A Cholesterol Recognition Motif in Human Phospholipid Scramblase 1

Human phospholipid scramblase 1 (SCR) catalyzes phospholipid transmembrane (flip-flop) motion. This protein is assumed to bind the membrane hydrophobic core through a transmembrane domain (TMD) as well as via covalently bound palmitoyl residues. Here, we explore the possible interaction of the SCR TMD with cholesterol by using a variety of experimental and computational biophysical approaches. Our findings indicate that SCR contains an amino acid segment at the C-terminal region that shows a remarkable affinity for cholesterol, although it lacks the CRAC sequence. Other 3-OH sterols, but not steroids lacking the 3-OH group, also bind this region of the protein. The newly identified cholesterol-binding region is located partly at the C-terminal portion of the TMD and partly in the first amino acid residues in the SCR C-terminal extracellular coil. This finding could be related to the previously described affinity of SCR for cholesterol-rich domains in membranes.     

重组人磷脂爬行酶1的原核表达及纯化

目的:建立重组人磷脂爬行酶1(hPLSCR1)原核表达及纯化工艺。方法:PCR扩增hPLSCR1编码基因并连接到原核表达载体pET-28a,转化大肠杆菌BL21(DE3)并进行诱导表达,Western印迹鉴定所表达蛋白;优化表达条件后,Ni2+柱亲和层析纯化重组蛋白。结果:构建了pET-28a-PLSCR1重组质粒,诱导表达后,经SDS-PAGE分析目的蛋白的表达量达32%,Ni2+金属螯合法纯化目的蛋白后纯度达到95%以上,Western印迹验证了融合蛋白的特异性。结论:建立了高效稳定的His-PLSCR1表达体系,获得大规模生产His-PLSCR1的分离纯化工艺,为进一步研究其蛋白功能奠定了基础。     

Phospholipid Scramblase 1 Modulates FcR-Mediated Phagocytosis in Differentiated Macrophages

Phospholipid Scramblase 1 (PLSCR1) was initially characterized as a type II transmembrane protein involved in bilayer movements of phospholipids across the plasma membrane leading to the cell surface exposure of phosphatidylserine, but other cellular functions have been ascribed to this protein in signaling processes and in the nucleus. In the present study, expression and functions of PLSCR1 were explored in specialized phagocytic cells of the monocyte/macrophage lineage. The expression of PLSCR1 was found to be markedly increased in monocyte-derived macrophages compared to undifferentiated primary monocytes. Surprisingly, this 3-fold increase in PLSCR1 expression correlated with an apparent modification in the membrane topology of the protein at the cell surface of differentiated macrophages. While depletion of PLSCR1 in the monocytic THP-1 cell-line with specific shRNA did not inhibit the constitutive cell surface exposure of phosphatidylserine observed in differentiated macrophages, a net increase in the FcR-mediated phagocytic activity was measured in PLSCR1-depleted THP-1 cells and in bone marrow-derived macrophages from PLSCR1 knock-out mice. Reciprocally, phagocytosis was down-regulated in cells overexpressing PLSCR1. Since endogenous PLSCR1 was recruited both in phagocytic cups and in phagosomes, our results reveal a specific role for induced PLSCR1 expression in the modulation of the phagocytic process in differentiated macrophages.     

Phospholipid Scramblase 1 regulates Toll-like receptor 9-mediated type I interferon production in plasmacytoid dendritic cells

Toll-like receptor 9 (TLR9) senses microbial DNA in the endosomes of plasmacytoid dendritic cells (pDCs) and triggers MyD88-dependent type I interferon (IFN) responses. To better understand TLR9 biology in pDCs, we established a yeast two-hybrid library for the identification of TLR9-interacting proteins. Here, we report that an IFN-inducible protein, phospholipid scramblase 1 (PLSCR1), interacts with TLR9 in pDCs. Knockdown of PLSCR1 expression by siRNA in human pDC cell line led to a 60-70% reduction of IFN-α responses following CpG-ODN (oligodeoxynucleotide) stimulation. Primary pDCs from PLSCR1-deficient mice produced lower amount of type 1 IFN than pDCs from the wild-type mice in response to CpG-ODN, herpes simplex virus and influenza A virus. Following CpG-A stimulation, there were much lower amounts of TLR9 in the early endosomes together with CpG-A in pDCs from PLSCR1-deficient mice. Our study demonstrates that PLSCR1 is a TLR9-interacting protein that plays an important role in pDC''s type 1 IFN responses by regulating TLR9 trafficking to the endosomal compartment.     

Phospholipid Scramblase 1 Is Secreted by a Lipid Raft-dependent Pathway and Interacts with the Extracellular Matrix Protein 1 in the Dermal Epidermal Junction Zone of Human Skin

We examined the interaction of ECM1 (extracellular matrix protein 1) using yeast two-hybrid screening and identified the type II transmembrane protein, PLSCR1 (phospholipid scramblase 1), as a binding partner. This interaction was then confirmed by in vitro and in vivo co-immunoprecipitation experiments, and additional pull-down experiments with GST-tagged ECM1a fragments localized this interaction to occur within the tandem repeat region of ECM1a. Furthermore, immunohistochemical staining revealed a partial overlap of ECM1 and PLSCR1 in human skin at the basal epidermal cell layer. Moreover, in human skin equivalents, both proteins are expressed at the basal membrane in a dermal fibroblast-dependent manner. Next, immunogold electron microscopy of ultrathin human skin sections showed that ECM1 and PLSCR1 co-localize in the extracellular matrix, and using antibodies against ECM1 or PLSCR1 cross-linked to magnetic immunobeads, we were able to demonstrate PLSCR1-ECM1 interaction in human skin extracts. Furthermore, whereas ECM1 is secreted by the endoplasmic/Golgi-dependent pathway, PLSCR1 release from HaCaT keratinocytes occurs via a lipid raft-dependent mechanism, and is deposited in the extracellular matrix. In summary, we here demonstrate that PLSCR1 interacts with the tandem repeat region of ECM1a in the dermal epidermal junction zone of human skin and provide for the first time experimental evidence that PLSCR1 is secreted by an unconventional secretion pathway. These data suggest that PLSCR1 is a multifunctional protein that can function both inside and outside of the cell and together with ECM1 may play a regulatory role in human skin.     

Cholesterol Translocation in a Phospholipid Membrane

Cholesterol (CHOL) molecules play a key role in modulating the rigidity of cell membranes and controlling intracellular transport and signal transduction. Using an all-atom molecular dynamics approach, we study the process of CHOL interleaflet transport (flip-flop) in a dipalmitoylphosphatidycholine (DPPC)-CHOL bilayer over a time period of 15 μs. We investigate the effect of the flip-flop process on mechanical stress across the bilayer and the role of CHOL in inducing molecular order in bilayer leaflets. The simulations are carried out at physiologically relevant CHOL concentration (30%), temperature (323 K), and pressure (1 bar). CHOL flip-flop events are observed with a rate constant of 3 × 10⁴ s⁻¹. Once a flip-flop event is triggered, a CHOL molecule takes an average of 73 nanoseconds to migrate from one bilayer leaflet to the other.     

Regulation of the Tyrosine Phosphorylation of Phospholipid Scramblase 1 in Mast Cells That Are Stimulated through the High-Affinity IgE Receptor

Engagement of high-affinity immunoglobulin E receptors (FcεRI) activates two signaling pathways in mast cells. The Lyn pathway leads to recruitment of Syk and to calcium mobilization whereas the Fyn pathway leads to phosphatidylinositol 3-kinase recruitment. Mapping the connections between both pathways remains an important task to be completed. We previously reported that Phospholipid Scramblase 1 (PLSCR1) is phosphorylated on tyrosine after cross-linking FcεRI on RBL-2H3 rat mast cells, amplifies mast cell degranulation, and is associated with both Lyn and Syk tyrosine kinases. Here, analysis of the pathway leading to PLSCR1 tyrosine phosphorylation reveals that it depends on the FcRγ chain. FcεRI aggregation in Fyn-deficient mouse bone marrow-derived mast cells (BMMC) induced a more robust increase in FcεRI-dependent tyrosine phosphorylation of PLSCR1 compared to wild-type cells, whereas PLSCR1 phosphorylation was abolished in Lyn-deficient BMMC. Lyn association with PLSCR1 was not altered in Fyn-deficient BMMC. PLSCR1 phosphorylation was also dependent on the kinase Syk and significantly, but partially, dependent on detectable calcium mobilization. Thus, the Lyn/Syk/calcium axis promotes PLSCR1 phosphorylation in multiple ways. Conversely, the Fyn-dependent pathway negatively regulates it. This study reveals a complex regulation for PLSCR1 tyrosine phosphorylation in FcεRI-activated mast cells and that PLSCR1 sits at a crossroads between Lyn and Fyn pathways.     

Cholesterol Induces Specific Spatial and Orientational Order in Cholesterol/Phospholipid Membranes

Background

In lipid bilayers, cholesterol facilitates the formation of the liquid-ordered phase and enables the formation of laterally ordered structures such as lipid rafts. While these domains have an important role in a variety of cellular processes, the precise atomic-level mechanisms responsible for cholesterol''s specific ordering and packing capability have remained unresolved.

Methodology/Principal Findings

Our atomic-scale molecular dynamics simulations reveal that this ordering and the associated packing effects in membranes largely result from cholesterol''s molecular structure, which differentiates cholesterol from other sterols. We find that cholesterol molecules prefer to be located in the second coordination shell, avoiding direct cholesterol-cholesterol contacts, and form a three-fold symmetric arrangement with proximal cholesterol molecules. At larger distances, the lateral three-fold organization is broken by thermal fluctuations. For other sterols having less structural asymmetry, the three-fold arrangement is considerably lost.

Conclusions/Significance

We conclude that cholesterol molecules act collectively in lipid membranes. This is the main reason why the liquid-ordered phase only emerges for Chol concentrations well above 10 mol% where the collective self-organization of Chol molecules emerges spontaneously. The collective ordering process requires specific molecular-scale features that explain why different sterols have very different membrane ordering properties: the three-fold symmetry in the Chol-Chol organization arises from the cholesterol off-plane methyl groups allowing the identification of raft-promoting sterols from those that do not promote rafts.     

The N-terminal RNA Recognition Motif of PfSR1 Confers Semi-specificity for Pyrimidines during RNA Recognition

Alternative splicing confers a complexity to the mRNA landscape of apicomplexans, resulting in a high proteomic diversity. The Plasmodium falciparum Ser/Arg-rich protein 1 (PfSR1) is the first protein to be confirmed as an alternative splicing factor in this class of parasitic protists [1]. A recent study [2] showed a purine bias in RNA binding among cognate RNA substrates of PfSR1. Here, we have investigated the role played by the amino-terminal RNA recognition motif (RRM1) of PfSR1 from the solution structure of its complex with ACAUCA RNA hexamer to understand how its mechanism of RNA recognition compares to human orthologs and to the C-terminal RRM. RNA binding by RRM1 is mediated through specific recognition of a cytosine base situated 5′ of one or more pyrimidine bases by a conserved tyrosine residue on β₁ and a glutamate residue on the β₄ strand. Affinity is conferred through insertion of a 3′ pyrimidine into a positively charged pocket. Retention of fast dynamics and ITC binding constants indicate the complex to be of moderate affinity. Using calorimetry and mapping of NMR chemical shift perturbations, we have also ascertained the purine preference of PfSR1 to be a property of the carboxy terminal pseudo-RRM (RRM2), which binds RNA non-canonically and with greater affinity compared to RRM1. Our findings show conclusive evidence of complementary RNA sequence recognition by the two RRMs, which may potentially aid PfSR1 in binding RNA with a high sequence specificity.     

Characterization of Putative Cholesterol Recognition/Interaction Amino Acid Consensus-Like Motif of Campylobacter jejuni Cytolethal Distending Toxin C

Cytolethal distending toxin (CDT) produced by Campylobacter jejuni comprises a heterotrimeric complex formed by CdtA, CdtB, and CdtC. Among these toxin subunits, CdtA and CdtC function as essential proteins that mediate toxin binding to cytoplasmic membranes followed by delivery of CdtB into the nucleus. The binding of CdtA/CdtC to the cell surface is mediated by cholesterol, a major component in lipid rafts. Although the putative cholesterol recognition/interaction amino acid consensus (CRAC) domain of CDT has been reported from several bacterial pathogens, the protein regions contributing to CDT binding to cholesterol in C. jejuni remain unclear. Here, we selected a potential CRAC-like region present in the CdtC from C. jejuni for analysis. Molecular modeling showed that the predicted functional domain had the shape of a hydrophobic groove, facilitating cholesterol localization to this domain. Mutation of a tyrosine residue in the CRAC-like region decreased direct binding of CdtC to cholesterol rather than toxin intermolecular interactions and led to impaired CDT intoxication. These results provide a molecular link between C. jejuni CdtC and membrane-lipid rafts through the CRAC-like region, which contributes to toxin recognition and interaction with cholesterol.     

Structure of the Human Cholesterol Transporter ABCG1

ABCG1 is an ATP binding cassette (ABC) transporter that removes excess cholesterol from peripheral tissues. Despite its role in preventing lipid accumulation and the development of cardiovascular and metabolic disease, the mechanism underpinning ABCG1-mediated cholesterol transport is unknown. Here we report a cryo-EM structure of human ABCG1 at 4 Å resolution in an inward-open state, featuring sterol-like density in the binding cavity. Structural comparison with the multidrug transporter ABCG2 and the sterol transporter ABCG5/G8 reveals the basis of mechanistic differences and distinct substrate specificity. Benzamil and taurocholate inhibited the ATPase activity of liposome-reconstituted ABCG1, whereas the ABCG2 inhibitor Ko143 did not. Based on the structural insights into ABCG1, we propose a mechanism for ABCG1-mediated cholesterol transport.     

Molecular Mechanism for the Interaction of Phospholipid with Cholesterol

PREVIOUS models^1–3 for the interaction of phospholipid with cholesterol have been based on the idea of highly specific one-to-one complexes of these molecules. But on the basis of our interpretation of recent nuclear magnetic resonance (NMR)⁴, electron spin resonance (ESR)⁵, calorimetric⁶ and X-ray⁷ data we propose a model in which steric interaction of the phospholipid hydrocarbon chains with the cholesterol molecule is the dominant feature. The validity of the model does not depend on a stoichiometric ratio, such as one-to-one, although it is consistent with such a ratio. The shape of the cholesterol molecule is such that variations in the length and degree of saturation of the phospholipid hydrocarbon chains can be readily accommodated within a stable bilayer structure.     

Phospholipid Chain Interactions with Cholesterol Drive Domain Formation in Lipid Membranes

Cholesterol is a key component of eukaryotic membranes, but its role in cellular biology in general and in lipid rafts in particular remains controversial. Model membranes are used extensively to determine the phase behavior of ternary mixtures of cholesterol, a saturated lipid, and an unsaturated lipid with liquid-ordered and liquid-disordered phase coexistence. Despite many different experiments that determine lipid-phase diagrams, we lack an understanding of the molecular-level driving forces for liquid phase coexistence in bilayers with cholesterol. Here, we use atomistic molecular dynamics computer simulations to address the driving forces for phase coexistence in ternary lipid mixtures. Domain formation is directly observed in a long-timescale simulation of a mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine, unsaturated 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, and cholesterol. Free-energy calculations for the exchange of the saturated and unsaturated lipids between the ordered and disordered phases give insight into the mixing behavior. We show that a large energetic contribution to domain formation is favorable enthalpic interactions of the saturated lipid in the ordered phase. This favorable energy for forming an ordered, cholesterol-rich phase is opposed by a large unfavorable entropy. Martini coarse-grained simulations capture the unfavorable free energy of mixing but do not reproduce the entropic contribution because of the reduced representation of the phospholipid tails. Phospholipid tails and their degree of unsaturation are key energetic contributors to lipid phase separation.     

A Short N-Terminal Peptide Motif on Flavivirus Nonstructural Protein NS1 Modulates Cellular Targeting and Immune Recognition

Flavivirus NS1 is a versatile nonstructural glycoprotein, with intracellular NS1 functioning as an essential cofactor for viral replication and cell surface and secreted NS1 antagonizing complement activation. Even though NS1 has multiple functions that contribute to virulence, the genetic determinants that regulate the spatial distribution of NS1 in cells among different flaviviruses remain uncharacterized. Here, by creating a panel of West Nile virus-dengue virus (WNV-DENV) NS1 chimeras and site-specific mutants, we identified a novel, short peptide motif immediately C-terminal to the signal sequence cleavage position that regulates its transit time through the endoplasmic reticulum and differentially directs NS1 for secretion or plasma membrane expression. Exchange of two amino acids within this motif reciprocally changed the cellular targeting pattern of DENV or WNV NS1. For WNV, this substitution also modulated infectivity and antibody-induced phagocytosis of infected cells. Analysis of a mutant lacking all three conserved N-linked glycosylation sites revealed an independent requirement of N-linked glycans for secretion but not for plasma membrane expression of WNV NS1. Collectively, our experiments define the requirements for cellular targeting of NS1, with implications for the protective host responses, immune antagonism, and association with the host cell sorting machinery. These studies also suggest a link between the effects of NS1 on viral replication and the levels of secreted or cell surface NS1.West Nile virus (WNV) is a single-stranded, positive-sense enveloped RNA Flavivirus that cycles in nature between birds and Culex mosquitoes. It is endemic in parts of Africa, Europe, the Middle East, and Asia, and outbreaks occur annually in North America. More than 29,000 human cases of severe WNV infection have been diagnosed in the United States since its entry in 1999, and millions have been infected and remain undiagnosed (9). Humans can develop a febrile illness that progresses to a flaccid paralysis, meningitis, or encephalitis syndrome (59). Dengue virus (DENV) is a genetically related flavivirus that is transmitted by Aedes aegypti and Aedes albopictus mosquitoes and causes clinical syndromes in humans, ranging from an acute self-limited febrile illness (dengue fever [DF]) to a severe and life-threatening vascular leakage and bleeding diathesis (dengue hemorrhagic fever/dengue shock syndrome [DHF/DSS]). Globally, DENV causes an estimated 50 million infections annually, resulting in 500,000 hospitalizations and ∼22,000 deaths (45).The ∼10.7-kb Flavivirus RNA genome is translated as a single polyprotein, which is then cleaved into three structural proteins (C, prM/M, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by virus- and host-encoded proteases (39). The multifunctional NS proteins include an RNA-dependent RNA polymerase and methyltransferase (NS5), a helicase and protease (NS3), accessory proteins that form part of the viral replication complex, and immune evasion molecules (33, 34). Flavivirus NS1 is a 48-kDa nonstructural glycoprotein with two or three N-linked glycans, depending on the flavivirus, and is absent from the virion. The Japanese encephalitis virus (JEV) serogroup (West Nile, Japanese, Murray Valley, and St. Louis encephalitis viruses) generate NS1 and NS1′ proteins, the latter of which is a product of a ribosomal frameshift event that occurs at a heptanucleotide motif located at the beginning of the NS2A gene (25, 47).NS1 is an essential gene as it is required for efficient viral RNA replication (34, 41, 44). In infected mammalian cells, NS1 is synthesized as a soluble monomer, dimerizes after posttranslational modification in the lumen of the endoplasmic reticulum (ER), and accumulates extracellularly as higher-order oligomers, including hexamers (16, 26, 64, 65). Soluble NS1 binds back to the plasma membrane of uninfected cells through interactions with sulfated glycosaminoglycans (5). In infected cells, NS1 is also directly transported to and expressed on the plasma membrane although it lacks a transmembrane domain or canonical targeting motif. The mechanism of cell surface expression of flavivirus NS1 in infected cells remains uncertain although some fraction may be linked through an atypical glycosyl-phosphatidylinositol anchor (30, 50) or lipid rafts (49).NS1 has been implicated in having pathogenic consequences in flavivirus infection. The high levels of NS1 in the serum of DENV-infected patients correlate with severe disease (4, 37). NS1 has been proposed to facilitate immune complex formation (4), elicit auto-antibodies that react with host matrix proteins (21), damage endothelial cells via antibody-dependent complement-mediated cytolysis (38), or directly enhance infection (1). Flavivirus NS1 also has direct immune evasion functions and antagonizes complement activation on cell surfaces and in solution. WNV NS1 attenuates the alternative pathway of complement activation by binding the complement-regulatory protein factor H (11, 36), and DENV, WNV, and YFV NS1 proteins bind C1s and C4 in a complex to promote efficient degradation of C4 to C4b (3).Although NS1 is absent from the virion, antibodies against it can protect against infection in vivo. Immunization with purified NS1 or passive administration of some anti-WNV, anti-yellow fever virus (YFV), and anti-DENV NS1 monoclonal antibodies (MAbs) protect mice against lethal virus challenge (12, 13, 17, 22, 27, 29, 31, 32, 56-58). Initial studies with isotype switch variants and F(ab′)₂ fragments of anti-YFV NS1 MAbs suggested that the Fc region of anti-NS1 MAbs was required for protection (58). Subsequent mechanistic studies with WNV NS1 indicated that only MAbs recognizing cell surface-associated NS1 trigger Fc-γ receptor I- and/or IV-mediated phagocytosis and clearance of infected cells (13).In this study, we identify a reciprocal relationship between the secretion and cell surface expression patterns of WNV and DENV NS1s. Using WNV-DENV NS1 chimeras and point mutants, we identified a novel short peptide motif immediately C-terminal to the signal sequence cleavage position that directs NS1 for secretion or to the plasma membrane. These studies begin to explain how NS1 regulates its localization to several cellular compartments (ER, cell surface, and extracellular space) and have implications for viral infectivity, association with the host cell sorting machinery, and protective immune responses.     

Phospholipid Remodeling and Cholesterol Availability Regulate Intestinal Stemness and Tumorigenesis

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Opposing Effects of a Tyrosine-Based Sorting Motif and a PDZ-Binding Motif Regulate Human T-Lymphotropic Virus Type 1 Envelope Trafficking

Human T-lymphotropic virus type 1 (HTLV-1) envelope (Env) glycoprotein mediates binding of the virus to its receptor on the surface of target cells and subsequent fusion of virus and cell membranes. To better understand the mechanisms that control HTLV-1 Env trafficking and activity, we have examined two protein-protein interaction motifs in the cytoplasmic domain of Env. One is the sequence YSLI, which matches the consensus YXXΦ motifs that are known to interact with various adaptor protein complexes; the other is the sequence ESSL at the C terminus of Env, which matches the consensus PDZ-binding motif. We show here that mutations that destroy the YXXΦ motif increased Env expression on the cell surface and increased cell-cell fusion activity. In contrast, mutation of the PDZ-binding motif greatly diminished Env expression in cells, which could be restored to wild-type levels either by mutating the YXXΦ motif or by silencing AP2 and AP3, suggesting that interactions with PDZ proteins oppose an Env degradation pathway mediated by AP2 and AP3. Silencing of the PDZ protein hDlg1 did not affect Env expression, suggesting that hDlg1 is not a binding partner for Env. Substitution of the YSLI sequence in HTLV-1 Env with YXXΦ elements from other cell or virus membrane-spanning proteins resulted in alterations in Env accumulation in cells, incorporation into virions, and virion infectivity. Env variants containing YXXΦ motifs that are predicted to have high-affinity interaction with AP2 accumulated to lower steady-state levels. Interestingly, mutations that destroy the YXXΦ motif resulted in viruses that were not infectious by cell-free or cell-associated routes of infection. Unlike YXXΦ, the function of the PDZ-binding motif manifests itself only in the producer cells; AP2 silencing restored the incorporation of PDZ-deficient Env into virus-like particles (VLPs) and the infectivity of these VLPs to wild-type levels.Human T-lymphotropic virus type 1 (HTLV-1) envelope (Env), like most retroviral envelopes, is synthesized as a precursor protein in the endoplasmic reticulum, forms trimers, and is cleaved by a cellular furin-like protease as it transits through the trans-Golgi network on its way to the plasma membrane (7, 21, 31). Cleavage of the HTLV-1 Env precursor generates a 46-kDa surface subunit (SU, gp46) and a 21-kDa transmembrane protein (TM, gp21) (8, 43). SU contains the receptor-binding domain and is linked by a disulfide bond to TM, which anchors Env to the membrane and mediates fusion of virus and cell membranes after receptor engagement (11, 28, 40, 51). TM consists of extracellular, membrane-spanning, and cytoplasmic domains (31); the last contains motifs that direct Env trafficking, membrane targeting, and virion incorporation. HTLV-1 is poorly transmitted as cell-free virus, and there is good evidence supporting a model in which virions are transmitted in a polarized fashion between lymphocytes that are in close contact (22, 30). Unlike murine leukemia virus (MLV) and Mason-Pfizer monkey virus (MPMV) Envs, in which the cytoplasmic domain (CD) is cleaved by the virus-encoded protease to activate fusogenic activity (3, 6, 19, 42), the HTLV-1 Env cytoplasmic domain is not cleaved and HTLV-1 Env exists on the cell surface in a highly fusogenic state. In many respects, HTLV-1 Env resembles versions of MLV or MPMV Envs that lack C-terminal amino acids, e.g., with elevated cell-cell fusion activity and low virion infectivity. It is not exactly clear how HTLV-1 Env is controlled such that virus infection can proceed without cell-cell fusion, but it is probable that Env trafficking plays an important role. The cytoplasmic domain of HTLV-1 Env is relatively short and contains two important trafficking motifs: a YXXΦ motif (YSLI), which is involved in membrane protein trafficking and basolateral sorting in polarized epithelial cells (10), and a PDZ-binding motif (ESSL), which can interact with numerous PDZ proteins but is not found in other retroviral Envs (2).The tyrosine-based sorting motif (YXXΦ, where Y is tyrosine, X is any amino acid, and Φ is a bulky hydrophobic amino acid) determines the trafficking and turnover of many membrane-spanning proteins in the cell (5, 39) and is present in most retroviral Env proteins (7). The YXXΦ motif interacts with the μ subunit of the heterotetrameric adaptor protein complexes AP1, AP2, AP3, and AP4. Each adaptor complex is involved in a specific trafficking pathway: AP1 and AP4 deliver cargo from the trans-Golgi network to the plasma membrane (13, 33, 48), AP2 directs the endocytosis of proteins from the cell surface, and AP3 is involved in lysosomal sorting (5, 12, 24, 35). Each type of μ subunit interacts with a distinct but overlapping type of tyrosine-based motif; the tyrosine and the Φ residues are most critical, but affinity is determined in large part by the variable amino acids at positions +1 and +2 relative to tyrosine and also by surrounding amino acids (5, 37). Furthermore, interactions between AP2 and the YXXΦ motif may be regulated by phosphorylation of μ2 (38, 47), by localized changes in phosphoinositide concentration, or by interactions between AP2 and docking factors (47). Although most retroviral Env proteins contain YXXΦ-sorting motifs, the sequences of the motifs and their roles in Env trafficking and function appear to vary widely among different retroviruses. For example, mutation of the YXXΦ motif in MLV Env interferes with basolateral targeting of Env and diminishes viral pathogenesis in vivo but has little effect on Env accumulation at the plasma membrane (9, 16, 23, 25, 29). Mutations in the YXXΦ motif in MPMV Env are similar to those in MLV Evn and also were reported to affect Env incorporation into virions (45). Mutation of the YXXΦ motif in HTLV-1 Env was previously shown to decrease Env endocytosis, increase cell-cell fusion, increase Env incorporation into virions, abolish basolateral targeting, and decrease virus infectivity (1, 10).The most abundant protein-protein interaction domains in mammalian cells are the PDZ domains; more than 400 PDZ proteins are encoded in the human genome. PDZ domains are modular, recognize short C-terminal peptide motifs, and are often found in multiple copies or in combination with other protein interaction domains (36, 46, 50). PDZ proteins have the ability to form supramolecular scaffolds that coordinate signaling, synapse formation, cell polarity, and trafficking of interacting proteins (26, 44, 53). With respect to the last, it is important to note that PDZ proteins can delay the internalization of G protein-coupled receptors, ion channels, and membrane transporters (17, 41, 49, 52). Among retroviral Env proteins, only HTLV and simian T-lymphotropic virus (STLV) Envs contain putative PDZ-binding motifs. A yeast two-hybrid screen using the HTLV-1 Env cytoplasmic domain (CD) as bait identified the PDZ protein hDlg (human homolog of disc large protein) as a potential binding partner (2). In vitro pulldown experiments showed that a glutathione S-transferase (GST)-EnvCD fusion protein interacted with several PDZ proteins from cell lysates, one of which was hDlg. In one study, mutation of the PDZ-binding motif in HTLV-1 Env inhibited cell-cell fusion (2); in another study, hDlg small interfering RNA (siRNA) silencing caused a modest reduction in syncytium formation (54). Neither study examined how the PDZ-binding motif controls Env expression, membrane targeting, trafficking, or virus infectivity. Thus, it is still unclear which PDZ proteins interact with HTLV-1 Env in vivo and how those interactions affect Env trafficking and activity.In this paper, functional interactions between the YXXΦ motif and the PDZ-binding motif in the cytoplasmic domain of HTLV-1 Env were investigated by mutagenesis of Env and by siRNA silencing of potential cellular interacting proteins. The YXXΦ motif in HTLV-1 Env appears to interact primarily with AP2 and AP3, which regulate Env endocytosis and lysosomal degradation, respectively. Mutations that ablated the YXXΦ motif increased Env accumulation on the cell surface. The PDZ-binding motif at the C terminus of Env appears to delay Env turnover. Mutation of the PDZ-binding element diminished Env accumulation in cells to very low levels, indicating that loss of the PDZ-binding motif accelerates Env degradation. Expression of Env with a mutated PDZ-binding motif could be restored to normal levels by also mutating the YXXΦ motif or by silencing AP2 or AP3. The ability of the PDZ-binding motif to alter the activity of the YXXΦ motif depends on the particular sequence of the latter. The attenuating effect of the PDZ-binding motif on Env endocytosis could be overcome by substitution of the YSLI motif in HTLV-1 Env with YXXΦ elements from other cell or virus proteins that are predicted to have higher affinities for AP2 than the YSLI motif of HTLV-1 Env.