into oleic acid via SCD activity provides a mechanistic insight into how the cumulus cells protect the oocyte against toxicity by saturated fatty acid.

Characterization of bovine embryos cultured under conditions appropriate for sustaining human naïve pluripotency.

In mammalian preimplantation development, pluripotent cells are set aside from cells that contribute to extra-embryonic tissues. Although the pluripotent cell population of mouse and human embryos can be cultured as embryonic stem cells, little is known about the pathways involved in formation of a bovine pluripotent cell population, nor how to maintain these cells in vitro. The objective of this study was to determine the transcriptomic profile related to bovine pluripotency. Therefore, in vitro derived embryos were cultured in various culture media that recently have been reported capable of maintaining the naïve pluripotent state of human embryonic cells. Gene expression profiles of embryos cultured in these media were compared using microarray analysis and quantitative RT-PCR. Compared to standard culture conditions, embryo culture in 'naïve' media reduced mRNA expression levels of the key pluripotency markers NANOG and POU5F1. A relatively high percentage of genes with differential expression levels were located on the X-chromosome. In addition, reduced XIST expression was detected in embryos cultured in naïve media and female embryos contained fewer cells with H3K27me3 foci, indicating a delay in X-chromosome inactivation. Whole embryos cultured in one of the media, 5iLA, could be maintained until 23 days post fertilization. Together these data indicate that 'naïve' conditions do not lead to altered expression of known genes involved in pluripotency. Interestingly, X-chromosome inactivation and development of bovine embryos were dependent on the culture conditions.

Efficient Payload Delivery by a Bispecific Antibody-Drug Conjugate Targeting HER2 and CD63.

Antibody-drug conjugates (ADC) are designed to be stable in circulation and to release potent cytotoxic drugs intracellularly following antigen-specific binding, uptake, and degradation in tumor cells. Efficient internalization and routing to lysosomes where proteolysis can take place is therefore essential. For many cell surface proteins and carbohydrate structures on tumor cells, however, the magnitude of these processes is insufficient to allow for an effective ADC approach. We hypothesized that we could overcome this limitation by enhancing lysosomal ADC delivery via a bispecific antibody (bsAb) approach, in which one binding domain would provide tumor specificity, whereas the other binding domain would facilitate targeting to the lysosomal compartment. We therefore designed a bsAb in which one binding arm specifically targeted CD63, a protein that is described to shuttle between the plasma membrane and intracellular compartments, and combined it in a bsAb with a HER2 binding arm, which was selected as model antigen for tumor-specific binding. The resulting bsHER2xCD63his demonstrated strong binding, internalization and lysosomal accumulation in HER2-positive tumor cells, and minimal internalization into HER2-negative cells. By conjugating bsHER2xCD63his to the microtubule-disrupting agent duostatin-3, we were able to demonstrate potent cytotoxicity of bsHER2xCD63his-ADC against HER2-positive tumors, which was not observed with monovalent HER2- and CD63-specific ADCs. Our data demonstrate, for the first time, that intracellular trafficking of ADCs can be improved using a bsAb approach that targets the lysosomal membrane protein CD63 and provide a rationale for the development of novel bsADCs that combine tumor-specific targeting with targeting of rapidly internalizing antigens. Mol Cancer Ther; 15(11); 2688-97. ©2016 AACR.

Disease Modeling and Gene Therapy of Copper Storage Disease in Canine Hepatic Organoids.

The recent development of 3D-liver stem cell cultures (hepatic organoids) opens up new avenues for gene and/or stem cell therapy to treat liver disease. To test safety and efficacy, a relevant large animal model is essential but not yet established. Because of its shared pathologies and disease pathways, the dog is considered the best model for human liver disease. Here we report the establishment of a long-term canine hepatic organoid culture allowing undifferentiated expansion of progenitor cells that can be differentiated toward functional hepatocytes. We show that cultures can be initiated from fresh and frozen liver tissues using Tru-Cut or fine-needle biopsies. The use of Wnt agonists proved important for canine organoid proliferation and inhibition of differentiation. Finally, we demonstrate that successful gene supplementation in hepatic organoids of COMMD1-deficient dogs restores function and can be an effective means to cure copper storage disease.

High turnover of tissue factor enables efficient intracellular delivery of antibody-drug conjugates.

Antibody-drug conjugates (ADC) are emerging as powerful cancer treatments that combine antibody-mediated tumor targeting with the potent cytotoxic activity of toxins. We recently reported the development of a novel ADC that delivers the cytotoxic payload monomethyl auristatin E (MMAE) to tumor cells expressing tissue factor (TF). By carefully selecting a TF-specific antibody that interferes with TF:FVIIa-dependent intracellular signaling, but not with the procoagulant activity of TF, an ADC was developed (TF-011-MMAE/HuMax-TF-ADC) that efficiently kills tumor cells, with an acceptable toxicology profile. To gain more insight in the efficacy of TF-directed ADC treatment, we compared the internalization characteristics and intracellular routing of TF with the EGFR and HER2. Both in absence and presence of antibody, TF demonstrated more efficient internalization, lysosomal targeting, and degradation than EGFR and HER2. By conjugating TF, EGFR, and HER2-specific antibodies with duostatin-3, a toxin that induces potent cytotoxicity upon antibody-mediated internalization but lacks the ability to induce bystander killing, we were able to compare cytotoxicity of ADCs with different tumor specificities. TF-ADC demonstrated effective killing against tumor cell lines with variable levels of target expression. In xenograft models, TF-ADC was relatively potent in reducing tumor growth compared with EGFR- and HER2-ADCs. We hypothesize that the constant turnover of TF on tumor cells makes this protein specifically suitable for an ADC approach.

Itraconazole inhibits enterovirus replication by targeting the oxysterol-binding protein.

Itraconazole (ITZ) is a well-known antifungal agent that also has anticancer activity. In this study, we identify ITZ as a broad-spectrum inhibitor of enteroviruses (e.g., poliovirus, coxsackievirus, enterovirus-71, rhinovirus). We demonstrate that ITZ inhibits viral RNA replication by targeting oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4). Consistently, OSW-1, a specific OSBP/ORP4 antagonist, also inhibits enterovirus replication. Knockdown of OSBP inhibits virus replication, whereas overexpression of OSBP or ORP4 counteracts the antiviral effects of ITZ and OSW-1. ITZ binds OSBP and inhibits its function, i.e., shuttling of cholesterol and phosphatidylinositol-4-phosphate between membranes, thereby likely perturbing the virus-induced membrane alterations essential for viral replication organelle formation. ITZ also inhibits hepatitis C virus replication, which also relies on OSBP. Together, these data implicate OSBP/ORP4 as molecular targets of ITZ and point to an essential role of OSBP/ORP4-mediated lipid exchange in virus replication that can be targeted by antiviral drugs.