Dileucine Motif Research Articles

Regulatory role of sorting nexin 5 in protein stability and vesicular targeting of vesicular acetylcholine transporter to synaptic vesicle-like vesicles in PC12 cells

Accurate targeting of vesicular acetylcholine transporter (VAChT) to synaptic vesicles (SVs) is indispensable for efficient cholinergic transmission. Previous studies have suggested that the dileucine motif within the C-terminus of the transporter is sufficient for its targeting to SVs. However, the cytosolic machinery underlying specific regulation of VAChT trafficking and targeting to SVs is still unclear. Here we used the C-terminus of VAChT as a bait in a yeast two-hybrid screen to identify sorting nexin 5 (SNX5) as its novel interacting protein. SNX5 was detected in the SVs enriched LP2 subcellular fraction of rat brain homogenate and showed strong colocalization with VAChT in both brain sections and PC12 cells. Binding assays suggested that the C-terminal domain of VAChT can interact with both BAR and PX domain of SNX5. Depletion of SNX5 enhanced the degradation of VAChT and the process was mediated through the lysosomal pathway. More importantly, we found that, in PC12 cells, the depletion of SNX5 expression significantly decreased the synaptic vesicle-like vesicles (SVLVs) localization of VAChT. Therefore, the results suggest that SNX5 is a novel regulator for both stability and SV targeting of VAChT.

TASK channels: channelopathies, trafficking, and receptor-mediated inhibition

TWIK-related acid-sensitive K+ (TASK) channels contribute to the resting membrane potential in various kinds of cells, such as brain neurons, smooth muscle cells, and endocrine cells. Loss-of-function mutations at multiple sites in the KCNK3 gene encoding for TASK1 channels are one of the causes of pulmonary arterial hypertension in humans, whereas a mutation at only one site is reported for TASK3 channels, resulting in a syndrome of mental retardation, hypotonia, and facial dysmorphism. TASK channels are subject to regulation by G protein-coupled receptors (GPCRs). Two mechanisms have been proposed for the GPCR-mediated inhibition of TASK channels: a change in gating and channel endocytosis. The most feasible mechanism for altered gating is diacylglycerol binding to a site in the C-terminus, which is shared by TASK1 and TASK3. The inhibition of channel function by endocytosis requires the presence of a tyrosine residue subjected to phosphorylation by the non-receptor tyrosine kinase Src and a dileucine motif in the C-terminus of TASK1. Therefore, homomeric TASK1 and heteromeric TASK1-TASK3 channels, but not homomeric TASK3, are internalized by GPCR stimulation. Tyrosine phosphorylation by Src is expected to result in a conformational change in the C-terminus, allowing for AP-2, an adaptor protein for clathrin, to bind to the dileucine motif. It is likely that a raft membrane domain is a platform where TASK1 is located and the signaling molecules protein kinase C, Pyk2, and Src are recruited in sequence in response to GPCR stimulation.

SUN-138 Functional Characterization of Tumor-Associated Germline TMEM127 Variants Reveals Novel Insights into Membrane Topology and Protein Trafficking

TMEM127, a tumor suppressor gene in hereditary pheochromocytomas and paragangliomas, encodes for a poorly characterized, ubiquitous, transmembrane (TM) protein with no substantial identity to any other protein and no identifiable functional domains other than putative TM domains. The function and regulation of TMEM127 remain poorly defined which limits prediction of pathogenicity of germline TMEM127 variants. Characterizing structure-function features of TMEM127 is relevant for understanding how dysfunction of this tumor suppressor can lead to inherited tumors. Moreover, a better understanding of TMEM127 functional domains and critical residues will increase our ability to identify pathogenic variants which will improve interpretation of genetic screening results and help guide clinical decisions for patients and their families. In this study, we investigated subcellular localization and steady-state levels of patient-derived, tumor-associated, germline TMEM127 variants (n=21; 16 missense and 5 truncating/frameshift/indel variants). We used confocal immunofluorescence and immunoblot analysis of GFP-TMEM127 constructs expressed in HEK293T cells. Wild-type (WT) TMEM127 localized predominantly to endo-lysosomal vesicles (punctate) with some plasma membrane localization. The localization of variant TMEM127 proteins displayed three distinct patterns: punctate (similar to WT), diffuse (cytoplasmic), and plasma membrane only. All diffuse proteins, and a few punctate proteins, decreased at a faster rate than the WT suggesting instability. Variants resulting in diffuse proteins occurred within TM domains and indicated that membrane binding ability was lost. This observation, supported by in silico analysis and selective permeability assays, led us to conclude that TMEM127 is a four-TM protein, not a three-TM protein, as previously predicted, with a novel TM domain in the N-terminus. One variant with predominantly plasma membrane localization indicated that membrane binding ability was maintained but internalization capability was lost. We identified an atypical, extended acidic dileucine motif in the C-terminal which is responsible for TMEM127 internalization and showed that it is mediated by clathrin. Our findings provide novel insights into structure-function features of TMEM127 which will allow for better understanding of its physiological role and improved prediction of pathogenic variants.

Human mastadenovirus-B (HAdV-B)-specific E3-CR1β and E3-CR1γ glycoproteins interact with each other and localize at the plasma membrane of non-polarized airway epithelial cells

The E3 region of all simian and human types classified within species Human mastadenovirus B (HAdV-B) encodes two unique highly conserved ORFs of unknown function designated E3-CR1β and E3-CR1γ. We generated a HAdV-3 mutant encoding small epitope tags at the N-termini of both E3-CR1β and E3-CR1γ (HAdV-3 N-tag wt) and a double knock out (HAdV-3 N-tag DKO) mutant virus that does not express either protein. Our studies show that HAdV-3 E3-CR1β and E3-CR1γ are type I transmembrane proteins that are produced predominantly at late times post infection, are glycosylated, co-localize at the plasma membrane of non-polarized epithelial cells, and interact with each other. At their extreme C-termini HAdV-B E3-CR1β and E3-CR1γ possess a conserved di-leucine motif followed by a class II PDZ domain binding motif (PBM). HAdV-3 E3-CR1β and E3-CR1γ are dispensable for virus growth, progeny release, spread, and plaque formation in A549 cells.

PAK Kinases Target Sortilin and Modulate Its Sorting.

The multifunctional type 1 receptor sortilin is involved in endocytosis and intracellular transport of ligands. The short intracellular domain of sortilin binds several cytoplasmic adaptor proteins (e.g., the AP-1 complex and GGA1 to -3), most of which target two well-defined motifs: a C-terminal acidic cluster dileucine motif and a YXXΦ motif in the proximal third of the domain. Both motifs contribute to endocytosis as well as Golgi-endosome trafficking of sortilin. The C-terminal acidic cluster harbors a serine residue, which is subject to phosphorylation by casein kinase. Phosphorylation of this serine residue is known to modulate adaptor binding to sortilin. Here, we show that the cytoplasmic domain of sortilin also engages Rac-p21-activated kinases 1 to 3 (PAK1-3) via a binding segment that includes a tyrosine-based motif, also encompassing a serine residue. We further demonstrate that PAK1-3 specifically phosphorylate this serine residue and that this phosphorylation alters the affinity for AP-1 binding and consequently changes the intracellular localization of sortilin as a result of modulated trafficking. Our findings suggest that trafficking of ligands bound to sortilin is in part regulated by group A PAK kinases, which are downstream effectors of Rho GTPases and are known to affect a variety of processes by remodeling the cytoskeleton and by promoting gene transcription and cell survival.

Integration of JAK/STAT receptor-ligand trafficking, signalling and gene expression in Drosophila melanogaster cells.

The JAK/STAT pathway is an essential signalling cascade required for multiple processes during development and for adult homeostasis. A key question in understanding this pathway is how it is regulated in different cell contexts. Here, we have examined how endocytic processing contributes to signalling by the single cytokine receptor in Drosophila melanogaster cells, Domeless. We identify an evolutionarily conserved di-leucine (di-Leu) motif that is required for Domeless internalisation and show that endocytosis is required for activation of a subset of Domeless targets. Our data indicate that endocytosis both qualitatively and quantitatively regulates Domeless signalling. STAT92E, the single STAT transcription factor in Drosophila, appears to be the target of endocytic regulation, and our studies show that phosphorylation of STAT92E on Tyr704, although necessary, is not always sufficient for target transcription. Finally, we identify a conserved residue, Thr702, which is essential for Tyr704 phosphorylation. Taken together, our findings identify previously unknown aspects of JAK/STAT pathway regulation likely to play key roles in the spatial and temporal regulation of signalling in vivo.

Exploration of two major boron transport genes BOR1 and NIP5;1 in the genomes of different plants

Boron (B) is an essential plant micronutrient but studies regarding its transport are still limited to a few plants. This work identified two major B transport sequences in plants, NIP5;1 boric acid channel protein and BOR1 transporter. 80 BOR1 and 34 NIP5;1 homologs were identified in 18 different plant genomes. BOR1 homologs had a HCO3-transporter domain, 649–737 amino-acid residues with mainly basic nature, putative 8–11 transmembrane domains (TMDs) and 11–13 exons. NIP5;1 homologs had a MIP family domain, 294–311 amino-acid residues with basic nature, 5–6 putative TMDs and 3–5 exons. Tyrosine-based motif, acidic di-leucine motif and lysine residue, reported for polarity, vacuolar sorting and B-dependent degradation, were identified in BOR1 homologs. Two NPA motifs and an ar/R selectivity filter with AIGR residues, reportedly essential in B transport, were also found in NIP5;1 homologs. Two NPA motifs in AtNIP5;1 and OsNIP3;1 homologs were NPS and NPV, whereas in sequences homologous to AtNIP6;1 were NPA/V. Besides, ar/R selectivity filters were identified with A(N/S/T)IGR residues in NIP5;1 and NIP3;1 homologs. The BOR1 and NIP5;1 model structures were mainly conserved. Under different perturbations, Arabidopsis thaliana NIP5;1 and NIP6;1 genes demonstrated similar expression patterns although they act in different tissues, suggesting a common regulatory mechanism, whereas BOR1 showed a different expression pattern. BOR1 was substantially expressed in primary root, radicle and flower; NIP5;1 in primary root and roots, and NIP6;1 in petiole. NIP5;1, 6;1 and BOR1 expression in other plant organs implied their involvement in different pathways in addition to B uptake and its mobilization.

The ABCG2 multidrug transporter is a pump gated by a valve and an extracellular lid

The human ATP-binding cassette transporter ABCG2 is a key to anticancer resistance and physiological detoxification. However, the molecular mechanism of substrate transport remains enigmatic. A hydrophobic di-leucine motif in the ABCG2 core separates a large intracellular cavity from a smaller upper cavity. We show that the di-leucine motif acts as a valve that controls drug extrusion. Moreover, the extracellular structure engages the re-entry helix and all extracellular loops to form a roof architecture on top of the upper cavity. Disulfide bridges and a salt bridge limit roof flexibility, but provide a lid-like function to control drug release. We propose that drug translocation from the central to the upper cavities through the valve is driven by a squeezing motion, suggesting that ABCG2 operates similar to a peristaltic pump. Finally, the roof contains essential residues, offering therapeutic options to block ABCG2 by either targeting the valve or essential residues in the roof.

Function of the cargo sorting dileucine motif in a cytomegalovirus immune evasion protein.

As an immune evasion mechanism, cytomegaloviruses (CMVs) have evolved proteins that interfere with cell surface trafficking of MHC class-I (MHC-I) molecules to tone down recognition by antiviral CD8 T cells. This interference can affect the trafficking of recently peptide-loaded MHC-I from the endoplasmic reticulum to the cell surface, thus modulating the presentation of viral peptides, as well as the recycling of pre-existing cell surface MHC-I, resulting in reduction of the level of overall MHC-I cell surface expression. Murine cytomegalovirus (mCMV) was paradigmatic in that it led to the discovery of this immune evasion strategy of CMVs. Members of its m02-m16 gene family code for type-I transmembrane glycoproteins, proven or predicted, most of which carry cargo sorting motifs in their cytoplasmic, C-terminal tail. For the m06 gene product m06 (gp48), the cargo has been identified as being MHC-I, which is linked by m06 to cellular adapter proteins AP-1A and AP-3A through the dileucine motif EPLARLL. Both APs are involved in trans-Golgi network (TGN) cargo sorting and, based on transfection studies, their engagement by the dileucine motif was proposed to be absolutely required to prevent MHC-I exposure at the cell surface. Here, we have tested this prediction in an infection system with the herein newly described recombinant virus mCMV-m06AA, in which the dileucine motif is destroyed by replacing EPLARLL with EPLARAA. This mutation has a phenotype in that the transition of m06-MHC-I complexes from early endosomes (EE) to late endosomes (LE)/lysosomes for degradation is blocked. Consistent with the binding of the MHC-I α-chain to the luminal domain of m06, the m06-mediated disposal of MHC-I did not require the β2m chain of mature MHC-I. Unexpectedly, however, disconnecting MHC-I cargo from AP-1A/3A by the motif mutation in m06 had no notable rescuing impact on overall cell surface MHC-I, though it resulted in some improvement of the presentation of viral antigenic peptides by recently peptide-loaded MHC-I. Thus, the current view on the mechanism by which m06 mediates immune evasion needs to be revised. While the cargo sorting motif is critically involved in the disposal of m06-bound MHC-I in the endosomal/lysosomal pathway at the stage of EE to LE transition, this motif-mediated disposal is not the critical step by which m06 causes immune evasion. We rather propose that engagement of AP-1A/3A by the cargo sorting motif in m06 routes the m06-MHC-I complexes into the endosomal pathway and thereby detracts them from the constitutive cell surface transport.

A dileucine motif in the COOH-terminal domain of NKCC1 targets the cotransporter to the plasma membrane

Na+-K+-2Cl- cotransporter-1 (NKCC1) mediates the electroneutral transport of Na+, K+, and Cl- and is normally localized to the basolateral membrane of polarized epithelial cells. We recently reported the first known solute carrier family 12 member 2 ( SLC12A2) mutation (we call NKCC1-DFX) that causes epithelial dysfunction in an undiagnosed disease program case. The heterozygous mutation leads to truncation of the COOH-terminal tail of the cotransporter, resulting in both mutant and wild-type cotransporters being mistrafficked to the apical membrane of polarized epithelial cells. Here we demonstrate by using consecutive truncations and site-directed mutagenesis of the COOH-terminal domain of NKCC1 that truncation of NKCC1 COOH domain uncouples the cotransporter from the lateral membrane. We identify a dileucine motif that, when mutated, leads to cotransporter accumulation in the cytoplasm and mistrafficking to the apical/subapical region of epithelial cells, thereby recapitulating the phenotype observed with the patient mutation. We show that truncation deletion and LL substitution mutants are trafficked out of the endoplasmic reticulum and trans-Golgi network but accumulate in early and late endosomes where they are degraded.

CALHM1/CALHM3 channel is intrinsically sorted to the basolateral membrane of epithelial cells including taste cells

The CALHM1/CALHM3 channel in the basolateral membrane of polarized taste cells mediates neurotransmitter release. However, mechanisms regulating its localization remain unexplored. Here, we identified CALHM1/CALHM3 in the basolateral membrane of type II taste cells in discrete puncta localized close to afferent nerve fibers. As in taste cells, CALHM1/CALHM3 was present in the basolateral membrane of model epithelia, although it was distributed throughout the membrane and did not show accumulation in puncta. We identified canonical basolateral sorting signals in CALHM1 and CALHM3: tyrosine-based and dileucine motifs. However, basolateral sorting remained intact in mutated channels lacking those signals, suggesting that non-canonical signals reside elsewhere. Our study demonstrates intrinsic basolateral sorting of CALHM channels in polarized cells, and provides mechanistic insights.

A novel function for the ER retention signals in the C-terminus of kainate receptor subunit, GluK5

Classically, endoplasmic reticulum (ER) retention signals in secreted integral membrane proteins impose the requirement to assemble with other cognate subunits to form functional assemblies before they can exit the ER. We report that GluK5 has two ER retention signals in its cytoplasmic C-terminus: an arginine-based signal and a di-leucine motif previously thought to be an endocytic motif. GluK5 assembles with GluK2, but surprisingly GluK2 association does little to block the ER retention signals. We find instead that the ER retention signals are blocked by two proteins involved in intracellular trafficking, SAP97 and CASK. We show that SAP97, in the presence of CASK and the receptor complex, assumes an extended conformation. In the extended conformation, SAP97 makes its SH3 and GuK domains available to bind and sterically mask the ER retention signals in the GluK5 C-terminus. SAP97 and CASK are also necessary for sorting receptor cargoes into the local dendritic secretory pathway in neurons. We show that the ER retention signals of GluK5 play a vital role in sorting the receptor complex in the local dendritic secretory pathway in neurons. These data suggest a new role for ER retention signals in trafficking integral membrane proteins in neurons. SignificanceWe present evidence that the ER retention signals in the kainate receptors containing GluK5 impose a requirement for sorting into local dendritic secretory pathways in neurons, as opposed to traversing the somatic Golgi apparatus. There are two ER retention signals in the C-terminus of GluK5. We show that both are blocked by physical association with SAP97 and CASK. The SH3 and GuK domains of SAP97, in the presence of CASK, bind directly to each ER retention signal and form a complex. These results support an entirely new function for ER retention signals in the C-termini of neuronal receptors, such as NMDA and kainate receptors, and define a mechanism for selective entry of receptors into local secretory pathways.

Peptides derived from cadherin juxtamembrane region inhibit platelet function.

The juxtamembrane domains (JMD) of transmembrane proteins are rich in critical peptide sequences that participate in dynamic cell signalling events. Synthetic JMD peptides derived from cadherin cell adhesion proteins have previously been shown to modulate platelet function. In this study, we aimed to develop functional bioactive agents from bioinformatically identified critical peptide sequences. We synthesized overlapping 12–15 amino acid peptides from E- and N-cadherin JMD and assessed their effect on platelet aggregation and platelet ATP secretion. Peptides derived from close to the membrane proximal region inhibit platelet function. Sequential deletion of amino acids from the N- and C-termini of the inhibitory E-cadherin peptides identified the short K756EPLLP763 motif as a critical bioactive sequence. Alanine scanning studies further identified that the di-leucine (LL) motif and positively charged lysine (K) are crucial for peptide activity. Moreover, scrambled peptides failed to show any effect on platelet activity. We conclude that peptides derived from JMD of E-cadherin provide potential lead peptides for the development of anti-thrombotic agents and to enable further understanding of the role of cadherins in platelet function.

Mutations in Disordered Regions Can Cause Disease by Creating Dileucine Motifs

Many disease-causing missense mutations affect intrinsically disordered regions (IDRs) of proteins, but the molecular mechanism of their pathogenicity is enigmatic. Here, we employ a peptide-based proteomic screen to investigate the impact of mutations in IDRs on protein-protein interactions. We find thatmutations in disordered cytosolic regions of three transmembrane proteins (GLUT1, ITPR1, and CACNA1H) lead to an increased clathrin binding. All three mutations create dileucine motifs known to mediate clathrin-dependent trafficking. Follow-up experiments on GLUT1 (SLC2A1), the glucose transporter causative of GLUT1 deficiency syndrome, revealed that the mutated protein mislocalizes to intracellular compartments. Mutant GLUT1 interacts with adaptor proteins (APs) invitro, and knocking down AP-2 reverts the cellular mislocalization and restores glucose transport. A systematic analysis of other known disease-causing variants revealed a significant and specific overrepresentation of gained dileucine motifs in structurally disordered cytosolic domains of transmembrane proteins. Thus, several mutations in disordered regions appear to cause "dileucineopathies."

SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue

SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation.

The C-terminus of varicella-zoster virus glycoprotein M contains trafficking motifs that mediate skin virulence in the SCID-human model of VZV pathogenesis

Knowledge about the function of varicella-zoster virus glycoprotein M is limited; the requirement of gM for skin and neural tropism are unknown. VZV gM contains two predicted YXXΦ trafficking motifs and a dileucine motif in the carboxyl-terminus. We constructed a recombinant VZV with gM truncated from the first YXXΦ and five additional viruses with YXXΦ tyrosine substitutions, alone and in combination with dileucine substitution. All recombinant viruses grew to high titer but mutation of the membrane-proximal YXXΦ motif reduced plaque size in cultured cells and altered gM localization. C-terminus truncation had a pronounced effect on virion morphogenesis and plaque size, but not on overall replication kinetics in vitro. Mutation of gM trafficking motifs and truncation attenuated replication in human skin xenografts in vivo; gM truncation did not alter neurotropism. Our results demonstrate that the gM C-terminus is dispensable for virus replication in cultured cells but is important for skin pathogenesis.

Sorting of Arabidopsis NRAMP3 and NRAMP4 depends on adaptor protein complex AP4 and a dileucine-based motif.

Adaptor protein complexes mediate cargo selection and vesicle trafficking to different cellular membranes in all eukaryotic cells. Information on the role of AP4 in plants is still limited. Here, we present the analyses of Arabidopsis thaliana mutants lacking different subunits of AP4. These mutants show abnormalities in their development and in protein sorting. We found that growth of roots and etiolated hypocotyls, as well as male fertility and trichome morphology are disturbed in ap4. Analyses of GFP-fusions transiently expressed in mesophyll protoplasts demonstrated that the tonoplast (TP) proteins MOT2, NRAMP3 and NRAMP4, but not INT1, are partially sorted to the plasma membrane (PM) in the absence of a functional AP4 complex. Moreover, alanine mutagenesis revealed that in wild-type plants, sorting of NRAMP3 and NRAMP4 to the TP requires an N-terminal dileucine-based motif. The NRAMP3 or NRAMP4 N-terminal domain containing the dileucine motif was sufficient to redirect the PM localized INT4 protein to the TP and to confer AP4-dependency on sorting of INT1. Our data show that correct sorting of NRAMP3 and NRAMP4 depends on both, an N-terminal dileucine-based motif as well as AP4.

Mapping the nuclear localization signal in the matrix protein of potato yellow dwarf virus.

The ability of the matrix (M) protein of potato yellow dwarf virus (PYDV) to remodel nuclear membranes is controlled by a di-leucine motif located at residues 223 and 224 of its primary structure. This function can be uncoupled from that of its nuclear localization signal (NLS), which is controlled primarily by lysine and arginine residues immediately downstream of the LL motif. In planta localization of green fluorescent protein fusions, bimolecular fluorescence complementation assays with nuclear import receptor importin-α1 and yeast-based nuclear import assays provided three independent experimental approaches to validate the authenticity of the M-NLS. The carboxy terminus of M is predicted to contain a nuclear export signal, which is belived to be functional, given the ability of M to bind the Arabidopsis nuclear export receptor 1 (XPO1). The nuclear shuttle activity of M has implications for the cell-to-cell movement of PYDV nucleocapsids, based upon its interaction with the N and Y proteins.

Interactions between the Hepatitis C Virus Nonstructural 2 Protein and Host Adaptor Proteins 1 and 4 Orchestrate Virus Release.

ABSTRACTHepatitis C virus (HCV) spreads via secreted cell-free particles or direct cell-to-cell transmission. Yet, virus-host determinants governing differential intracellular trafficking of cell-free- and cell-to-cell-transmitted virus remain unknown. The host adaptor proteins (APs) AP-1A, AP-1B, and AP-4 traffic in post-Golgi compartments, and the latter two are implicated in basolateral sorting. We reported that AP-1A mediates HCV trafficking during release, whereas the endocytic adaptor AP-2 mediates entry and assembly. We demonstrated that the host kinases AAK1 and GAK regulate HCV infection by controlling these clathrin-associated APs. Here, we sought to define the roles of AP-4, a clathrin-independent adaptor; AP-1A; and AP-1B in HCV infection. We screened for interactions between HCV proteins and the μ subunits of AP-1A, AP-1B, and AP-4 by mammalian cell-based protein fragment complementation assays. The nonstructural 2 (NS2) protein emerged as an interactor of these adaptors in this screening and by coimmunoprecipitations in HCV-infected cells. Two previously unrecognized dileucine-based motifs in the NS2 C terminus mediated AP binding and HCV release. Infectivity and coculture assays demonstrated that while all three adaptors mediate HCV release and cell-free spread, AP-1B and AP-4, but not AP-1A, mediate cell-to-cell spread. Live-cell imaging revealed HCV cotrafficking with AP-1A, AP-1B, and AP-4 and that AP-4 mediates HCV trafficking in a post-Golgi compartment. Lastly, HCV cell-to-cell spread was regulated by AAK1 and GAK and thus susceptible to treatment with AAK1 and GAK inhibitors. These data provide a mechanistic understanding of HCV trafficking in distinct release pathways and reveal a requirement for APs in cell-to-cell viral spread.

Role of the Dileucine Motif in Nef-Induced Trimerization of the ARF1:AP-1 Clathrin Adaptor Complex

Role of the Dileucine Motif in Nef-Induced Trimerization of the ARF1:AP-1 Clathrin Adaptor Complex