The Rab5-Rab11 Endosomal Pathway is Required for BDNF-Induced CREB Transcriptional Regulation in Hippocampal Neurons.

Brain-derived neurotrophic factor (BDNF) is a key regulator of the morphology and connectivity of central neurons. We have previously shown that BDNF/TrkB signaling regulates the activity and mobility of the GTPases Rab5 and Rab11, which in turn determine the postendocytic sorting of signaling TrkB receptors. Moreover, decreased Rab5 or Rab11 activity inhibits BDNF-induced dendritic branching. Whether Rab5 or Rab11 activity is important for local events only or for regulating nuclear signaling and gene expression is unknown. Here, we investigated, in rat hippocampal neuronal cultures derived from embryos of unknown sex, whether BDNF-induced signaling cascades are altered when early and recycling endosomes are disrupted by the expression of dominant-negative mutants of Rab5 and Rab11. The activity of both Rab5 and Rab11 was required for sustained activity of Erk1/2 and nuclear CREB phosphorylation, and increased transcription of a BDNF-dependent program of gene expression containing CRE binding sites, which includes activity-regulated genes such as Arc, Dusp1, c-fos, Egr1, and Egr2, and growth and survival genes such as Atf3 and Gem Based on our results, we propose that early and recycling endosomes provide a platform for the integration of neurotrophic signaling from the plasma membrane to the nucleus in neurons, and that this mechanism is likely to regulate neuronal plasticity and survival.SIGNIFICANCE STATEMENT BDNF is a neurotrophic factor that regulates plastic changes in the brain, including dendritic growth. The cellular and molecular mechanisms underlying this process are not completely understood. Our results uncover the cellular requirements that central neurons possess to integrate the plasma membrane into nuclear signaling in neurons. Our results indicate that the endosomal pathway is required for the signaling cascade initiated by BDNF and its receptors at the plasma membrane to modulate BDNF-dependent gene expression and neuronal dendritic growth mediated by the CREB transcription factor. CREB is a key transcription factor regulating circuit development and learning and memory.

P38α MAPK Signaling-A Robust Therapeutic Target for Rab5-Mediated Neurodegenerative Disease.

Multifactorial pathologies, involving one or more aggregated protein(s) and neuroinflammation are common in major neurodegenerative diseases, such as Alzheimer's disease and dementia with Lewy bodies. This complexity of multiple pathogenic drivers is one potential explanation for the lack of success or, at best, the partial therapeutic effects, respectively, with approaches that have targeted one specific driver, e.g., amyloid-beta, in Alzheimer's disease. Since the endosome-associated protein Rab5 appears to be a convergence point for many, if not all the most prominent pathogenic drivers, it has emerged as a major therapeutic target for neurodegenerative disease. Further, since the alpha isoform of p38 mitogen-activated protein kinase (p38α) is a major regulator of Rab5 activity and its effectors, a biology that is distinct from the classical nuclear targets of p38 signaling, brain-penetrant selective p38α kinase inhibitors provide the opportunity for significant therapeutic advances in neurogenerative disease through normalizing dysregulated Rab5 activity. In this review, we provide a brief summary of the role of Rab5 in the cell and its association with neurodegenerative disease pathogenesis. We then discuss the connection between Rab5 and p38α and summarize the evidence that through modulating Rab5 activity there are therapeutic opportunities in neurodegenerative diseases for p38α kinase inhibitors.

Role of Rab GTPases in HSV-1 infection: Molecular understanding of viral maturation and egress.

Most enveloped viruses exploit complex cellular pathways for assembly and egress from the host cell, and the large DNA virus Herpes simplex virus 1 (HSV-1) makes no exception, hijacking several cellular transport pathways for its glycoprotein trafficking and maturation, as well as for viral morphogenesis and egress according to the envelopment, de-envelopment and re-envelopment model. Importantly Rab GTPases, widely distributed master regulators of intracellular membrane trafficking pathways, have recently being tightly implicated in such process. Indeed, siRNA-mediated genetic ablation of specific Rab proteins differently affected HSV-1 production, suggesting a complex role of different Rab proteins in HSV-1 life cycle. In this review, we discuss how different Rabs can regulate HSV-1 assembly/egress and the potential therapeutic applications of such findings for the management of HSV-1 infections.

Rab5 Isoforms Specifically Regulate Different Modes of Endocytosis in Leishmania.

Differential functions of Rab5 isoforms in endocytosis are not well characterized. Here, we cloned, expressed, and characterized Rab5a and Rab5b from Leishmania and found that both of them are localized in the early endosome. To understand the role of LdRab5 isoforms in different modes of endocytosis in Leishmania, we generated transgenic parasites overexpressing LdRab5a, LdRab5b, or their dominant-positive (LdRab5a:Q93L and LdRab5b:Q80L) or dominant-negative mutants (LdRab5a:N146I and LdRab5b:N133I). Using LdRab5a or its mutants overexpressing parasites, we found that LdRab5a specifically regulates the fluid-phase endocytosis of horseradish peroxidase and also specifically induced the transport of dextran-Texas Red to the lysosomes. In contrast, cells overexpressing LdRab5b or its mutants showed that LdRab5b explicitly controls receptor-mediated endocytosis of hemoglobin, and overexpression of LdRab5b:WT enhanced the transport of internalized Hb to the lysosomes in comparison with control cells. To unequivocally demonstrate the role of Rab5 isoforms in endocytosis in Leishmania, we tried to generate null-mutants of LdRab5a and LdRab5b parasites, but both were lethal indicating their essential functions in parasites. Therefore, we used heterozygous LdRab5a(+/-) and LdRab5b(+/-) cells. LdRab5a(+/-) Leishmania showed 50% inhibition of HRP uptake, but hemoglobin endocytosis was uninterrupted. In contrast, about 50% inhibition of Hb endocytosis was observed in LdRab5b(+/-) cells without any significant effect on HRP uptake. Finally, we tried to identify putative LdRab5a and LdRab5b effectors. We found that LdRab5b interacts with clathrin heavy chain and hemoglobin receptor. However, LdRab5a failed to interact with the clathrin heavy chain, and interaction with hemoglobin receptor was significantly less. Thus, our results showed that LdRab5a and LdRab5b differentially regulate fluid phase and receptor-mediated endocytosis in Leishmania.

VipD is a Rab5-activated phospholipase A1 that protects Legionella pneumophila from endosomal fusion.

A crucial step in the elimination of invading microbes by macrophages is phagosomal maturation through heterotypic endosomal fusion. This process is controlled by the guanine nucleotide binding protein Rab5, which assembles protein microdomains that include the tethering protein early endosomal antigen (EEA) 1 and the phosphatidylinositol (PI) 3-kinase hVps34, which generates PI(3)P, a phospholipid required for membrane association of EEA1 and other fusion factors. During infection of macrophages, the pathogen Legionella pneumophila bypasses the microbicidal endosomal compartment by an unknown mechanism. Here, we show that the effector protein VipD from L. pneumophila exhibits phospholipase A1 activity that is activated only upon binding to endosomal Rab5 or Rab22. Within mammalian cells, VipD localizes to endosomes and catalyzes the removal of PI(3)P from endosomal membranes. EEA1 and other transport and fusion factors are consequently depleted from endosomes, rendering them fusion-incompetent. During host cell infection, VipD reduces exposure of L. pneumophila to the endosomal compartment and protects their surrounding vacuoles from acquiring Rab5. Thus, by catalyzing PI(3)P depletion in a Rab5-dependent manner, VipD alters the protein composition of endosomes thereby blocking fusion with Legionella-containing vacuoles.

Rab5-mediated VE-cadherin internalization regulates the barrier function of the lung microvascular endothelium.

The small GTPase Rab5 has been well defined to control the vesicle-mediated plasma membrane protein transport to the endosomal compartment. However, its function in the internalization of vascular endothelial (VE)-cadherin, an important component of adherens junctions, and as a result regulating the endothelial cell polarity and barrier function remain unknown. Here, we demonstrated that lipopolysaccharide (LPS) simulation markedly enhanced the activation and expression of Rab5 in human pulmonary microvascular endothelial cells (HPMECs), which is accompanied by VE-cadherin internalization. In parallel, LPS challenge also induced abnormal cell polarity and dysfunction of the endothelial barrier in HPMECs. LPS stimulation promoted the translocation of VE-cadherin from the plasma membrane to intracellular compartments, and intracellularly expressed VE-cadherin was extensively colocalized with Rab5. Small interfering RNA (siRNA)-mediated depletion of Rab5a expression attenuated the disruption of LPS-induced internalization of VE-cadherin and the disorder of cell polarity. Furthermore, knockdown of Rab5 inhibited the vascular endothelial hyperpermeability and protected endothelial barrier function from LPS injury, both in vitro and in vivo. These results suggest that Rab5 is a critical mediator of LPS-induced endothelial barrier dysfunction, which is likely mediated through regulating VE-cadherin internalization. These findings provide evidence, implicating that Rab5a is a potential therapeutic target for preventing endothelial barrier disruption and vascular inflammation.

Down-regulation of Rab5 decreases characteristics associated with maintenance of cell transformation.

The early endosomal protein Rab5 is highly expressed in tumor samples, although a causal relationship between Rab5 expression and cell transformation has not been established. Here, we report the functional effects of targeting endogenous Rab5 with specific shRNA sequences in different tumor cell lines. Rab5 down-regulation in B16-F10 cells decreased tumor formation by subcutaneous injection into C57/BL6 mice. Accordingly, Rab5 targeting in B16-F10 and A549, but not MDA-MB-231 cells was followed by decreased cell proliferation, increased apoptosis and decreased anchorage-independent growth. These findings suggest that Rab5 expression is required to maintain characteristics associated with cell transformation.

Exoenzyme S ADP-ribosylates Rab5 effector sites to uncouple intracellular trafficking.

Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylates multiple eukaryotic targets to promote cytopathology and bacterial colonization. ADP-ribosylation of the small GTPase Rab5 has previously been shown to block fluid-phase endocytosis and trafficking of plasma membrane receptors to the early endosomes as well as inhibit phagocytosis of the bacterium. In this study, ExoS is shown to be capable of ADP-ribosylating 6 candidate arginine residues that are located in the effector binding region or in the C terminus of Rab5. Two Rab5 derivatives were engineered, which contained Arg→Ala mutations at four Arg residues within the effector binding region (EF) or two Arg residues within the C-terminal tail (TL). Expression of Rab5(TL) does not affect the ability of ExoS to modify intracellular trafficking, while expression of Rab5(EF) rescued the ability of ExoS to inhibit intracellular trafficking. ADP-ribosylation of effector arginines likely uncouples Rab5 signaling to downstream effectors. This is a different mechanism for inhibition than observed for the ADP-ribosylation of Ras by ExoS, where ADP-ribosylated Ras loses the ability to bind guanine nucleotide exchange factor (GEF). Other experiments showed that expression of dominant negative Rab5(Ser34Asn) does not inhibit ExoS trafficking to the perinuclear region of intoxicated cells. This study provides insight into a mechanism for how ExoS ADP-ribosylation of Rab5 inhibits Rab5 function.

Three-dimensional tracking of Rab5- and Rab7-associated infection process of influenza virus.

Three-dimensional (3D) single-particle tracking (SPT) techniques have been widely reported. However, the 3D SPT technique remains poorly used for solving actual biological problems. In this work, a quantum dots (QDs)-based single-particle tracking technique is utilized to explore the Rab5- and Rab7-associated infection behaviors of influenza virus in three dimensions with a set of easily-attained equipment by the fast and accurate centroid method for 3D SPT. The experimental results indicate that Rab5 protein takes part in the virus infection process from the cell periphery to the perinuclear region, while Rab7 protein is mainly involved in the intermittent and confined movements of the virus in the perinuclear region. Evidently, the transition process of the virus-containing vesicles from early to late endosomes might occur during the intermittent movement in the perinuclear region. These findings reveal distinct dynamic behaviors of Rab5- and Rab7-positive endosomes in the course of the intracellular transport of viruses. This work is helpful in understanding the intracellular transport of cargoes.

Bovine ephemeral fever virus uses a clathrin-mediated and dynamin 2-dependent endocytosis pathway that requires Rab5 and Rab7 as well as microtubules.

The specific cell pathways involved in bovine ephemeral fever virus (BEFV) cell entry have not been determined. In this work, colocalization of the M protein of BEFV with clathrin or dynamin 2 was observed under a fluorescence microscope. To better understand BEFV entry, we carried out internalization studies with a fluorescently labeled BEFV by using a lipophilic dye, 3,30-dilinoleyloxacarbocyanine perchlorate (DiO), further suggesting that BEFV uses a clathrin-mediated endocytosis pathway. Our results suggest that clathrin-mediated and dynamin 2-dependent endocytosis is an important avenue of BEFV entry. Suppression of Rab5 or Rab7a through the use of a Rab5 dominant negative mutant and Rab7a short hairpin RNA (shRNA) demonstrated that BEFV requires both early and late endosomes for endocytosis and subsequent infection in MDBK and Vero cells. Treatment of BEFV-infected cells with nocodazole significantly decreased the M protein synthesis and viral yield, indicating that microtubules play an important role in BEFV productive infection, likely by mediating trafficking of BEFV-containing endosomes. Furthermore, BEFV infection was strongly blocked by different inhibitors of endosomal acidification, suggesting that virus enters host cells by clathrin-mediated and dynamin 2-dependent endocytosis in a pH-dependent manner.

A C-terminal tyrosine-based motif in the bile salt export pump directs clathrin-dependent endocytosis.

UNLABELLED: The liver-specific bile salt export pump (BSEP) is crucial for bile acid-dependent bile flow at the apical membrane. BSEP, a member of the family of structurally related adenosine triphosphate (ATP)-binding cassette (ABC) proteins, is composed of 12 transmembrane segments (TMS) and two large cytoplasmic nucleotide-binding domains (NBDs). The regulation of trafficking of BSEP to and from the cell surface is not well understood, but is believed to play an important role in cholestatic liver diseases such as primary familial intrahepatic cholestasis type 2 (PFIC2). To address this issue, BSEP endocytosis was studied by immunofluorescence and a cell surface enzyme-linked immunosorbent assay (ELISA) endocytosis reporter system using a chimera of the interleukin-2 receptor α (previously referred to as Tac) and the C-terminal tail of BSEP (TacCterm). An autonomous endocytosis motif in the carboxyl cytoplasmic terminus of BSEP was identified. We define this endocytic motif by site-directed mutagenesis as a canonical tyrosine-based motif (1310) YYKLV(1314) (YxxØ). When expressed in HEK293T cells, TacCterm is constitutively internalized via a dynamin- and clathrin-dependent pathway. Mutation of the Y(1310) Y(1311) amino acids in TacCterm and in full-length human BSEP blocks the internalization. Subsequent sequence analysis reveals this motif to be highly conserved between the closely related ABCB subfamily members that mediate ATP-dependent transport of broad substrate specificity. CONCLUSION: Our results indicate that constitutive internalization of BSEP is clathrin-mediated and dependent on the tyrosine-based endocytic motif at the C-terminal end of BSEP.

Rab5 regulates internalisation of P2X4 receptors and potentiation by ivermectin.

The P2X4 receptor is an ATP-gated ion channel expressed in neurons, endothelia and immune cells. Plasma membrane expression of P2X4 is regulated by dynamin-dependent endocytosis, and this study identifies a Rab5-dependent pathway of receptor internalisation. Expression of Rab5 constructs altered the distribution of P2X4 in HEK-293 cells, and both constitutive internalisation and agonist-induced desensitisation of P2X4 were increased by co-expression of wild-type Rab5 or constitutively active Rab5 (Q79L). Expression of inactive dynamin K44A and Rab5 S34N constructs abolished agonist-induced desensitisation, suggesting internalisation as the underlying mechanism. Blocking P2X4 internalisation in this way also abolished potentiation of ATP-induced currents by the allosteric modulator ivermectin. This suggests that the dynamin-Rab5 internalisation pathway is essential for the ivermectin potentiation effect. In agreement with this hypothesis, the co-expression of wild-type dynamin, wild-type Rab5 or active Rab5 (Q79L) could increase the potentiation of the ATP-induced P2X4 response by ivermectin. These findings highlight Rab5 GTPase as a key regulator of P2X4 receptor cell surface expression and internalisation.

The role of Rab5a GTPase in endocytosis and post-endocytic trafficking of the hCG-human luteinizing hormone receptor complex.

This study examined the role of Rab5a GTPase in regulating hCG-induced internalization and trafficking of the hCG-LH receptor complex in transfected 293T cells. Coexpression of wild-type Rab5a (WT) or constitutively active Rab5a (Q79L) with LHR significantly increased hCG-induced LHR internalization. Conversely, coexpression of dominant negative Rab5a (S34N) with LHR reduced internalization. Confocal microscopy showed LHR colocalizing with Rab5a (WT) and Rab5a (Q79L) in punctuate structures. Coexpression of Rab5a (WT) and Rab5a (Q79L) with LHR significantly increased colocalization of LHR in early endosomes. Conversely, dominant negative Rab5a (S34N) decreased this colocalization. While Rab5a stimulated internalization of LHR, it significantly decreased LHR recycling to the cell surface and increased degradation. Dominant negative Rab5a (S34N) increased LHR recycling and decreased degradation. These results suggest that Rab5a plays a role in LHR trafficking by facilitating internalization and fusion to early endosomes, increasing the degradation of internalized receptor resulting in a reduction in LHR recycling.

Clathrin-dependent APP endocytosis and Abeta secretion are highly sensitive to the level of plasma membrane cholesterol.

Several lines of evidence support a strong relationship between cholesterol and Alzheimer's disease pathogenesis. Membrane cholesterol is known to modulate amyloid precursor protein (APP) endocytosis and amyloid-beta (Abeta) secretion. Here we show in a human cell line model of endocytosis (HEK293 cells) that cholesterol exerts these effects in a dose-dependent and linear manner, over a wide range of concentrations (-40% to +40% variations of plasma membrane cholesterol induced by methyl-beta-cyclodextrin (MBCD) and MBCD-cholesterol complex respectively). We found that the gradual effect of cholesterol is inhibited by small interference RNA-mediated downregulation of clathrin. Modulation of clathrin-mediated APP endocytosis by cholesterol was further demonstrated using mutants of proteins involved in the formation of early endosomes (dynamin2, Eps15 and Rab5). Importantly we show that membrane proteins other than APP are not affected by cholesterol to the same extent. Indeed clathrin-dependent endocytosis of transferrin and cannabinoid1 receptors as well as internalization of surface proteins labelled with a biotin derivative (sulfo-NHS-SS-biotin) were not sensitive to variations of plasma membrane cholesterol from -40% to 40%. In conclusion clathrin-dependent APP endocytosis appears to be very sensitive to the levels of membrane cholesterol. These results suggest that cholesterol increase in AD could be responsible for the enhanced internalization of clathrin-, dynamin2-, Eps15- and Rab5-dependent endocytosis of APP and the ensuing overproduction of Abeta.

Vps9 domain-containing proteins: activators of Rab5 GTPases from yeast to neurons.

Endocytosis of cell surface receptors plays an important role in regulating cell signaling cascades. In some cases, internalization of an activated receptor attenuates the signaling process, while in other cases the clustering of activated receptors on early endosomal structures has been proposed to be essential for fully activating signaling cascades. Regulating the movement of receptors and other signaling proteins through the endocytic pathway, therefore, has a direct impact on cellular homeostasis. The small GTPase Rab5 is a crucial regulatory component of the endocytic pathway. Activation of Rab5 is mediated by GDP-GTP exchange factors (GEFs) that generate the Rab5-GTP complex. A large number of proteins have been identified that contain a specific, highly conserved domain (Vps9) that catalyzes nucleotide exchange on Rab5, linking the regulation of cell signaling cascades with intracellular receptor trafficking through the endocytic pathway.

Rab5 regulates motility of early endosomes on microtubules.

The small GTPase Rab5 regulates membrane docking and fusion in the early endocytic pathway. Here we reveal a new role for Rab5 in the regulation of endosome interactions with the microtubule network. Using Rab5 fused to green fluorescent protein we show that Rab5-positive endosomes move on microtubules in vivo. In vitro, Rab5 stimulates both association of early endosomes with microtubules and early-endosome motility towards the minus ends of microtubules. Moreover, similarly to endosome membrane docking and fusion, Rab5-dependent endosome movement depends on the phosphatidylinositol-3-OH kinase hVPS34. Thus, Rab5 functionally links regulation of membrane transport, motility and intracellular distribution of early endosomes.

Sorting nexin 3 (SNX3) is a component of a tubular endosomal network induced by Salmonella and involved in maturation of the Salmonella-containing vacuole.

Salmonella enterica serovar Typhimurium is an intracellular pathogen that grows within a modified endomembrane compartment, the Salmonella-containing vacuole (SCV). Maturation of nascent SCVs involves the recruitment of early endosome markers and the remodelling of phosphoinositides at the membrane of the vacuole, in particular the production of phosphatidylinositol 3-phosphate [PI(3)P]. Sorting nexins (SNXs) are a family of proteins characterized by the presence of a phox homology (PX) domain that binds to phosphoinositides and are involved in intracellular trafficking in eukaryotic cells. We therefore studied whether sorting nexins, particularly sorting nexin 3 (SNX3), play a role in Salmonella infection. We found that SNX3 transiently localized to SCVs at early times post invasion (10 min) and presented a striking tubulation phenotype in the vicinity of SCVs at later times (30-60 min). The bacterial effector SopB, which is known to promote PI(3)P production on SCVs, was required for the formation of SNX3 tubules. In addition, RAB5 was also required for the formation of SNX3 tubules. Depletion of SNX3 by siRNA impaired RAB7 and LAMP1 recruitment to the SCV. Moreover, the formation of Salmonella-induced filaments (Sifs) was altered by SNX3 knock-down. Therefore, SNX3 plays a significant role in regulating the maturation of SCVs.

Rab5a regulates surface expression of FcεRI and functional activation in mast cells.

Surface expression levels of high-affinity immunoglobulin E (IgE) receptors (FcεRI) on mast cells are regulated by constitutive internalization from the plasma membrane, which is thought to be an important determinant of FcεRI-mediated signaling potential. However, molecular mechanism of FcεRI trafficking has remained poorly understood. Rab proteins are small guanosine 5'-triphosphatases (GTPases) involved in the regulation of membrane traffic. In particular, Rab5 has been shown to regulate transport in the early endocytic pathway, whereas it is not known whether the FcεRI surface expression levels are regulated by Rab5. In this study, we investigated the role of individual Rab5 isoforms in mast cells by small interfering RNA knockdown method. Our results demonstrate that Rab5a knockdown enhanced FcεRI-dependent mast cell activation and upregulated FcεRI surface expression in its steady state. In contrast, Rab5c knockdown caused suppression of the activation. These findings revealed modulatory and individual roles of Rab5 isoforms in mast cell functions.

The Rab5 guanylate exchange factor Rin1 regulates endocytosis of the EphA4 receptor in mature excitatory neurons.

Ephrin signaling through Eph receptor tyrosine kinases regulates important morphogenetic events during development and synaptic plasticity in the adult brain. Although Eph-ephrin endocytosis is required for repulsive axon guidance, its role in postnatal brain and synaptic plasticity is unknown. Here, we show that Rin1, a postnatal brain-specific Rab5-GEF, is coexpressed with EphA4 in excitatory neurons and interacts with EphA4 in synaptosomal fractions. The interaction of Rin1 and EphA4 requires Rin1's SH2 domain, consistent with the view that Rin1 targets tyrosine phosphorylated receptors to Rab5 compartments. We find that Rin1 mediates EphA4 endocytosis in postnatal amygdala neurons after engagement of EphA4 with its cognate ligand ephrinB3. Rin1 was shown to suppress synaptic plasticity in the amygdala, a forebrain structure important for fear learning, possibly by internalizing synaptic receptors. We find that the EphA4 receptor is required for synaptic plasticity in the amygdala, raising the possibility that an underlying mechanism of Rin1 function in amygdala is to down-regulate EphA4 signaling by promoting its endocytosis.

Surfaceome CRISPR screen identifies OLFML3 as a rhinovirus-inducible IFN antagonist.

BACKGROUND: Rhinoviruses (RVs) cause more than half of common colds and, in some cases, more severe diseases. Functional genomics analyses of RVs using siRNA or genome-wide CRISPR screen uncovered a limited set of host factors, few of which have proven clinical relevance. RESULTS: Herein, we systematically compare genome-wide CRISPR screen and surface protein-focused CRISPR screen, referred to as surfaceome CRISPR screen, for their efficiencies in identifying RV host factors. We find that surfaceome screen outperforms the genome-wide screen in the success rate of hit identification. Importantly, using the surfaceome screen, we identify olfactomedin-like 3 (OLFML3) as a novel host factor of RV serotypes A and B, including a clinical isolate. We find that OLFML3 is a RV-inducible suppressor of the innate immune response and that OLFML3 antagonizes type I interferon (IFN) signaling in a SOCS3-dependent manner. CONCLUSION: Our study suggests that RV-induced OLFML3 expression is an important mechanism for RV to hijack the immune system and underscores surfaceome CRISPR screen in identifying viral host factors.