Evaluation of the reducing potential of PSMA-containing endosomes by FRET imaging

Aim:Ligand-targeted therapeutics are experiencing increasing use for treatment of human diseases due to their ability to concentrate a desired drug at a pathologic site while reducing accumulation in healthy tissues.For many ligand-targeted drug conjugates,a critical aspect of conjugate design lies in engineering release of the therapeutic payload to occur only after its internalization by targeted cells.Because disulfide bond reduction is frequently exploited to ensure intracellular drug release,an understanding of the redox properties of endocytic compartments can be critical to ligand-targeted drug design.While the redox properties of folate receptor trafficking endosomes have been previously reported,little is known about the trafficking of prostate-specific membrane antigen(PSMA),a receptor that is experiencing increasing use for drug targeting in humans.Methods:To obtain this information,we have constructed a PSMA-targeted fluorescence resonance energy transfer pair that reports on disulfide bond reduction by changing fluorescence from red to green.Results:We show here that this reporter exhibits rapid and selective uptake by PSMA-positive cells,and that reduction of its disulfide bond proceeds steadily but incompletely following internalization.The fact that maximal disulfide reduction reaches only~50%,even after 24 h incubation,suggests that roughly half of the conjugates must traffic through endosomes that display no reducing capacity.Conclusion:As the level of disulfide reduction differs between PSMA trafficked and previously published folate trafficked conjugates,it also follows that not all internalizing receptors are translocated through similar intracellular compartments.Taken together,these data suggest that the efficiency of disulfide bond reduction must be independently analyzed for each receptor trafficking pathway when disulfide bond reduction is exploited for intracellular drug release.

Langerin-mediated internalization of a modified peptide routes antigens to early endosomes and enhances cross-presentation by human Langerhans cells

The potential of the skin immune system to generate immune responses is well established, and the skin is actively exploited as a vaccination site. Human skin contains several antigen-presenting cell subsets with specialized functions. In particular, the capacity to cross-present exogenous antigens to CD8^＋ T cells is of interest for the design of effective immunotherapies against viruses or cancer. Here, we show that primary human Langerhans cells （LCs） were able to cross-present a synthetic long peptide （SLP） to CD8^＋ T cells. In addition, modification of this SLP using antibodies against the receptor langerin, but not dectin-1, further enhanced the cross-presenting capacity of LCs through routing of internalized antigens to less proteolytic early endosome antigen 1 ^＋ early endosomes. The potency of LCs to enhance CD8^＋ T-cell responses could be further increased through activation of LCs with the toll-like receptor 3 ligand polyinosinic：polycytidylic acid （phC）. Altogether, the data provide evidence that human LCs are able to cross-present antigens after langerin-mediated internalization. Furthermore, the potential for antigen modification to target LCs specifically provides a rationale for generating effective anti-tumor or anti-viral cytotoxic T lymphocyte responses.

Interleukin-6 secretion is limited by self-signaling in endosomes

Cells producing cytokines often express the receptor for the same cytokine, which makes them prone to autocrine signaling. How cytokine release and signaling are regulated in the same cell is not understood? In this study, we demonstrate that signaling by exogenous and self-synthesized inflammatory cytokine interleukin-6 (IL-6) within endosomal compartments acts as a cellular brake that limits the synthesis of IL-6. Our data show that IL-6 is internalized by dendritic cells and signals from endosomal compartments containing the IL-6 receptor. Newly synthesized IL-6 also traffics via these endosomal compartments and signals in transit to the plasma membrane. This allows activation of STAT3 which in turn limits toll-like receptor 4 stimulant lipopolysaccharide (LPS) triggered transcription of IL-6. Long-term exposure to LPS removes this brake via inhibition of STAT3 by increased expression of suppressor of cytokine signaling 3 and results in fully fledged IL-6 production. This transient regulation could prevent excessive IL-6 production during early infections.

Endosomes and Microtubles are Required for Productive Infection in Aquareovirus

Grass carp reovirus(GCRV),the genus Aquareovirus in family Reoviridae,is viewed as the most pathogenic aquareovirus.To understand the molecular mechanism of how aquareovirus initiates productive infection,the roles of endosome and microtubule in cell entry of GCRV are investigated by using quantum dots(QDs)-tracking in combination with biochemical approaches.We found that GCRV infection and viral protein synthesis were significantly inhibited by pretreating host cells with endosome acidification inhibitors NH4Cl,chloroquine and bafilomycin A1(Bafi).Confocal images indicated that GCRV particles could colocalize with Rab5,Rab7 and lysosomes in host cells.Further ultrastructural examination validated that viral particle was found in late endosomes.Moreover,disruption of microtubules with nocodazole clearly blocked GCRV entry,while no inhibitory effects were observed with cytochalasin D treated cells in viral infection,hinting that intracellular transportation of endocytic uptake in GCRV infected cells is via microtubules but not actin filament.Notably,viral particles were observed to transport along microtubules by using QD-labeled GCRV.Altogether,our results suggest that GCRV can use endosomes and microtubules to initiate productive infection.

Converging links between adult-onset neurodegenerative Alzheimer’s disease and early life neurodegenerative neuronal ceroid lipofuscinosis?

Evidence from genetics and from analyzing cellular and animal models have converged to suggest links between neurodegenerative disorders of early and late life.Here,we summarize emerging links between the most common late life neurodegenerative disease,Alzheimer’s disease,and the most common early life neurodegenerative diseases,neuronal ceroid lipofuscinoses.Genetic studies reported an overlap of clinically diagnosed Alzheimer’s disease and mutations in genes known to cause neuronal ceroid lipofuscinoses.Accumulating data strongly suggest dysfunction of intracellular trafficking mechanisms and the autophagy-endolysosome system in both types of neurodegenerative disorders.This suggests shared cytopathological processes underlying these different types of neurodegenerative diseases.A better understanding of the common mechanisms underlying the different diseases is important as this might lead to the identification of novel targets for therapeutic concepts,the transfer of therapeutic strategies from one disease to the other and therapeutic approaches tailored to patients with specific mutations.Here,we review dysfunctions of the endolysosomal autophagy pathway in Alzheimer’s disease and neuronal ceroid lipofuscinoses and summarize emerging etiologic and genetic overlaps.

Important relationships between Rab and MICAL proteins in endocytic trafficking

The internalization of essential nutrients,lipids and receptors is a crucial process for all eukaryotic cells.Accordingly,endocytosis is highly conserved across cell types and species.Once internalized,small cargocontaining vesicles fuse with early endosomes(also known as sorting endosomes),where they undergo segregation to distinct membrane regions and are sorted and transported on through the endocytic pathway.Although the mechanisms that regulate this sorting are still poorly understood,some receptors are directed to late endosomes and lysosomes for degradation,whereas other receptors are recycled back to the plasma membrane;either directly or through recycling endosomes.The Rab family of small GTP-binding proteins plays crucial roles in regulating these trafficking pathways.Rabs cycle from inactive GDP-bound cytoplasmic proteins to active GTP-bound membraneassociated proteins,as a consequence of the activity of multiple specific GTPase-activating proteins(GAPs) and GTP exchange factors(GEFs).Once bound to GTP,Rabs interact with a multitude of effector proteins that carry out Rab-specific functions.Recent studies have shown that some of these effectors are also interaction partners for the C-terminal Eps15 homology(EHD) proteins,which are also intimately involved in endocytic regulation.A particularly interesting example of common Rab-EHD interaction partners is the MICALlike protein,MICAL-L1.MICAL-L1 and its homolog,MICAL-L2,belong to the larger MICAL family of proteins,and both have been directly implicated in regulating endocytic recycling of cell surface receptors and junctional proteins,as well as controlling cytoskeletal rearrangement and neurite outgrowth.In this review,we summarize the functional roles of MICAL and Rab proteins,and focus on the significance of their interactions and the implications for endocytic transport.

A multi-functional nanoplatform for efficacy tumor theranostic applications

Nanomaterials with multiple functions have become more and more popular in the domain of cancer research. MoS2 has a great potential in photothermal therapy, X-ray/CT imaging and drug delivery. In this study, a water soluble MoS2 nanosystem(MoS2-PEG) was synthesized and explored in drug delivery, photothermal therapy(PTT) and X-ray imaging.Doxorubicin(DOX) was loaded onto MoS2-PEG with a high drug loading efficiency(~69%)and obtained a multifunctional drug delivery system(MoS2-PEG/DOX). As the drug delivery, MoS2-PEG/DOX could efficiently cross the cell membranes, and escape from the endosome via NIR light irradiation, lead to more apoptosis in MCF-7 cells, and afford higher antitumor efficacy without obvious toxic effects to normal organs owing to its prolonged blood circulation and 11.6-fold higher DTX uptake of tumor than DOX. Besides, MoS2-PEG/DOX not only served as a drug delivery system, but also as a powerful PTT agent for thermal ablation of tumor and a strong X-ray contrast agent for tumor diagnosis. In the in vitro and in vivo studies, MoS2-PEG/DOX exhibited excellent tumor-targeting efficacy, outstanding synergistic anti-cancer effect of photothermal and chemotherapy and X-ray imaging property,demonstrating that MoS2-PEG/DOX had a great potential for simultaneous diagnosis and photothermal-chemotherapy in cancer treatment.

Taking out the garbage:cathepsin D and calcineurin in neurodegeneration

Cellular homeostasis requires a tightly controlled balance between protein synthesis, folding and degradation. Especially long-lived, post-mitotic cells such as neurons depend on an efficient proteostasis system to maintain cellular health over decades. Thus, a functional decline of processes contributing to protein degradation such as autophagy and general lysosomal proteolytic capacity is connected to several age-associated neurodegenerative disorders, including Parkinson＇s, Alzheimer＇s and Huntington＇s diseases. These so called proteinopathies are characterized by the accumulation and misfolding of distinct proteins, subsequently driving cellular demise. We recently linked efficient lysosomal protein breakdown via the protease cathep- sin D to the Ca2＋/calmodulin-dependent phosphatase calcineurin. In a yeast model for Parkinson＇s disease, functional calcineurin was required for proper trafficking of cathepsin D to the lysosome and for recycling of its endosomal sorting receptor to allow further rounds of shuttling. Here, we discuss these findings in relation to present knowledge about the involvement of cathepsin D in proteinopathies in general and a possible connection between this protease, calcineurin signalling and endosomal sorting in particular. As dysregulation of Ca2＋ homeostasis as well as lysosomal impairment is connected to a plethora of neurode- generative disorders, this novel interplay might very well impact pathologies beyond Parkinson＇s disease.

Elucidation of the early infection machinery of hepatitis B virus by using bio-nanocapsule

Currently, hepatitis B virus(HBV), upon attaching to human hepatocytes, is considered to interact first with heparan sulfate proteoglycan(HSPG) via an antigenic loop of HBV envelope S protein. Then, it is promptly transferred to the sodium taurocholate cotransporting polypeptide(NTCP) via the myristoylated N-terminal sequence of pre-S1 region(from Gly-2 to Gly-48, HBV genotype D), and it finally enters the cell by endocytosis. However, it is not clear how HSPG passes HBV to NTCP and how NTCP contributes to the cellular entry of HBV. Owing to the poor availability and the difficulty of manipulations, including fluorophore encapsulation, it has been nearly impossible to perform biochemical and cytochemical analyses using a substantial amount of HBV. A bio-nanocapsule(BNC), which is a hollow nanoparticle consisting of HBV envelope L protein, was efficiently synthesized in Saccharomyces cerevisiae. Since BNC could encapsulate payloads(drugs, genes, proteins) and specifically enter human hepatic cells utilizing HBV-derived infection machinery, it could be used as a model of HBV infection to elucidate the early infection machinery. Recently, it was demonstrated that the N-terminal sequence of pre-S1 region(from Asn-9 to Gly-24) possesses low p H-dependent fusogenic activity, which might play a crucial role in the endosomal escape of BNC payloads and in the uncoating process of HBV. In this minireview, we describe a model in which each domain of the HBV L protein contributes to attachment onto human hepatic cells through HSPG, initiation of endocytosis, interaction with NTCP in endosomes, and consequent provocation of membrane fusion followed by endosomal escape.

CDK11 negatively regulates Wnt/β-catenin signaling in the endosomal compartment by affecting microtubule stability

Objectives:Improper activation of Wnt/β-catenin signaling has been implicated in human diseases.Beyond the well-studied glycogen synthase kinase 3p(GSK3p)and casein kinase 1(CK1),other kinases affecting Wnt/β-catenin signaling remain to be defined.Methods:To identify the kinases that modulate Wnt/β-catenin signaling,we applied a kinase small interfering RNA(siRNA)library screen approach.Luciferase assays,immunoblotting,and real-time polymerase chain reaction(PCR)were performed to confirm the regulation o f the Wnt/β-catenin signaling pathway by cyclin-dependent kinase 11(CDK11)and to investigate the underlying mechanism.Confocal immunofluorescence,coimmunoprecipitation(co-IP),and scratch wound assays were used to demonstrate colocalization,detect protein interactions,and explore the function of CDK11.Results:CDK11 was found to be a significant candidate kinase participating in the negative control of Wnt/P-catenin signaling.Down-regulation of CDK11 led to the accumulation of Wnt/β-catenin signaling receptor complexes,in a manner dependent on intact adenomatosis polyposis coli(APC)protein.Further analysis showed that CDK11 modulation of Wnt/P-catenin signaling engaged the endolysosomal machinery,and CDK11 knockdown enhanced the colocalization of Wnt/β-catenin signaling receptor complexes with early endosomes and decreased colocalization with lysosomes.Mechanistically,CDK11 was found to function in Wnt/β-catenin signaling by regulating microtubule stability.Depletion of CDK11 down-regulated acetyl-a-tubulin.Moreover,co-IP assays demonstrated that CDK11 interacts with the a-tubulin deacetylase SIRT2,whereas SIRT2 down-regulation in CDK11-depleted cells reversed the accumulation of Wnt/(3-catenin signaling receptor complexes.CDK11 was found to suppress cell migration through altered W nt/β-catenin signaling.Conclusions:CDK11 is a negative modulator of Wnt/β-catenin signaling that stabilizes microtubules,thus resulting in the dysregulation of receptor complex trafficking from early endosomes to lysosomes.

pH-responsive magnesium- and carbonate-substituted apatite nano-crystals for efficient and cell-targeted delivery of transgenes

The short half-lives due to the enzymatic degradation in blood, the lack of tissue targetability and the incapability to passively diffuse across the plasma membrane and smoothly traffic across the harsh intracelluar environment are the major shortcomings for nucleic acid-based potential therapeutics, such as recombinant plasmid and antisense oligonucleotides or small interferring RNA (siRNA). Plasmid DNA containing a gene of interest could have immense impact as a promising therapeutic drug for treating genetic as well as acquired human diseases at the molecular level with high level of efficacy and precision. Thus both viral and non-viral synthetic vectors have been developed in the past decades to address the aforementioned challenges of naked DNA. While in the viral particles plasmid DNA is integrated into the viral genome, in most non-viral cases the DNA being anionic in nature is electrostatically associated with a cationic lipid or polymer forming lipoplex or polyplex, respectively, or a cationized inorganic gold, silica or iron oxide particle. Due to the potential immunogenicity and carcinogenicity issues with the viral particles, non-viral vectors have drawn much more attention for the clinical evaluation. However, the main concern of using non-biodegradable particles, specially the inorganic ones, is the adverse effects owing to their long term interactions with body components. We have recently developed biodegradable pH-sensitive inorganic nanoparticles of Mg/CaPi and carbonate apatite for efficient transgene delivery to primary, cancer and embryonic stem cells, by virtue of their high affinity binding with the DNA, ability to contact the cell membrane by ionic or ligand-receptor interactions and fast dissolution kinectis in endosomal acidic pH facilitating release of the DNA from the dissolving particles and also from the endosomes.

Synthesis, Biophysical Characterization and <i>in Vitro</i>Transfection Activity of Novel Bivalent Amine Cationic Lipids in the Absence of Dioleoylphosphatidylethanolamine (DOPE)

In this paper, a novel series of bis [(aminoethyl)]-amine cationic lipid derivatives have been synthesized and identified to purity by NMR and Elemental analysis. B16-F0 cells were transfected with cationic lipid/pEGFP-N1 and cationic lipid/&#223-gal lipoplexes complexed at +/&#45 charge ratios of 1:1, 2:1, and 4:1. Dimyristoyl derivative showed highest activity at charge ratio 2:1 and both dimyristoyl and dioleoyl derivatives showed similar &#223-gal activity at charge ratios 4:1. In 40 mM tris buffer pH 7.2 the dioleoyl derivative was able to fully complex with and retard pDNA at charge ratios above 2:1. None of the other lipid derivatives, dilauroyl, dimyristoyl, dipalmitoyl and distearoyl were able to fully neutralize the plasmid DNA at charge ratios similar to those used in the transfection experiment. The gel-to-liquid phase transition temperatures for dimyristoyl, dipalmitoyl and distearoyl were determined by a fluorescence anisotropy method to be 27.5&degC, 32.5&degC and 39&degC, respectively. A gel-to-liquid crystalline phase transition temperature below 37&degC, appears to be the crucial property that cationic lipids have to possess in order to mediate high levels of in vitro transfection activity in the absence of other helper lipids.

主旨报告

PL-1 Presynaptic Endosomal Cathepsin D Regulates the Biogenesis of GABAergic Synaptic Vesicles DUAN Shu-min Zhejiang University School of Medicine,Hangzhou,310058,China Dr. Shumin Duan is a professor at Zhejiang University School of Medicine,China. He got his Ph. D. from Kyushu University in Japan in 1991 and received a postdoctoral training at University of Hawaii and University of California at San Francisco during 1997-1999. His research interests include the function and the mechanisms of neuronglia interactions in health and disease and neural circuit mechanisms of brain functions. He is an academician of Chinese Academy of Sciences and a member of the Academy of Sciences for the Developing World (TWAS). He is the president of Chinese Society for Neuroscience and a council member of International Brain Research Organization. He serves as the Editor-in-Chief of Neuroscience Bulletin and an editorial board member in several international neuroscience journals.

Perturbation of autophagy pathways in murine alveolar macrophage by 2D TMDCs is chalcogen-dependent

Increasing risks of incidental and occupational exposures to two-dimensional transition metal dichalcogenides(2D TMDCs)due to their broad application in various areas raised their public health concerns.While the composition-dependent cytotoxicity of 2D TMDCs has been well-recognized,how the outer chalcogenide atoms and inner transition metal atoms differentially contribute to their perturbation on cell homeostasis at non-lethal doses remains to be identified.In the present work,we compared the autophagy induction and related mechanisms in response to WS_(2),NbS_(2),WSe_(2)and Nb Se_(2)nanosheets exposures in MH-S murine alveolar macrophages.All these 2D TMDCs had comparable physicochemical properties,overall cytotoxicity and capability in triggering autophagy in MH-S cells,but showed outer chalcogen-dependent subcellular localization and activation of autophagy pathways.Specifically,WS_(2)and NbS_(2)nanosheets adhered on the cell surface and internalized in the lysosomes,and triggered m TOR-dependent activation of autophagy.Meanwhile,WSe_(2)and Nb Se_(2)nanosheets had extensive distribution in cytoplasm of MH-S cells and induced autophagy in an m TOR-independent manner.Furthermore,the 2D TMDCs-induced perturbation on autophagy aggravated the cytotoxicity of respirable benzo[a]pyrene.These findings provide a deeper insight into the potential health risk of environmental 2D TMDCs from the perspective of homeostasis perturbation.

Stay in touch with the endoplasmic reticulum

The endoplasmic reticulum(ER),which is composed of a continuous network of tubules and sheets,forms the most widely distributed membrane system in eukaryotic cells.As a result,it engages a variety of organelles by establishing membrane contact sites(MCSs).These contacts regulate organelle positioning and remodeling,including fusion and fission,facilitate precise lipid exchange,and couple vital signaling events.Here,we systematically review recent advances and converging themes on ER-involved organellar contact.The molecular basis,cellular influence,and potential physiological functions for ER/nuclear envelope contacts with mitochondria,Golgi,endosomes,lysosomes,lipid droplets,autophagosomes,and plasma membrane are summarized.

M6PR interacts with the HA2 subunit of influenza A virus to facilitate the fusion of viral and endosomal membranes

Influenza A virus(IAV) commandeers numerous host cellular factors for successful replication. However, very few host factors have been revealed to be involved in the fusion of viral envelope and late endosomal membranes. In this study, we identified cation-dependent mannose-6-phosphate receptor(M6PR) as a crucial host factor for the replication of IAV. We found that siRNA knockdown of M6PR expression significantly reduced the growth titers of different subtypes of IAV, and that the inhibitory effect of M6PR siRNA treatment on IAV growth was overcome by the complement of exogenously expressed M6PR. When A549 cells were treated with siRNA targeting M6PR,the nuclear accumulation of viral nucleoprotein(NP) was dramatically inhibited at early timepoints post-infection, indicating that M6PR engages in the early stage of the IAV replication cycle. By investigating the role of M6PR in the individual entry and post-entry steps of IAV replication, we found that the downregulation of M6PR expression had no effect on attachment, internalization, early endosome trafficking,or late endosome acidification. However, we found that M6PR expression was critical for the fusion of viral envelope and late endosomal membranes. Of note, M6PR interacted with the hemagglutinin(HA) protein of IAV, and further studies showed that the lumenal domain of M6PR and the ectodomain of HA2 mediated the interaction and directly promoted the fusion of the viral and late endosomal membranes,thereby facilitating IAV replication. Together, our findings highlight the importance of the M6PR–HA interaction in the fusion of viral and late endosomal membranes during IAV replication.

Interconnection of cellular autophagy and endosomal vesicle trafficking and its role in hepatitis B virus replication and release

Hepatitis B virus(HBV)produces and releases various particle types,including complete virions,subviral particles with envelope proteins,and naked capsids.Recent studies demonstrate that HBV exploits distinct intracellular membrane trafficking pathways,including the endosomal vesicle trafficking and autophagy pathway,to assemble and release viral and subviral particles.Herein,we summarize the findings about the distinct roles of autophagy and endosomal membrane trafficking and the interaction of both pathways in HBV replication,assembly,and release.

Interplay of transport vesicles during plant-fungal pathogen interaction

Vesicle trafficking is an essential cellular process upon which many physiological processes of eukaryotic cells rely.It is usually the‘language’of communication among the components of the endomembrane system within a cell,between cells and between a cell and its external environment.Generally,cells have the potential to internalize membrane-bound vesicles from external sources by endocytosis.Plants constantly interact with both mutualistic and pathogenic microbes.A large part of this interaction involves the exchange of transport vesicles between the plant cells and the microbes.Usually,in a pathogenic interaction,the pathogen releases vesicles containing bioactive molecules that can modulate the host immunity when absorbed by the host cells.In response to this attack,the host cells similarly mobilize some vesicles containing pathogenesis-related compounds to the pathogen infection site to destroy the pathogen,prevent it from penetrating the host cell or annul its influence.In fact,vesicle trafficking is involved in nearly all the strategies of phytopathogen attack subsequent plant immune responses.However,this field of plant-pathogen interaction is still at its infancy when narrowed down to plant-fungal pathogen interaction in relation to exchange of transport vesicles.Herein,we summarized some recent and novel findings unveiling the involvement of transport vesicles as a crosstalk in plant-fungal phytopathogen interaction,discussed their significance and identified some knowledge gaps to direct future research in the field.The roles of vesicles trafficking in the development of both organisms are also established.

Lipid carriers for mRNA delivery

Messenger RNA(mRNA)is the template for protein biosynthesis and is emerging as an essential active molecule to combat various diseases,including viral infection and cancer.Especially,mRNA-based vaccines,as a new type of vaccine,have played a leading role in fighting against the current global pandemic of COVID-19.However,the inherent drawbacks,including large size,negative charge,and instability,hinder its use as a therapeutic agent.Lipid carriers are distinguishable and promising vehicles for mRNA delivery,owning the capacity to encapsulate and deliver negatively charged drugs to the targeted tissues and release cargoes at the desired time.Here,we first summarized the structure and properties of different lipid carriers,such as liposomes,liposome-like nanoparticles,solid lipid nanoparticles,lipid-polymer hybrid nanoparticles,nanoemulsions,exosomes and lipoprotein particles,and their applications in delivering mRNA.Then,the development of lipid-based formulations as vaccine delivery systems was discussed and highlighted.Recent advancements in the mRNA vaccine of COVID-19 were emphasized.Finally,we described our future vision and perspectives in this field.

Spastin is required for human immunodeficiency virus-1 efficient replication through cooperation with the endosomal sorting complex required for transport(ESCRT)protein

Human immunodeficiency virus-1(HIV-1)encodes simply 15 proteins and thus depends on multiple host cellular factors for virus reproduction.Spastin,a microtubule severing protein,is an identified HIV-1 dependency factor,but the mechanism regulating HIV-1 is unclear.Here,the study showed that knockdown of spastin inhibited the production of the intracellular HIV-1 Gag protein and new virions through enhancing Gag lysosomal degradation.Further investigation showed that increased sodium tolerance 1(IST1),the subunit of endosomal sorting complex required for transport(ESCRT),could interact with the MIT domain of spastin to regulate the intracellular Gag production.In summary,spastin is required for HIV-1 replication,while spastin-IST1 interaction facilitates virus production by regulating HIV-1 Gag intracellular trafficking and degradation.Spastin may serve as new target for HIV-1 prophylactic and therapy.