Exploring the triad: VPS35, neurogenesis, and neurodegenerative diseases.

Vacuolar protein sorting 35 (VPS35), a critical component of the retromer complex, plays a pivotal role in the pathogenesis of neurodegenerative diseases (NDs). It is involved in protein transmembrane sorting, facilitating the transport from endosomes to the trans-Golgi network (TGN) and plasma membrane. Recent investigations have compellingly associated mutations in the VPS35 gene with neurodegenerative disorders such as Parkinson's and Alzheimer's disease. These genetic alterations are implicated in protein misfolding, disrupted autophagic processes, mitochondrial dysregulation, and synaptic impairment. Furthermore, VPS35 exerts a notable impact on neurogenesis by influencing neuronal functionality, protein conveyance, and synaptic performance. Dysregulation or mutation of VPS35 may escalate the progression of neurodegenerative conditions, underscoring its pivotal role in safeguarding neuronal integrity. This review comprehensively discusses the role of VPS35 and its functional impairments in NDs. Furthermore, we provide an overview of the impact of VPS35 on neurogenesis and further explore the intricate relationship between neurogenesis and NDs. These research advancements offer novel perspectives and valuable insights for identifying potential therapeutic targets in the treatment of NDs.

TDP-43-stratified single-cell proteomic profiling of postmortem human spinal motor neurons reveals protein dynamics in amyotrophic lateral sclerosis.

Unbiased proteomics has been employed to interrogate central nervous system (CNS) tissues (brain, spinal cord) and fluid matrices (CSF, plasma) from amyotrophic lateral sclerosis (ALS) patients; yet, a limitation of conventional bulk tissue studies is that motor neuron (MN) proteome signals may be confounded by admixed non-MN proteins. Recent advances in trace sample proteomics have enabled quantitative protein abundance datasets from single human MNs (Cong et al., 2020b). In this study, we leveraged laser capture microdissection (LCM) and nanoPOTS (Zhu et al., 2018c) single-cell mass spectrometry (MS)-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control donor spinal cord tissues, leading to the identification of 2515 proteins across MNs samples (>900 per single MN) and quantitative comparison of 1870 proteins between disease groups. Furthermore, we studied the impact of enriching/stratifying MN proteome samples based on the presence and extent of immunoreactive, cytoplasmic TDP-43 inclusions, allowing identification of 3368 proteins across MNs samples and profiling of 2238 proteins across TDP-43 strata. We found extensive overlap in differential protein abundance profiles between MNs with or without obvious TDP-43 cytoplasmic inclusions that together point to early and sustained dysregulation of oxidative phosphorylation, mRNA splicing and translation, and retromer-mediated vesicular transport in ALS. Our data are the first unbiased quantification of single MN protein abundance changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein abundance changes in human neurologic diseases.

Beneficial Effect of a Small Pharmacologic Chaperone on the Established Alzheimer's Disease Phenotype.

BACKGROUND: The endosomal retromer complex system is a key controller for trafficking of proteins. Downregulation of its recognition core proteins, such as VPS35, is present in Alzheimer's disease (AD) brain, whereas its normalization prevents the development of AD pathology in a transgenic model with amyloid-ß deposits and tau tangles. OBJECTIVE: Assess the effect of targeting VPS35 after the AD pathology and memory impairments have developed. METHODS: Twelve-month-old triple transgenic mice were treated with a small pharmacological chaperone, TPT-172, or vehicle for 14 weeks. At the end of this period, the effect of the drug on their phenotype was evaluated. RESULTS: While control mice had a decline of learning and memory, the group receiving the chaperone did not. Moreover, when compared with controls the treated mice had significantly less amyloid-ß peptides and phosphorylated tau, elevation of post-synaptic protein, and reduction in astrocytes activation. CONCLUSION: Taken together, our findings demonstrate that pharmacologic stabilization of the retromer recognition core is beneficial also after the AD-like pathologic phenotype is established.

RAB21 controls autophagy and cellular energy homeostasis by regulating retromer-mediated recycling of SLC2A1/GLUT1.

The endosomal system maintains cellular homeostasis by coordinating multiple vesicular trafficking events, and the retromer complex plays a critical role in endosomal cargo recognition and sorting. Here, we demonstrate an essential role for the small GTPase RAB21 in regulating retromer-mediated recycling of the glucose transporter SLC2A1/GLUT1 and macroautophagy/autophagy. RAB21 depletion mis-sorts SLC2A1 to lysosomes and affects glucose uptake, thereby activating the AMPK-ULK1 pathway to increase autophagic flux. RAB21 depletion also increases lysosome function. Notably, RAB21 depletion does not overtly affect retrograde transport of IGF2R/CI-M6PR or WLS from endosomes to the trans-Golgi network. We speculate that RAB21 regulates fission of retromer-decorated endosomal tubules, as RAB21 depletion causes accumulation of the SNX27-containing retromer complex on enlarged endosomes at the perinuclear region. Functionally, RAB21 depletion sensitizes cancer cells to energy stress and inhibits tumor growth in vivo, suggesting an oncogenic role for RAB21. Overall, our study illuminates the role of RAB21 in regulating endosomal dynamics and maintaining cellular energy homeostasis and suggests RAB21 as a potential metabolic target for cancer therapy.

The regulatory role of the Aspergillus flavus core retromer complex in aflatoxin metabolism.

Aflatoxins are a series of highly toxic and carcinogenic secondary metabolites that are synthesized by Aspergillus species. The degradation of aflatoxin enzymes is an important regulatory mechanism which modulates mycotoxin producing. The retromer complex is responsible for the retrograde transport of specific biomolecules and the vacuolar fusion in the intracellular transport. Late endosomal-associated GTPase (Rab7) has been shown to be a downstream effector protein of the retromer complex. A deficiency in the retromer complex or Rab7 results in several cellular trafficking problems in yeast and humans, like protein abnormal accumulation. However, whether retromer dysfunction is involved in aflatoxin synthesis remains unclear. Here, we report that the core retromer complex, which comprises three vacuolar protein sorting-associated proteins (AflVps26-AflVps29-AflVps35), is essential for the development of dormant and resistant fungal forms such as conidia (asexual reproductive spore) and sclerotia (hardened fungal mycelium), as well as aflatoxin production and pathogenicity, in Aspergillus flavus. In particular, we show the AflVps26-AflVps29-AflVps35 complex is negatively correlated with aflatoxin exportation. Structural simulation, site-specific mutagenesis, and coimmunoprecipitation experiments showed that interactions among AflVps26, AflVps29, and AflVps35 played crucial roles in the retromer complex executing its core functions. We further found an intrinsic connection between AflRab7 and the retromer involved in vesicle-vacuole fusion, which in turn affected the accumulation of aflatoxin synthesis-associated enzymes, suggesting that they work together to regulate the production of toxins. Overall, these results provide mechanistic insights that contribute to our understanding of the regulatory role of the core retromer complex in aflatoxin metabolism.

Understanding the contributions of VPS35 and the retromer in neurodegenerative disease.

Perturbations of the endolysosomal pathway have been suggested to play an important role in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease (PD) and Alzheimer's disease (AD). Specifically, VPS35 and the retromer complex play an important role in the endolysosomal system and are implicated in the pathophysiology of these diseases. A single missense mutation in VPS35, Asp620Asn (D620N), is known to cause late-onset, autosomal dominant familial PD. In this review, we focus on the emerging role of the PD-linked D620N mutation in causing retromer dysfunction and dissect its implications in neurodegeneration. Additionally, we will discuss how VPS35 and the retromer are linked to AD, amyotrophic lateral sclerosis, and primary tauopathies. Interestingly, reduced levels of VPS35 and other retromer components have been observed in post-mortem brain tissue, suggesting a role for the retromer in the pathophysiology of these diseases. This review will provide a comprehensive dive into the mechanisms of VPS35 dysfunction in neurodegenerative diseases. Furthermore, we will highlight outstanding questions in the field and the retromer as a therapeutic target for neurodegenerative disease at large.

Plant ESCRT protein ALIX coordinates with retromer complex in regulating receptor-mediated sorting of soluble vacuolar proteins.

Vacuolar proteins play essential roles in plant physiology and development, but the factors and the machinery regulating their vesicle trafficking through the endomembrane compartments remain largely unknown. We and others have recently identified an evolutionarily conserved plant endosomal sorting complex required for transport (ESCRT)-associated protein apoptosis-linked gene-2 interacting protein X (ALIX), which plays canonical functions in the biogenesis of the multivesicular body/prevacuolar compartment (MVB/PVC) and in the sorting of ubiquitinated membrane proteins. In this study, we elucidate the roles and underlying mechanism of ALIX in regulating vacuolar transport of soluble proteins, beyond its conventional ESCRT function in eukaryotic cells. We show that ALIX colocalizes and physically interacts with the retromer core subunits Vps26 and Vps29 in planta. Moreover, double-mutant analysis reveals the genetic interaction of ALIX with Vps26 and Vps29 for regulating trafficking of soluble vacuolar proteins. Interestingly, depletion of ALIX perturbs membrane recruitment of Vps26 and Vps29 and alters the endosomal localization of vacuolar sorting receptors (VSRs). Taken together, ALIX functions as a unique retromer core subcomplex regulator by orchestrating receptor-mediated vacuolar sorting of soluble proteins.

The understudied links of the retromer complex to age-related pathways.

Neuronal aging is associated with numerous diseases resulting in memory impairment and functional decline. A common hallmark of these disorders is the accumulation of intracellular and extracellular protein aggregates. The retromer complex plays a central role in sorting proteins by marking them for reuse rather than degradation. Retromer dysfunction has been shown to induce protein aggregates and neurodegeneration, suggesting that it may be important for age-related neuronal decline and disease progression. Despite this, little is known about how aging influences retromer stability and the proteins with which it interacts. Detailed insights into age-dependent changes in retromer structure and function could provide valuable information towards treating and preventing many age-related neurodegenerative disorders. Here, we visit age-related pathways which interact with retromer function that ought to be further explored to determine its role in age-related neurodegeneration.

Phospholipase D and retromer promote recycling of TRPL ion channel via the endoplasmic reticulum.

Plasma membrane protein trafficking is of fundamental importance for cell function and cell integrity of neurons and includes regulated protein recycling. In this work, we report a novel role of the endoplasmic reticulum (ER) for protein recycling as discovered in trafficking studies of the ion channel TRPL in photoreceptor cells of Drosophila. TRPL is located within the rhabdomeric membrane from where it is endocytosed upon light stimulation and stored in the cell body. Conventional immunohistochemistry as well as stimulated emission depletion super-resolution microscopy revealed TRPL storage at the ER after illumination, suggesting an unusual recycling route of TRPL. Our results also imply that both phospholipase D (PLD) and retromer complex are required for correct recycling of TRPL to the rhabdomeric membrane. Loss of PLD activity in PLD3.1 mutants results in enhanced degradation of TRPL. In the retromer mutant vps35MH20 , TRPL is trapped in a Rab5-positive compartment. Evidenced by epistatic analysis in the double mutant PLD3.1 vps35MH20 , PLD activity precedes retromer function. We propose a model in which PLD and retromer function play key roles in the transport of TRPL to an ER enriched compartment.

Retromer Complex and PI3K Complex II-Related Genes Mediate the Yeast (Saccharomyces cerevisiae) Sodium Metabisulfite Resistance Response.

Sodium metabisulfite (Na2S2O5) is widely used as a preservative in the food and wine industry. However, it causes varying degrees of cellular damage to organisms. In order to improve our knowledge regarding its cyto-toxicity, a genome-wide screen using the yeast single deletion collection was performed. Additionally, a total of 162 Na2S2O5-sensitive strains and 16 Na2S2O5-tolerant strains were identified. Among the 162 Na2S2O5 tolerance-related genes, the retromer complex was the top enriched cellular component. Further analysis demonstrated that retromer complex deletion leads to increased sensitivity to Na2S2O5, and that Na2S2O5 can induce mislocalization of retromer complex proteins. Notably, phosphatidylinositol 3-monophosphate kinase (PI3K) complex II, which is important for retromer recruitment to the endosome, might be a potential regulator mediating retromer localization and the yeast Na2S2O5 tolerance response. Na2S2O5 can decrease the protein expressions of Vps34, which is the component of PI3K complex. Therefore, Na2S2O5-mediated retromer redistribution might be caused by the effects of decreased Vps34 expression levels. Moreover, both pharmaceutical inhibition of Vps34 functions and deletions of PI3K complex II-related genes affect cell tolerance to Na2S2O5. The results of our study provide a global picture of cellular components required for Na2S2O5 tolerance and advance our understanding concerning Na2S2O5-induced cytotoxicity effects.

VPS35 Downregulation Alters Degradation Pathways in Neuronal Cells.

BACKGROUND: The vacuolar protein sorting 35 (VPS35) is the main component of the retromer recognition core complex system which regulates intracellular cargo protein sorting and trafficking. Downregulation of VPS35 has been linked to the pathogenesis of neurodegenerative disorders such Alzheimer's and Parkinson's diseases via endosome dysregulation. OBJECTIVE: Here we show that the genetic manipulation of VPS35 affects intracellular degradation pathways. METHODS: A neuronal cell line expressing human APP Swedish mutant was used. VPS35 silencing was performed treating cells with VPS35 siRNA or Ctr siRNA for 72 h. RESULTS: Downregulation of VPS35 was associated with alteration of autophagy flux and intracellular accumulation of acidic and ubiquitinated aggregates suggesting that dysfunction of the retromer recognition core leads to a significant alteration in both pathways. CONCLUSION: Taken together, our data demonstrate that besides cargo sorting and trafficking, VPS35 by supporting the integral function of the retromer complex system plays an important role also as a critical regulator of intracellular degradation pathways.

Expression of Low Level of VPS35-mCherry Fusion Protein Diminishes Vps35 Depletion Induced Neuron Terminal Differentiation Deficits and Neurodegenerative Pathology, and Prevents Neonatal Death.

Vps35 (vacuolar protein sorting 35) is a key component of retromer that consists of Vps35, Vps26, and Vps29 trimers, and sortin nexin dimers. Dysfunctional Vps35/retromer is believed to be a risk factor for development of various neurodegenerative diseases. Vps35Neurod6 mice, which selectively knock out Vps35 in Neurod6-Cre+ pyramidal neurons, exhibit age-dependent impairments in terminal differentiation of dendrites and axons of cortical and hippocampal neurons, neuro-degenerative pathology (i.e., increases in P62 and Tdp43 (TAR DNA-binding protein 43) proteins, cell death, and reactive gliosis), and neonatal death. The relationships among these phenotypes and the underlying mechanisms remain largely unclear. Here, we provide evidence that expression of low level of VPS35-mCherry fusion protein in Vps35Neurod6 mice could diminish the phenotypes in an age-dependent manner. Specifically, we have generated a conditional transgenic mouse line, LSL-Vps35-mCherry, which expresses VPS35-mCherry fusion protein in a Cre-dependent manner. Crossing LSL-Vps35-mCherry with Vps35Neurod6 to obtain TgVPS35-mCherry, Vps35Neurod6 mice prevent the neonatal death and diminish the dendritic morphogenesis deficit and gliosis at the neonatal, but not the adult age. Further studies revealed that the Vps35-mCherry transgene expression was low, and the level of Vps35 mRNA comprised only ~5-7% of the Vps35 mRNA of control mice. Such low level of VPS35-mCherry could restore the amount of other retromer components (Vps26a and Vps29) at the neonatal age (P14). Importantly, the neurodegenerative pathology presented in the survived adult TgVps35-mCherry; Vps35Neurod6 mice. These results demonstrate the sufficiency of low level of VPS35-mCherry fusion protein to diminish the phenotypes in Vps35Neurod6 mice at the neonatal age, verifying a key role of neuronal Vps35 in stabilizing retromer complex proteins, and supporting the view for Vps35 as a potential therapeutic target for neurodegenerative diseases.

A new functional role of mitochondria-anchored protein ligase in peroxisome morphology in mammalian cells.

Mitochondria and peroxisomes are metabolically interconnected and functionally active subcellular organelles. These two dynamic organelles, share a number of common biochemical functions such as ß-oxidation of fatty acids and detoxification of peroxides. The biogenesis and morphology of both these organelles in the mammalian cells is controlled by common transcription factors like PGC1α, and by a common fission machinery comprising of fission proteins like DRP1, Mff, and hFis1, respectively. In addition, the outer membrane mitochondria-anchored protein ligase (MAPL), the first mitochondrial SUMO E3 ligase with a RING-finger domain, also regulates mitochondrial morphology inducing mitochondrial fragmentation upon its overexpression. This fragmentation is dependent on both the RING domain of MAPL and the presence of the mitochondrial fission GTPase dynamin-related protein-1 (DRP1). Earlier studies have demonstrated that mitochondrial-derived vesicles are formed independently of the known mitochondrial fission GTPase, DRP1 are enriched for MAPL and are targeted to peroxisomes. The current study shows that MAPL regulates morphology of peroxisomes in a cell-type specific manner. Fascinatingly, the peroxisome elongation caused either due to silencing of DRP1 or by addition of polyunsaturated fatty acid, docosahexaenoic acid was blocked by overexpressing MAPL in mammalian cell lines. Furthermore, the transfection and colocalisation studies of MAPL with peroxisome membrane marker, PMP70, in different cell lines clearly revealed a cell-type specificity of transport of MAPL to peroxisomes. Previous work has placed the Vps35 (retromer component) as vital for delivery of MAPL to peroxisomes, placing the retromer as critical for the formation of MAPL-positive mitochondrial-derived vesicles. The results of polyethylene glycol-based cell-cell fusion assay signified that the enrichment of MAPL in peroxisomes is through vesicles and a retromer dependent phenomenon. Thus, a novel function for MAPL in peroxisomes is established to regulate peroxisome elongation and morphology under growth conditions and thus possibly modulate peroxisome fission.

Depletion of VPS35 attenuates metastasis of hepatocellular carcinoma by restraining the Wnt/PCP signaling pathway.

Vesicle Protein Sorting 35 (VPS35) is a novel oncogene that promotes tumor growth through the PI3K/AKT signaling in hepatocellular carcinoma (HCC). However, the role of VPS35 in HCC metastasis and the underlying mechanisms remain largely unclear. In this study, we observed that overexpression of VPS35 enhanced hepatoma cell invasion and metastasis by inducing epithelial-mesenchymal transition (EMT)-related gene expression. Conversely, knockout of VPS35 significantly inhibited hepatoma cell migration and invasion. Furthermore, depletion of VPS35 decreased the lung metastasis of HCC in nude mice. By transcriptome analysis, we determined that VPS35 promoted HCC metastasis by activating the Wnt/non-canonical planar cell polarity (PCP) pathway. Mechanistically, VPS35 activated the PCP pathway by regulating membrane sorting and trafficking of Frizzled-2 (FZD2) and ROR1 in hepatoma cells. Collectively, our results indicate that VPS35 promotes HCC metastasis via enhancing the Wnt/PCP signaling, thus providing a potential prognostic marker and therapeutic target for HCC.

Toward Understanding the Molecular Role of SNX27/Retromer in Human Health and Disease.

Aberrations in membrane trafficking pathways have profound effects in cellular dynamics of cellular sorting processes and can drive severe physiological outcomes. Sorting nexin 27 (SNX27) is a metazoan-specific sorting nexin protein from the PX-FERM domain family and is required for endosomal recycling of many important transmembrane receptors. Multiple studies have shown SNX27-mediated recycling requires association with retromer, one of the best-known regulators of endosomal trafficking. SNX27/retromer downregulation is strongly linked to Down's Syndrome (DS) via glutamate receptor dysfunction and to Alzheimer's Disease (AD) through increased intracellular production of amyloid peptides from amyloid precursor protein (APP) breakdown. SNX27 is further linked to addiction via its role in potassium channel trafficking, and its over-expression is linked to tumorigenesis, cancer progression, and metastasis. Thus, the correct sorting of multiple receptors by SNX27/retromer is vital for normal cellular function to prevent human diseases. The role of SNX27 in regulating cargo recycling from endosomes to the cell surface is firmly established, but how SNX27 assembles with retromer to generate tubulovesicular carriers remains elusive. Whether SNX27/retromer may be a putative therapeutic target to prevent neurodegenerative disease is now an emerging area of study. This review will provide an update on our molecular understanding of endosomal trafficking events mediated by the SNX27/retromer complex on endosomes.

Dysregulation of the Retromer Complex in Brain Endothelial Cells Results in Accumulation of Phosphorylated Tau.

INTRODUCTION: Transport through endothelial cells of the blood-brain barrier (BBB) involves a complex group of structures of the endo-lysosome system such as early and late endosomes, and the retromer complex system. Studies show that neuronal dysregulation of the vacuolar protein sorting 35 (VPS35), the main component of the retromer complex recognition core, results in altered protein trafficking and degradation and is involved in neurodegeneration. Since the functional role of VPS35 in endothelial cells has not been fully investigated, in the present study we aimed at characterizing the effect of its downregulation on these pathways. METHODS: Genetic silencing of VPS35 in human brain endothelial cells; measurement of retromer complex system proteins, autophagy and ubiquitin-proteasome systems. RESULTS: VPS35-downregulated endothelial cells had increased expression of LC3B2/1 and more ubiquitinated products, markers of autophagy flux and impaired proteasome activity, respectively. Additionally, compared with controls VPS35 downregulation resulted in significant accumulation of tau protein and its phosphorylated isoforms. DISCUSSION: Our findings demonstrate that in brain endothelial cells retromer complex dysfunction by influencing endosome-lysosome degradation pathways results in altered proteostasis. Restoration of the retromer complex system function should be considered a novel therapeutic approach to rescue endothelial protein transport.

VPS35, the core component of the retromer complex, and Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease that is common in middle-aged and elderly people, and its onset is related to multiple factors, such as heredity, environment, and age. The vesicle protein sorting 35 (VPS35) gene was found to be a late-onset autosomal dominant familial PD (PARK17) causative gene. The protein encoded by this gene is located in the endosome and aggregates with other membrane proteins to form a retromer complex, which participates in the membrane protein cycle between the endosome and the Golgi network. Increasing evidence shows that VPS35 may participate in the pathogenesis of PD by affecting autophagy, mitochondria, neurosynaptic transmission, dopamine signaling pathways, and so forth, and it can interact with other disease-causing genes of familial PD. This article aimed to review the functions of VPS35 and the mechanism of its mutations in PD that have been discovered in recent years.

An update on cellular and molecular determinants of Parkinson's disease with emphasis on the role of the retromer complex.

Parkinson's disease (PD) is a highly prevalent neurodegenerative condition. The disease involves the progressive degeneration of dopaminergic neurons located in the substantia nigra pars compacta. Among late-onset, familial forms of Parkinson are cases with mutations in the PARK17 locus encoding the vacuolar protein sorting 35 (Vps35), a subunit of the retromer complex. The retromer complex is composed of a heterotrimeric protein core (Vps26-Vps35-Vps29). The best-known role of retromer is the retrieval of cargoes from endosomes to the Golgi complex or the plasma membrane. However, recent literature indicates that retromer performs roles associated with lysosomal and mitochondrial functions and degradative pathways such as autophagy. A common point mutation affecting the retromer subunit Vps35 is D620N, which has been linked to the alterations in the aforementioned cellular processes as well as with neurodegeneration. Here, we review the main aspects of the malfunction of the retromer complex and its implications for PD pathology. Besides, we highlight several controversies still awaiting clarification.

The Role of VPS35 in the Pathobiology of Parkinson's Disease.

The vacuolar protein sorting 35 (VPS35) gene located on chromosome 16 has recently emerged as a cause of late-onset familial Parkinson's disease (PD) (PARK17). The gene encodes a 796-residue protein nearly ubiquitously expressed in human tissues. The protein localizes on endosomes where it assembles with other peripheral membrane proteins to form the retromer complex. How VPS35 mutations induce dopaminergic neuron degeneration in humans is still unclear. Because the retromer complex recycles the receptors that mediate the transport of hydrolase to lysosome, it has been suggested that VPS35 mutations lead to impaired lysosomal and autophagy function. Recent studies also demonstrated that VPS35 and the retromer complex influence mitochondrial homeostasis, suggesting that VPS35 mutations elicit mitochondrial dysfunction. More recent studies have identified a key role of VPS35 in neurotransmission, whilst others reported a functional interaction between VPS35 and other genes associated with familial PD, including α-SYNUCLEIN-PARKIN-LRRK2. Here, we review the biological role of VPS35 protein, the VPS35 mutations identified in human PD patients, and the potential molecular mechanism by which VPS35 mutations can induce progressive neurodegeneration in PD.

The retromer is co-opted to deliver lipid enzymes for the biogenesis of lipid-enriched tombusviral replication organelles.

Biogenesis of viral replication organelles (VROs) is critical for replication of positive-strand RNA viruses. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely related carnation Italian ringspot virus (CIRV) hijack the retromer to facilitate building VROs in the surrogate host yeast and in plants. Depletion of retromer proteins, which are needed for biogenesis of endosomal tubular transport carriers, strongly inhibits the peroxisome-associated TBSV and the mitochondria-associated CIRV replication in yeast and in planta. In vitro reconstitution revealed the need for the retromer for the full activity of the viral replicase. The viral p33 replication protein interacts with the retromer complex, including Vps26, Vps29, and Vps35. We demonstrate that TBSV p33-driven retargeting of the retromer into VROs results in delivery of critical retromer cargoes, such as 1) Psd2 phosphatidylserine decarboxylase, 2) Vps34 phosphatidylinositol 3-kinase (PI3K), and 3) phosphatidylinositol 4-kinase (PI4Kα-like). The recruitment of these cellular enzymes by the co-opted retromer is critical for de novo production and enrichment of phosphatidylethanolamine phospholipid, phosphatidylinositol-3-phosphate [PI(3)P], and phosphatidylinositol-4-phosphate [PI(4)P] phosphoinositides within the VROs. Co-opting cellular enzymes required for lipid biosynthesis and lipid modifications suggest that tombusviruses could create an optimized lipid/membrane microenvironment for efficient VRO assembly and protection of the viral RNAs during virus replication. We propose that compartmentalization of these lipid enzymes within VROs helps tombusviruses replicate in an efficient milieu. In summary, tombusviruses target a major crossroad in the secretory and recycling pathways via coopting the retromer complex and the tubular endosomal network to build VROs in infected cells.