Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity.

Metabolic coordination between neurons and astrocytes is critical for the health of the brain. However, neuron-astrocyte coupling of lipid metabolism, particularly in response to neural activity, remains largely uncharacterized. Here, we demonstrate that toxic fatty acids (FAs) produced in hyperactive neurons are transferred to astrocytic lipid droplets by ApoE-positive lipid particles. Astrocytes consume the FAs stored in lipid droplets via mitochondrial ß-oxidation in response to neuronal activity and turn on a detoxification gene expression program. Our findings reveal that FA metabolism is coupled in neurons and astrocytes to protect neurons from FA toxicity during periods of enhanced activity. This coordinated mechanism for metabolizing FAs could underlie both homeostasis and a variety of disease states of the brain.

Protein Quality Control and Lipid Droplet Metabolism.

Lipid droplets (LDs) are endoplasmic reticulum-derived organelles that consist of a core of neutral lipids encircled by a phospholipid monolayer decorated with proteins. As hubs of cellular lipid and energy metabolism, LDs are inherently involved in the etiology of prevalent metabolic diseases such as obesity and nonalcoholic fatty liver disease. The functions of LDs are regulated by a unique set of associated proteins, the LD proteome, which includes integral membrane and peripheral proteins. These proteins control key activities of LDs such as triacylglycerol synthesis and breakdown, nutrient sensing and signal integration, and interactions with other organelles. Here we review the mechanisms that regulate the composition of the LD proteome, such as pathways that mediate selective and bulk LD protein degradation and potential connections between LDs and cellular protein quality control.

An oncogenic JMJD6-DGAT1 axis tunes the epigenetic regulation of lipid droplet formation in clear cell renal cell carcinoma.

Characterized by intracellular lipid droplet accumulation, clear cell renal cell carcinoma (ccRCC) is resistant to cytotoxic chemotherapy and is a lethal disease. Through an unbiased siRNA screen of 2-oxoglutarate (2-OG)-dependent enzymes, which play a critical role in tumorigenesis, we identified Jumonji domain-containing 6 (JMJD6) as an essential gene for ccRCC tumor development. The downregulation of JMJD6 abolished ccRCC colony formation in vitro and inhibited orthotopic tumor growth in vivo. Integrated ChIP-seq and RNA-seq analyses uncovered diacylglycerol O-acyltransferase 1 (DGAT1) as a critical JMJD6 effector. Mechanistically, JMJD6 interacted with RBM39 and co-occupied DGAT1 gene promoter with H3K4me3 to induce DGAT1 expression. JMJD6 silencing reduced DGAT1, leading to decreased lipid droplet formation and tumorigenesis. The pharmacological inhibition (or depletion) of DGAT1 inhibited lipid droplet formation in vitro and ccRCC tumorigenesis in vivo. Thus, the JMJD6-DGAT1 axis represents a potential new therapeutic target for ccRCC.

Amelioration of hepatic steatosis by dietary essential amino acid-induced ubiquitination.

Nonalcoholic fatty liver disease (NAFLD) is a global health concern with no approved drugs. High-protein dietary intervention is currently the most effective treatment. However, its underlying mechanism is unknown. Here, using Drosophila oenocytes, the specialized hepatocyte-like cells, we find that dietary essential amino acids ameliorate hepatic steatosis by inducing polyubiquitination of Plin2, a lipid droplet-stabilizing protein. Leucine and isoleucine, two branched-chain essential amino acids, strongly bind to and activate the E3 ubiquitin ligase Ubr1, targeting Plin2 for degradation. We further show that the amino acid-induced Ubr1 activity is necessary to prevent steatosis in mouse livers and cultured human hepatocytes, providing molecular insight into the anti-NAFLD effects of dietary protein/amino acids. Importantly, split-intein-mediated trans-splicing expression of constitutively active UBR2, an Ubr1 family member, significantly ameliorates obesity-induced and high fat diet-induced hepatic steatosis in mice. Together, our results highlight activation of Ubr1 family proteins as a promising strategy in NAFLD treatment.

Choline kinase alpha 2 acts as a protein kinase to promote lipolysis of lipid droplets.

Lipid droplets are important for cancer cell growth and survival. However, the mechanism underlying the initiation of lipid droplet lipolysis is not well understood. We demonstrate here that glucose deprivation induces the binding of choline kinase (CHK) α2 to lipid droplets, which is sequentially mediated by AMPK-dependent CHKα2 S279 phosphorylation and KAT5-dependent CHKα2 K247 acetylation. Importantly, CHKα2 with altered catalytic domain conformation functions as a protein kinase and phosphorylates PLIN2 at Y232 and PLIN3 at Y251. The phosphorylated PLIN2/3 dissociate from lipid droplets and are degraded by Hsc70-mediated autophagy, thereby promoting lipid droplet lipolysis, fatty acid oxidation, and brain tumor growth. In addition, levels of CHKα2 S279 phosphorylation, CHKα2 K247 acetylation, and PLIN2/3 phosphorylation are positively correlated with one another in human glioblastoma specimens and are associated with poor prognosis in glioblastoma patients. These findings underscore the role of CHKα2 as a protein kinase in lipolysis and glioblastoma development.

MIGA2 Links Mitochondria, the ER, and Lipid Droplets and Promotes De Novo Lipogenesis in Adipocytes.

Physical contact between organelles is vital to the function of eukaryotic cells. Lipid droplets (LDs) are dynamic organelles specialized in lipid storage that interact physically with mitochondria in several cell types. The mechanisms coupling these organelles are, however, poorly understood, and the cell-biological function of their interaction remains largely unknown. Here, we discover in adipocytes that the outer mitochondrial membrane protein MIGA2 links mitochondria to LDs. We identify an amphipathic LD-targeting motif and reveal that MIGA2 binds to the membrane proteins VAP-A or VAP-B in the endoplasmic reticulum (ER). We find that in adipocytes MIGA2 is involved in promoting triglyceride (TAG) synthesis from non-lipid precursors. Our data indicate that MIGA2 links reactions of de novo lipogenesis in mitochondria to TAG production in the ER, thereby facilitating efficient lipid storage in LDs. Based on its presence in many tissues, MIGA2 is likely critical for lipid and energy homeostasis in a wide spectrum of cell types.

Spartin-mediated lipid transfer facilitates lipid droplet turnover.

Lipid droplets (LDs) are organelles critical for energy storage and membrane lipid homeostasis, whose number and size are carefully regulated in response to cellular conditions. The molecular mechanisms underlying lipid droplet biogenesis and degradation, however, are not well understood. The Troyer syndrome protein spartin (SPG20) supports LD delivery to autophagosomes for turnover via lipophagy. Here, we characterize spartin as a lipid transfer protein whose transfer ability is required for LD degradation. Spartin copurifies with phospholipids and neutral lipids from cells and transfers phospholipids in vitro via its senescence domain. A senescence domain truncation that impairs lipid transfer in vitro also impairs LD turnover in cells while not affecting spartin association with either LDs or autophagosomes, supporting that spartin's lipid transfer ability is physiologically relevant. Our data indicate a role for spartin-mediated lipid transfer in LD turnover.

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.

DGAT1 activity synchronises with mitophagy to protect cells from metabolic rewiring by iron depletion.

Mitophagy removes defective mitochondria via lysosomal elimination. Increased mitophagy coincides with metabolic reprogramming, yet it remains unknown whether mitophagy is a cause or consequence of such state changes. The signalling pathways that integrate with mitophagy to sustain cell and tissue integrity also remain poorly defined. We performed temporal metabolomics on mammalian cells treated with deferiprone, a therapeutic iron chelator that stimulates PINK1/PARKIN-independent mitophagy. Iron depletion profoundly rewired the metabolome, hallmarked by remodelling of lipid metabolism within minutes of treatment. DGAT1-dependent lipid droplet biosynthesis occurred several hours before mitochondrial clearance, with lipid droplets bordering mitochondria upon iron chelation. We demonstrate that DGAT1 inhibition restricts mitophagy in vitro, with impaired lysosomal homeostasis and cell viability. Importantly, genetic depletion of DGAT1 in vivo significantly impaired neuronal mitophagy and locomotor function in Drosophila. Our data define iron depletion as a potent signal that rapidly reshapes metabolism and establishes an unexpected synergy between lipid homeostasis and mitophagy that safeguards cell and tissue integrity.

Lipid droplet dynamics are essential for the development of the malaria parasite Plasmodium falciparum.

Lipid droplets (LDs) are organelles that are central to lipid and energy homeostasis across all eukaryotes. In the malaria-causing parasite Plasmodium falciparum the roles of LDs in lipid acquisition from its host cells and their metabolism are poorly understood, despite the high demand for lipids in parasite membrane synthesis. We systematically characterised LD size, composition and dynamics across the disease-causing blood infection. Applying split fluorescence emission analysis and three-dimensional (3D) focused ion beam-scanning electron microscopy (FIB-SEM), we observed a decrease in LD size in late schizont stages. LD contraction likely signifies a switch from lipid accumulation to lipid utilisation in preparation for parasite egress from host red blood cells. We demonstrate connections between LDs and several parasite organelles, pointing to potential functional interactions. Chemical inhibition of triacylglyerol (TAG) synthesis or breakdown revealed essential LD functions for schizogony and in counteracting lipid toxicity. The dynamics of lipid synthesis, storage and utilisation in P. falciparum LDs might provide a target for new anti-malarial intervention strategies.

Detecting material state changes in the nucleolus by label-free digital holographic microscopy.

Ribosome biogenesis is initiated in the nucleolus, a multiphase biomolecular condensate formed by liquid-liquid phase separation. The nucleolus is a powerful disease biomarker and stress biosensor whose morphology reflects function. Here we have used digital holographic microscopy (DHM), a label-free quantitative phase contrast microscopy technique, to detect nucleoli in adherent and suspension human cells. We trained convolutional neural networks to detect and quantify nucleoli automatically on DHM images. Holograms containing cell optical thickness information allowed us to define a novel index which we used to distinguish nucleoli whose material state had been modulated optogenetically by blue-light-induced protein aggregation. Nucleoli whose function had been impacted by drug treatment or depletion of ribosomal proteins could also be distinguished. We explored the potential of the technology to detect other natural and pathological condensates, such as those formed upon overexpression of a mutant form of huntingtin, ataxin-3, or TDP-43, and also other cell assemblies (lipid droplets). We conclude that DHM is a powerful tool for quantitatively characterizing nucleoli and other cell assemblies, including their material state, without any staining.

Interactome Analysis Identifies the Role of BZW2 in Promoting Endoplasmic Reticulum-Mitochondria Contact and Mitochondrial Metabolism.

Understanding the molecular functions of less-studied proteins is an important task of life science research. Despite reports of basic leucine zipper and W2 domain-containing protein 2 (BZW2) promoting cancer progression first emerging in 2017, little is known about its molecular function. Using a quantitative proteomic approach to identify its interacting proteins, we found that BZW2 interacts with both endoplasmic reticulum (ER) and mitochondrial proteins. We thus hypothesized that BZW2 localizes to and promotes the formation of ER-mitochondria contact sites and that such localization would promote calcium transport from ER to the mitochondria and promote ATP production. Indeed, we found that BZW2 localized to ER-mitochondria contact sites and that BZW2 knockdown decreased ER-mitochondria contact, mitochondrial calcium levels, and ATP production. These findings provide key insights into molecular functions of BZW2, the potential role of BZW2 in cancer progression, and highlight the utility of interactome data in understanding the function of less-studied proteins.

Rotavirus-mediated DGAT1 degradation: A pathophysiological mechanism of viral-induced malabsorptive diarrhea.

Gastroenteritis is among the leading causes of mortality globally in infants and young children, with rotavirus (RV) causing ~258 million episodes of diarrhea and ~128,000 deaths annually in infants and children. RV-induced mechanisms that result in diarrhea are not completely understood, but malabsorption is a contributing factor. RV alters cellular lipid metabolism by inducing lipid droplet (LD) formation as a platform for replication factories named viroplasms. A link between LD formation and gastroenteritis has not been identified. We found that diacylglycerol O-acyltransferase 1 (DGAT1), the terminal step in triacylglycerol synthesis required for LD biogenesis, is degraded in RV-infected cells by a proteasome-mediated mechanism. RV-infected DGAT1-silenced cells show earlier and increased numbers of LD-associated viroplasms per cell that translate into a fourfold-to-fivefold increase in viral yield (P < 0.05). Interestingly, DGAT1 deficiency in children is associated with diarrhea due to altered trafficking of key ion transporters to the apical brush border of enterocytes. Confocal microscopy and immunoblot analyses of RV-infected cells and DGAT1-/- human intestinal enteroids (HIEs) show a decrease in expression of nutrient transporters, ion transporters, tight junctional proteins, and cytoskeletal proteins. Increased phospho-eIF2α (eukaryotic initiation factor 2 alpha) in DGAT1-/- HIEs, and RV-infected cells, indicates a mechanism for malabsorptive diarrhea, namely inhibition of translation of cellular proteins critical for nutrient digestion and intestinal absorption. Our study elucidates a pathophysiological mechanism of RV-induced DGAT1 deficiency by protein degradation that mediates malabsorptive diarrhea, as well as a role for lipid metabolism, in the pathogenesis of gastroenteritis.

The ubiquitin-protein ligase MIEL1 localizes to peroxisomes to promote seedling oleosin degradation and lipid droplet mobilization.

Lipid droplets are organelles conserved across eukaryotes that store and release neutral lipids to regulate energy homeostasis. In oilseed plants, fats stored in seed lipid droplets provide fixed carbon for seedling growth before photosynthesis begins. As fatty acids released from lipid droplet triacylglycerol are catabolized in peroxisomes, lipid droplet coat proteins are ubiquitinated, extracted, and degraded. In Arabidopsis seeds, the predominant lipid droplet coat protein is OLEOSIN1 (OLE1). To identify genes modulating lipid droplet dynamics, we mutagenized a line expressing mNeonGreen-tagged OLE1 expressed from the OLE1 promoter and isolated mutants with delayed oleosin degradation. From this screen, we identified four miel1 mutant alleles. MIEL1 (MYB30-interacting E3 ligase 1) targets specific MYB transcription factors for degradation during hormone and pathogen responses [D. Marino et al., Nat. Commun. 4, 1476 (2013); H. G. Lee and P. J. Seo, Nat. Commun. 7, 12525 (2016)] but had not been implicated in lipid droplet dynamics. OLE1 transcript levels were unchanged in miel1 mutants, indicating that MIEL1 modulates oleosin levels posttranscriptionally. When overexpressed, fluorescently tagged MIEL1 reduced oleosin levels, causing very large lipid droplets. Unexpectedly, fluorescently tagged MIEL1 localized to peroxisomes. Our data suggest that MIEL1 ubiquitinates peroxisome-proximal seed oleosins, targeting them for degradation during seedling lipid mobilization. The human MIEL1 homolog (PIRH2; p53-induced protein with a RING-H2 domain) targets p53 and other proteins for degradation and promotes tumorigenesis [A. Daks et al., Cells 11, 1515 (2022)]. When expressed in Arabidopsis, human PIRH2 also localized to peroxisomes, hinting at a previously unexplored role for PIRH2 in lipid catabolism and peroxisome biology in mammals.

Paxillin family proteins Hic-5 and LPXN promote lipid storage by regulating the ubiquitination degradation of CIDEC.

Many metabolic diseases are caused by disorders of lipid homeostasis. CIDEC, a lipid droplet (LD)-associated protein, plays a critical role in controlling LD fusion and lipid storage. However, regulators of CIDEC remain largely unknown. Here, we established a homogeneous time-resolved fluorescence (HTRF)-based high-throughput screening method and identified LPXN as a positive regulatory candidate for CIDEC. LPXN and Hic-5, the members of the Paxillin family, are focal adhesion adaptor proteins that contribute to the recruitment of specific kinases and phosphatases, cofactors, and structural proteins, participating in the transduction of extracellular signals into intracellular responses. Our data showed that Hic-5 and LPXN significantly increased the protein level of CIDEC and enhanced CIDEC stability not through triacylglycerol synthesis and FAK signaling pathways. Hic-5 and LPXN reduced the ubiquitination of CIDEC and inhibited its proteasome degradation pathway. Furthermore, Hic-5 and LPXN enlarged LDs and promoted lipid storage in adipocytes. Therefore, we identified Hic-5 and LPXN as novel regulators of CIDEC. Our current findings also suggest intervention with Hic-5 and LPXN might ameliorate ectopic fat storage by enhancing the lipid storage capacity of white adipose tissues.

Senp7 deficiency impairs lipid droplets maturation in white adipose tissues via Plin4 deSUMOylation.

Lipid metabolism is important for the maintenance of physiological homeostasis. Several members of the small ubiquitin-like modifier (SUMO)-specific protease (SENP) family have been reported as the regulators of lipid homeostasis. However, the function of Senp7 in lipid metabolism remains unclear. In this study, we generated both conventional and adipocyte-specific Senp7 KO mice to characterize the role of Senp7 in lipid metabolism homeostasis. Both Senp7-deficient mice displayed reduced white adipose tissue mass and decreased size of adipocytes. By analyzing the lipid droplet morphology, we demonstrated that the lipid droplet size was significantly smaller in Senp7-deficient adipocytes. Mechanistically, Senp7 could deSUMOylate the perilipin family protein Plin4 to promote the lipid droplet localization of Plin4. Our results reveal an important role of Senp7 in the maturation of lipid droplets via Plin4 deSUMOylation.

The lipid droplet assembly complex consists of seipin and four accessory factors in budding yeast.

Seipin, a crucial protein for cellular lipid droplet (LD) assembly, oligomerizes at the interface between the endoplasmic reticulum and LDs to facilitate neutral lipid packaging. Using proximity labeling, we identified four proteins-Ldo45, Ldo16, Tgl4, and Pln1-that are recruited to the vicinity of yeast seipin, the Sei1-Ldb16 complex, exclusively when seipin function is intact, hence termed seipin accessory factors. Localization studies identified Tgl4 at the endoplasmic reticulum-LD contact site, in contrast to Ldo45, Ldo16, and Pln1 at the LD surface. Cells with compromised seipin function resulted in uneven distribution of these proteins with aberrant LDs, supporting a central role of seipin in orchestrating their association with the LD. Overexpression of any seipin accessory factor causes LD aggregation and affects a subset of LD protein distribution, highlighting the importance of their stoichiometry. Although single factor mutations show minor LD morphology changes, the combined mutations have additive effects. Lastly, we present evidence that seipin accessory factors assemble and interact with seipin in the absence of neutral lipids and undergo dynamical rearrangements during LD formation induction, with Ldo45 acting as a central hub recruiting other factors to interact with the seipin complex.

The Saccharomyces cerevisiae Spo7 basic tail is required for Nem1-Spo7/Pah1 phosphatase cascade function in lipid synthesis.

The Saccharomyces cerevisiae Nem1-Spo7 protein phosphatase complex dephosphorylates and thereby activates Pah1 at the nuclear/endoplasmic reticulum membrane. Pah1, a phosphatidate phosphatase catalyzing the dephosphorylation of phosphatidate to produce diacylglycerol, is one of the most highly regulated enzymes in lipid metabolism. The diacylglycerol produced in the lipid phosphatase reaction is utilized for the synthesis of triacylglycerol that is stored in lipid droplets. Disruptions of the Nem1-Spo7/Pah1 phosphatase cascade cause a plethora of physiological defects. Spo7, the regulatory subunit of the Nem1-Spo7 complex, is required for the Nem1 catalytic function and interacts with the acidic tail of Pah1. Spo7 contains three conserved homology regions (CR1-3) that are important for the interaction with Nem1, but its region for the interaction with Pah1 is unknown. Here, by deletion and site-specific mutational analyses of Spo7, we revealed that the C-terminal basic tail (residues 240-259) containing five arginine and two lysine residues is important for the Nem1-Spo7 complex-mediated dephosphorylation of Pah1 and its cellular function (triacylglycerol synthesis, lipid droplet formation, maintenance of nuclear/endoplasmic reticulum membrane morphology, and cell growth at elevated temperatures). The glutaraldehyde cross-linking analysis of synthetic peptides indicated that the Spo7 basic tail interacts with the Pah1 acidic tail. This work advances our understanding of the Spo7 function and the Nem1-Spo7/Pah1 phosphatase cascade in yeast lipid synthesis.

Molecular characterization of Rft1, an ER membrane protein associated with congenital disorder of glycosylation RFT1-CDG.

The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multistep pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 congenital disorders of glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multispanning membrane protein located in the ER, with its N and C termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.

The class III phosphatidylinositol 3-kinase VPS34 supports EV71 replication by promoting viral replication organelle formation.

Enterovirus 71 (EV71) belongs to the family of Picornaviridae; it could cause a variety of illnesses and pose a great threat to public health worldwide. Currently, there is no specific drug treatment for this virus, and a better understanding of virus-host interaction is crucial for novel antiviral development. Here, we find that the class III phosphatidylinositol 3-kinase, VPS34, is an essential host factor for EV71 infection. VPS34 inhibition with either shRNA or specific chemical inhibitor significantly reduces EV71 infection. Meanwhile, EV71 infection upregulates phosphatidylinositol 3-phosphate (PI3P) production in viral replication organelles (ROs), while the depletion of PI3P by phosphatase overexpression inhibits EV71 infection. In addition, the PI3P-binding protein, double FYVE-containing protein 1 (DFCP1), is also required for an efficient replication of EV71. DFCP1 could interact with viral 2C protein and facilitate viral association with lipid droplets (LDs), which are important lipid sources for viral RO biogenesis. Taken together, these results indicate that EV71 virus exploits the VPS34-PI3P-DFCP1-LDs pathway to promote viral RO formation and viral infection, and they also illuminate novel targets for antiviral development.IMPORTANCEEnterovirus 71 (EV71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD) and other serious complications, which are big threats to children under 5 years old. Unravelling the interactions between virus and the host cells will open new avenues in antiviral research. Here, we found the class III phosphatidylinositol 3-kinase, VPS34, and its effector, double FYVE-containing protein 1 (DFCP1), were essential for EV71 infection, both of which could support EV71 viral replication by enhancing the biogenesis of viral replication organelles (ROs). As DFCP1 localizes to lipid droplets, hijacking of these host factors will enable viral utilization of lipids from LDs for the generation of membrane structures during RO biogenesis. In addition, the VPS34 kinase inhibitor was found to be potent against EV71 infection; therefore, this study also brings up a novel target for future anti-EV71 drug development.