BST2/Tetherin enhances entry of human cytomegalovirus

Interferon-induced BST2/Tetherin prevents budding of vpu-deficient HIV-1 by tethering mature viral particles to the plasma membrane. BST2 also inhibits release of other enveloped viruses including Ebola virus and Kaposi's sarcoma associated herpesvirus (KSHV), indicating that BST2 is a broadly acting antiviral host protein. Unexpectedly however, recovery of human cytomegalovirus (HCMV) from supernatants of BST2-expressing human fibroblasts was increased rather than decreased. Furthermore, BST2 seemed to enhance viral entry into cells since more virion proteins were released into BST2-expressing cells and subsequent viral gene expression was elevated. A significant increase in viral entry was also observed upon induction of endogenous BST2 during differentiation of the pro-monocytic cell line THP-1. Moreover, treatment of primary human monocytes with siRNA to BST2 reduced HCMV infection, suggesting that BST2 facilitates entry of HCMV into cells expressing high levels of BST2 either constitutively or in response to exogenous stimuli. Since BST2 is present in HCMV particles we propose that HCMV entry is enhanced via a reverse-tethering mechanism with BST2 in the viral envelope interacting with BST2 in the target cell membrane. Our data suggest that HCMV not only counteracts the well-established function of BST2 as inhibitor of viral egress but also employs this anti-viral protein to gain entry into BST2-expressing hematopoietic cells, a process that might play a role in hematogenous dissemination of HCMV.     

Matrix‐glycoprotein interactions required for budding of a plant nucleorhabdovirus and induction of inner nuclear membrane invagination

Nucleorhabdoviruses such as Sonchus yellow net virus (SYNV) replicate in the nuclei and undergo morphogenesis at the inner nuclear membrane (IM) in plant cells. Mature particles are presumed to form by budding of the Matrix (M) protein‐nucleocapsid complexes through host IMs to acquire host phospholipids and the surface glycoproteins (G). To address mechanisms underlying nucleorhabdovirus budding, we generated recombinant SYNV G mutants containing a truncated amino‐terminal (NT) or carboxyl‐terminal (CT) domain. Electron microscopy and sucrose gradient centrifugation analyses showed that the CT domain is essential for virion morphogenesis whereas the NT domain is also required for efficient budding. SYNV infection induces IM invaginations that are thought to provide membrane sites for virus budding. We found that in the context of viral infections, interactions of the M protein with the CT domain of the membrane‐anchored G protein mediate M protein translocation and IM invagination. Interestingly, tethering the M protein to endomembranes, either by co‐expression with a transmembrane G protein CT domain or by artificial fusion with the G protein membrane targeting sequence, induces IM invagination in uninfected cells. Further evidence to support functions of G‐M interactions in virus budding came from dominant negative effects on SYNV‐induced IM invagination and viral infections that were elicited by expression of a soluble version of the G protein CT domain. Based on these data, we propose that cooperative G‐M interactions promote efficient SYNV budding.     

C-terminal hydrophobic region in human bone marrow stromal cell antigen 2 (BST-2)/tetherin protein functions as second transmembrane motif

BST-2/CD317/HM1.24/tetherin is a host factor that inhibits the release of HIV-1 and other enveloped viruses. Structurally, tetherin consists of an N-terminal transmembrane (TM) region, a central coiled coil motif, and a putative C-terminal glycosylphosphatidylinositol (GPI) anchor motif. A current working model proposes that BST-2 inhibits virus release by physically tethering viral particles to the cell surface via its TM motif and GPI anchor. Here we analyzed the functional importance of the C-terminal GPI anchor motif in BST-2. We replaced the GPI anchor motif in BST-2 with the TM regions of several surface markers and found that the TM motifs of CD40 and transferrin receptor, but not that of CD45, could functionally substitute for a GPI anchor in BST-2. Conversely, replacing the TM region of CD4 by the putative GPI anchor signal of human BST-2 resulted in proper membrane targeting and surface expression of the chimeric protein, indicating that the BST-2 GPI anchor signal can function as a bona fide TM region. In fact, attempts to demonstrate GPI anchor modification of human BST-2 by biochemical methods failed. Our results demonstrate that the putative C-terminal GPI anchor motif in human BST-2 fulfills the requirements of a bona fide TM motif, leading us to propose that human BST-2 may in fact contain a second TM segment rather than a GPI anchor.     

Structural and biophysical analysis of BST-2/tetherin ectodomains reveals an evolutionary conserved design to inhibit virus release

BST-2/tetherin is a host antiviral molecule that functions to potently inhibit the release of enveloped viruses from infected cells. In return, viruses have evolved antagonists to this activity. BST-2 traps budding virions by using two separate membrane-anchoring regions that simultaneously incorporate into the host and viral membranes. Here, we detailed the structural and biophysical properties of the full-length BST-2 ectodomain, which spans the two membrane anchors. The 1.6-Å crystal structure of the complete mouse BST-2 ectodomain reveals an ∼145-Å parallel dimer in an extended α-helix conformation that predominantly forms a coiled coil bridged by three intermolecular disulfides that are required for stability. Sequence analysis in the context of the structure revealed an evolutionarily conserved design that destabilizes the coiled coil, resulting in a labile superstructure, as evidenced by solution x-ray scattering displaying bent conformations spanning 150 and 180 Å for the mouse and human BST-2 ectodomains, respectively. Additionally, crystal packing analysis revealed possible curvature-sensing tetrameric structures that may aid in proper placement of BST-2 during the genesis of viral progeny. Overall, this extended coiled-coil structure with inherent plasticity is undoubtedly necessary to accommodate the dynamics of viral budding while ensuring separation of the anchors.     

Coiled‐coil registry shifts in the F684I mutant of Bicaudal D result in cargo‐independent activation of dynein motility

The dynein adaptor Drosophila Bicaudal D (BicD) is auto‐inhibited and activates dynein motility only after cargo is bound, but the underlying mechanism is elusive. In contrast, we show that the full‐length BicD/F684I mutant activates dynein processivity even in the absence of cargo. Our X‐ray structure of the C‐terminal domain of the BicD/F684I mutant reveals a coiled‐coil registry shift; in the N‐terminal region, the two helices of the homodimer are aligned, whereas they are vertically shifted in the wild‐type. One chain is partially disordered and this structural flexibility is confirmed by computations, which reveal that the mutant transitions back and forth between the two registries. We propose that a coiled‐coil registry shift upon cargo‐binding activates BicD for dynein recruitment. Moreover, the human homolog BicD2/F743I exhibits diminished binding of cargo adaptor Nup358, implying that a coiled‐coil registry shift may be a mechanism to modulate cargo selection for BicD2‐dependent transport pathways.     

Structural basis for paramyxovirus-mediated membrane fusion

Paramyxoviruses are responsible for significant human mortality and disease worldwide, but the molecular mechanisms underlying their entry into host cells remain poorly understood. We have solved the crystal structure of a fragment of the simian parainfluenza virus 5 fusion protein (SV5 F), revealing a 96 A long coiled coil surrounded by three antiparallel helices. This structure places the fusion and transmembrane anchor of SV5 F in close proximity with a large intervening domain at the opposite end of the coiled coil. Six amino acids, potentially part of the fusion peptide, form a segment of the central coiled coil, suggesting that this structure extends into the membrane. Deletion mutants of SV5 F indicate that putative flexible tethers between the coiled coil and the viral membrane are dispensable for fusion. The lack of flexible tethers may couple a final conformational change in the F protein directly to the fusion of two bilayers.     

Limiting Respiratory Viral Infection by Targeting Antiviral and Immunological Functions of BST‐2/Tetherin: Knowledge and Gaps

Recent findings regarding the cellular biology and immunology of BST‐2 (also known as tetherin) indicate that its function could be exploited as a universal replication inhibitor of enveloped respiratory viruses (e.g., influenza, respiratory syncytial virus, etc.). BST‐2 inhibits viral replication by preventing virus budding from the plasma membrane and by inducing an antiviral state in cells adjacent to infection via unique inflammatory signaling mechanisms. This review presents the first comprehensive summary of what is currently known about BST‐2 anti‐viral function against respiratory viruses, how these viruses construct countermeasures to antagonize BST‐2, and how BST‐2 function might be targeted to develop therapies to treat respiratory virus infections. The authors address the current gaps in knowledge, including the need for mechanistic understanding of BST‐2 antagonism by respiratory viruses, that should be bridged to achieve that goal.

     

Mapping of the chaperone AcrH binding regions of translocators AopB and AopD and characterization of oligomeric and metastable AcrH‐AopB‐AopD complexes in the type III secretion system of Aeromonas hydrophila

In the type III secretion system (T3SS) of Aeromonas hydrophila, AcrH acts as a chaperone for translocators AopB and AopD. AcrH forms a stable 1:1 monomeric complex with AopD, whereas the 1:1 AcrH‐AopB complex exists mainly as a metastable oligomeric form and only in minor amounts as a stable monomeric form. Limited protease digestion shows that these complexes contain highly exposed regions, thus allowing mapping of intact functional chaperone binding regions of AopB and AopD. AopD uses the transmembrane domain (DF1, residues 16–147) and the C‐terminal amphipathic helical domain (DF2, residues 242–296) whereas AopB uses a discrete region containing the transmembrane domain and the putative N‐terminal coiled coil domain (BF1, residues 33–264). Oligomerization of the AcrH‐AopB complex is mainly through the C‐terminal coiled coil domain of AopB, which is dispensable for chaperone binding. The three proteins, AcrH, AopB, and AopD, can be coexpressed to form an oligomeric and metastable complex. These three proteins are also oligomerized mainly through the C‐terminal domain of AopB. Formation of such an oligomeric and metastable complex may be important for the proper formation of translocon of correct topology and stoichiometry on the host membrane.     

Dimerization of the transmembrane domain of human tetherin in membrane mimetic environments

Tetherin/Bst-2 is a cell surface protein that can act as a restriction factor against a number of enveloped viruses, including HIV-1. It acts by tethering new virus particles to the host cell membrane, promoting their internalization and degradation. Tetherin is a type II membrane protein, with an N-terminal transmembrane domain, an extracellular coiled-coil domain, and a C-terminal GPI anchor. This double membrane anchor is important for anti-HIV activity, as is dimerization of the coiled-coil domain, but despite recent crystal structures of the coiled-coil ectodomains of human and mouse tetherin, the topology of tetherin with respect to host and viral membranes has yet to be determined. The tetherin transmembrane domain is also thought to mediate interactions with the HIV-1 encoded integral membrane protein Vpu, which is an antagonist of tetherin, through direct binding to the transmembrane region of Vpu. Using a combination of SDS-PAGE, size exclusion chromatography, and pyrene excimer fluorescence, we show that in the absence of the coiled-coil domain the transmembrane domain of human tetherin forms parallel homodimers in membrane mimetic environments. Transmembrane domain dimerization does not require disulfide bond formation and is favored in TFE, SDS micelles, and POPC liposomes. This observation has implications for functional models of tetherin, suggesting that both transmembrane domains in the dimeric molecule are inserted into the same lipid bilayer, rather than into opposing membranes.     

Molecular Mechanism of BST2/Tetherin Downregulation by K5/MIR2 of Kaposi's Sarcoma-Associated Herpesvirus

K3/MIR1 and K5/MIR2 of Kaposi''s sarcoma-associated herpesvirus (KSHV) are viral members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family and contribute to viral immune evasion by directing the conjugation of ubiquitin to immunostimulatory transmembrane proteins. In a quantitative proteomic screen for novel host cell proteins downregulated by viral immunomodulators, we previously observed that K5, as well as the human immunodeficiency virus type 1 (HIV-1) immunomodulator VPU, reduced steady-state levels of bone marrow stromal cell antigen 2 (BST2; also called CD317 or tetherin), suggesting that BST2 might be a novel substrate of K5 and VPU. Recent work revealed that in the absence of VPU, HIV-1 virions are tethered to the plasma membrane in BST2-expressing HeLa cells. By targeting BST2, K5 might thus similarly overcome an innate antiviral host defense mechanism. Here we establish that despite its type II transmembrane topology and carboxy-terminal glycosylphosphatidylinositol (GPI) anchor, BST2 represents a bona fide target of K5 that is downregulated during primary infection by and reactivation of KSHV. Upon exit of the protein from the endoplasmic reticulum, lysines in the short amino-terminal domain of BST2 are ubiquitinated by K5, resulting in rapid degradation of BST2. Ubiquitination of BST2 is required for degradation, since BST2 lacking cytosolic lysines was K5 resistant and ubiquitin depletion by proteasome inhibitors restored BST2 surface expression. Thus, BST2 represents the first type II transmembrane protein targeted by K5 and the first example of a protein that is both ubiquitinated and GPI linked. We further demonstrate that KSHV release is decreased in the absence of K5 in a BST2-dependent manner, suggesting that K5 contributes to the evasion of intracellular antiviral defense programs.Bone marrow stromal cell antigen 2 (BST2) was recently identified as a host cell restriction factor that prevents the release of retroviral and filoviral particles from infected host cells (23). Human immunodeficiency virus type 1 (HIV-1) counteracts this antiviral function of BST2 by expressing the viral auxiliary protein VPU (41, 53). In the absence of VPU, virus particles are prevented from budding off the cellular membrane in cells that express BST2, resulting in virions being tethered to the plasma membrane. BST2 was therefore renamed tetherin (41), although questions still remain as to whether BST2 acts as the actual tether and whether BST2-dependent tethering occurs in all BST2-expressing cell types (36). Independently, BST2 was shown to be induced by type I and type II interferons (IFNs) (7), suggesting that BST2 is part of the innate antiviral response triggered in infected cells.Using a quantitative membrane proteomic approach, we observed that BST2 is underrepresented in plasma membranes from cells expressing not only VPU (14) but also the K5 protein of Kaposi''s sarcoma-associated herpesvirus (KSHV) (4). K5 is a viral homologue of a family of cellular transmembrane ubiquitin ligases, termed membrane-associated RING-CH (MARCH) proteins (3), that mediate the ubiquitination of the cytoplasmic portion of transmembrane proteins (reviewed in reference 40). Each member of this family targets a subset of cellular membrane proteins with both unique and shared specificities (4, 56). One of the functions of cellular MARCH proteins is to modulate antigen presentation by mediating the ubiquitin-dependent turnover of major histocompatibility complex (MHC) class II molecules in dendritic cells, B cells, and monocytes/macrophages (43, 52). In contrast, viral homologues of MARCH proteins encoded by KSHV, murine herpesvirus 68, and the leporipoxvirus myxomavirus all share the ability to mediate the destruction of MHC-I (reviewed in reference 16) but not MHC-II molecules. Thus, one of the functions of the viral proteins is to promote viral escape from immune clearance by CD8⁺ T lymphocytes (50). Furthermore, each viral MARCH homologue specifically eliminates additional host cell proteins, so each plays multiple roles in viral pathogenesis. KSHV carries two viral MARCH proteins, K3 and K5, also known as MIR1 and MIR2, which both support viral escape from T-cell, NK-cell, and NKT-cell recognition by eliminating the corresponding ligands from the surfaces of infected cells (reviewed in reference 10). In endothelial cells (ECs), K5 additionally downregulates EC-specific adhesion molecules that play an essential role in the formation of adhesive platforms and adherens junctions (31, 32). Since Kaposi''s sarcoma is a tumor of EC origin, K5 might thus also contribute to tumorigenesis by disrupting normal EC barrier function and by modulating the interaction of ECs with inflammatory leukocytes.The downregulation of BST2 by K5 further suggests that K5 also counteracts innate antiviral responses, which might benefit KSHV. However, most transmembrane proteins targeted by viral or cellular MARCH proteins are type I transmembrane proteins that belong to the immunoglobulin superfamily. In contrast, BST2 is a type II transmembrane protein that is also glycosylphosphatidylinositol (GPI) anchored (25). Thus, BST2 has a short cytoplasmic amino terminus followed by an outside-in transmembrane domain, a large glycosylated extracellular portion, and a GPI anchor. The additional propensity of BST2 to form homodimers (44) was speculated to be crucial for the tethering function of BST2 in that self-association of BST2 molecules in the viral envelope with plasma membrane BST2 could prevent viral exit (19). The unusual topology of BST2 and its multimerization raised the question of whether BST2 is a bona fide target of K5 or whether its downregulation is a downstream effect of K5 eliminating other transmembrane proteins. Additionally, it is not clear whether BST2 would be downregulated in the context of a normal viral infection and, particularly, whether virally expressed K5 would be able to overcome the high expression levels of BST2 observed upon IFN induction. We now demonstrate that KSHV efficiently downregulates IFN-induced BST2 both during primary infection and upon reactivation from latency in ECs. IFN-induced BST2 is ubiquitinated by K5 upon exiting the endoplasmic reticulum (ER) and is rapidly degraded by a pathway that is sensitive to proteasome inhibitors but resistant to inhibitors of lysosomal acidification. These data suggest that despite its unusual topology, BST2 is directly targeted by K5. We further demonstrate that BST2 reduces KSHV release upon inhibition of K5 expression by small interfering RNA (siRNA), suggesting that BST2 is part of the IFN-induced innate immune response to KSHV. Thus, in addition to contributing to viral evasion of cellular immune responses and remodeling EC function, K5 also counteracts the innate immune defense of the host cell.     

ProP‐ProP and ProP‐phospholipid interactions determine the subcellular distribution of osmosensing transporter ProP in Escherichia coli

Osmosensing transporter ProP protects bacteria from osmotically induced dehydration by mediating the uptake of zwitterionic osmolytes. ProP activity is a sigmoidal function of the osmolality. ProP orthologues share an extended, cytoplasmic C‐terminal domain. Orthologues with and without a C‐terminal, α‐helical coiled‐coil domain respond similarly to the osmolality. ProP concentrates at the poles and septa of Escherichia coli cells in a cardiolipin (CL)‐dependent manner. The roles of phospholipids and the C‐terminal domain in subcellular localization of ProP were explored. Liposome association of peptides representing the C‐terminal domains of ProP orthologues and variants in vitro was compared with subcellular localization of the corresponding orthologues and variants in vivo. In the absence of coiled‐coil formation, the C‐terminal domain bound liposomes and ProP concentrated at the cell poles in a CL‐independent manner. The presence of the coiled‐coil replaced those phenomena with CL‐dependent binding and localization. The effects of amino acid replacements on lipid association of the C‐terminal peptide fully recapitulated their effects on the subcellular localization of ProP. These data suggest that polar localization of ProP results from association of its C‐terminal domain with the anionic lipid‐enriched membrane at the cell poles. The coiled‐coil domain present on only some orthologues renders that phenomenon CL‐dependent.     

More than one door - Budding of enveloped viruses through cellular membranes

Enveloped viruses exit their host cell by budding from a cellular membrane and thereby spread from one cell to another. Virus budding in general involves the distortion of a cellular membrane away from the cytoplasm, envelopment of the viral capsid by one or more lipid bilayers that are enriched in viral membrane glycoproteins, and a fission event that separates the enveloped virion from the cellular membrane. While it was initially thought that virus budding is always driven by viral transmembrane proteins interacting with the inner structural proteins, it is now clear that the driving force may be different depending on the virus. Research over the past years has shown that viral components specifically interact with host cell lipids and proteins, thereby adopting cellular functions and pathways to facilitate virus release. This review summarizes the current knowledge of the cellular membrane systems that serve as viral budding sites and of the viral and cellular factors involved in budding. One of the best studied cellular machineries required for virus egress is the ESCRT complex, which will be described in more detail.     

Interdomain salt‐bridges in the Ebola virus protein VP40 and their role in domain association and plasma membrane localization

The Ebola virus protein VP40 is a transformer protein that possesses an extraordinary ability to accomplish multiple functions by transforming into various oligomeric conformations. The disengagement of the C‐terminal domain (CTD) from the N‐terminal domain (NTD) is a crucial step in the conformational transformations of VP40 from the dimeric form to the hexameric form or octameric ring structure. Here, we use various molecular dynamics (MD) simulations to investigate the dynamics of the VP40 protein and the roles of interdomain interactions that are important for the domain–domain association and dissociation, and report on experimental results of the behavior of mutant variants of VP40. The MD studies find that various salt‐bridge interactions modulate the VP40 domain dynamics by providing conformational specificity through interdomain interactions. The MD simulations reveal a novel salt‐bridge between D45‐K326 when the CTD participates in a latch‐like interaction with the NTD. The D45‐K326 salt‐bridge interaction is proposed to help domain–domain association, whereas the E76‐K291 interaction is important for stabilizing the closed‐form structure. The effects of the removal of important VP40 salt‐bridges on plasma membrane (PM) localization, VP40 oligomerization, and virus like particle (VLP) budding assays were investigated experimentally by live cell imaging using an EGFP‐tagged VP40 system. It is found that the mutations K291E and D45K show enhanced PM localization but D45K significantly reduced VLP formation.     

Structural basis for coronavirus-mediated membrane fusion. Crystal structure of mouse hepatitis virus spike protein fusion core

The surface transmembrane glycoprotein is responsible for mediating virion attachment to cell and subsequent virus-cell membrane fusion. However, the molecular mechanisms for the viral entry of coronaviruses remain poorly understood. The crystal structure of the fusion core of mouse hepatitis virus S protein, which represents the first fusion core structure of any coronavirus, reveals a central hydrophobic coiled coil trimer surrounded by three helices in an oblique, antiparallel manner. This structure shares significant similarity with both the low pH-induced conformation of influenza hemagglutinin and fusion core of HIV gp41, indicating that the structure represents a fusion-active state formed after several conformational changes. Our results also indicate that the mechanisms for the viral fusion of coronaviruses are similar to those of influenza virus and HIV. The coiled coil structure has unique features, which are different from other viral fusion cores. Highly conserved heptad repeat 1 (HR1) and HR2 regions in coronavirus spike proteins indicate a similar three-dimensional structure among these fusion cores and common mechanisms for the viral fusion. We have proposed the binding regions of HR1 and HR2 of other coronaviruses and a structure model of their fusion core based on our mouse hepatitis virus fusion core structure and sequence alignment. Drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation may be applied to the inhibition of a number of emerging infectious diseases, including severe acute respiratory syndrome.     

C‐terminal juxtamembrane region of full‐length M2 protein forms a membrane surface associated amphipathic helix

The influenza A M2 protein is a 97‐residue integral membrane protein involved in viral budding and proton conductance. Although crystal and NMR structures exist of truncated constructs of the protein, there is disagreement between models and only limited structural data are available for the full‐length protein. Here, the structure of the C‐terminal juxtamembrane region (sites 50–60) is investigated in the full‐length M2 protein using site‐directed spin‐labeling electron paramagnetic resonance (EPR) spectroscopy in lipid bilayers. Sites 50–60 were chosen for study because this region has been shown to be critical to the role the M2 protein plays in viral budding. Continuous wave EPR spectra and power saturation data in the presence of paramagnetic membrane soluble oxygen are consistent with a membrane surface associated amphipathic helix. Comparison between data from the C‐terminal juxtamembrane region in full‐length M2 protein with data from a truncated M2 construct demonstrates that the line shapes and oxygen accessibilities are remarkably similar between the full‐length and truncated form of the protein.     

The influenza fusion peptide promotes lipid polar head intrusion through hydrogen bonding with phosphates and N‐terminal membrane insertion depth

Influenza infection requires fusion between the virus envelope and a host cell endosomal membrane. The influenza hemagglutinin fusion peptide (FP) is essential to viral membrane fusion. It was recently proposed that FPs would fuse membranes by increasing lipid tail protrusion, a membrane fusion transition state. The details of how FPs induce lipid tail protrusion, however, remain to be elucidated. To decipher the molecular mechanism by which FPs promote lipid tail protrusion, we performed molecular dynamics simulations of the wild‐type (WT) FP, fusogenic mutant F9A, and nonfusogenic mutant W14A in model bilayers. This article presents the peptide–lipid interaction responsible for lipid tail protrusion and a related lipid perturbation, polar head intrusion, where polar heads are sunk under the membrane surface. The backbone amides from the four N‐terminal peptide residues, deeply inserted in the membrane, promoted both perturbations through H bonding with lipid phosphates. Polar head intrusion correlated with peptides N‐terminal insertion depth and activity: the N‐termini of WT and F9A were inserted deeper into the membrane than nonfusogenic W14A. Based on these results, we propose that FP‐induced polar head intrusion would complement lipid tail protrusion in catalyzing membrane fusion by reducing repulsions between juxtaposed membranes headgroups. The presented model provides a framework for further research on membrane fusion and influenza antivirals. Proteins 2014; 82:2118–2127. © 2014 Wiley Periodicals, Inc.     

Differential backbone dynamics of companion helices in the extended helical coiled‐coil domain of a bacterial chemoreceptor

Cytoplasmic domains of transmembrane bacterial chemoreceptors are largely extended four‐helix coiled coils. Previous observations suggested the domain was structurally dynamic. We probed directly backbone dynamics of this domain of the transmembrane chemoreceptor Tar from Escherichia coli using site‐directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Spin labels were positioned on solvent‐exposed helical faces because EPR spectra for such positions reflect primarily polypeptide backbone movements. We acquired spectra for spin‐labeled, intact receptor homodimers solubilized in detergent or inserted into native E. coli lipid bilayers in Nanodiscs, characterizing 16 positions distributed throughout the cytoplasmic domain and on both helices of its helical hairpins, one amino terminal to the membrane‐distal tight turn (N‐helix), and the other carboxyl terminal (C‐helix). Detergent solubilization increased backbone dynamics for much of the domain, suggesting that loss of receptor activities upon solubilization reflects wide‐spread destabilization. For receptors in either condition, we observed an unanticipated difference between the N‐ and C‐helices. For bilayer‐inserted receptors, EPR spectra from sites in the membrane‐distal protein‐interaction region and throughout the C‐helix were typical of well‐structured helices. In contrast, for approximately two‐thirds of the N‐helix, from its origin as the AS‐2 helix of the membrane‐proximal HAMP domain to the beginning of the membrane‐distal protein‐interaction region, spectra had a significantly mobile component, estimated by spectral deconvolution to average approximately 15%. Differential helical dynamics suggests a four‐helix bundle organization with a pair of core scaffold helices and two more dynamic partner helices. This newly observed feature of chemoreceptor structure could be involved in receptor function.     

Interaction of Golgin‐84 with the COG Complex Mediates the Intra‐Golgi Retrograde Transport

The coiled‐coil Golgi membrane protein golgin‐84 functions as a tethering factor for coat protein I (COPI) vesicles. Protein interaction analyses have revealed that golgin‐84 interacts with another tether, the conserved oligomeric Golgi (COG) complex, through its subunit Cog7. Therefore, we explored the function of golgin‐84 as the tether for COPI vesicles of intra‐Golgi retrograde traffic. First, glycosylic maturation of both plasma membrane (CD44) and lysosomal (lamp1) glycoproteins was distorted in golgin‐84 knockdown (KD) cells. The depletion of golgin‐84 caused fragmentation of the Golgi with the mislocalization of Golgi resident proteins, resulting in the accumulation of vesicles carrying intra‐Golgi soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) and cis‐Golgi membrane protein GPP130. Similar observations were obtained by diminution of the COG complex, suggesting a strong correlation between the two tethers. Indeed, COG complex‐dependent (CCD) vesicles that accumulate in Cog3 or Cog7 KD cells carried golgin‐84. Surprisingly, the interaction between golgin‐84 and another candidate tethering partner CASP (CDP/cut alternatively spliced product) decreased in Cog3 KD cells. These results indicate that golgin‐84 on COPI vesicles interact with the COG complex before SNARE assembly, suggesting that the interaction of golgin‐84 with COG plays an important role in the tethering process of intra‐Golgi retrograde vesicle traffic.     

The cholesterol‐binding motif of the HIV‐1 glycoprotein gp41 regulates lateral sorting and oligomerization

Enveloped viruses often use membrane lipid rafts to assemble and bud, augment infection and spread efficiently. However, the molecular bases and functional consequences of the partitioning of viral glycoproteins into microdomains remain intriguing questions in virus biology. Here, we measured Foerster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM‐FRET) to study the role of distinct membrane proximal regions of the human immunodeficiency virus glycoprotein gp41 for lipid raft partitioning in living Chinese hamster ovary cells (CHO‐K1). Gp41 was labelled with a fluorescent protein at the exoplasmic face of the membrane, preventing any interference of the fluorophore with the proposed role of the transmembrane and cytoplasmic domains in lateral organization of gp41. Raft localization was deduced from interaction with an established raft marker, a fluorescently tagged glycophosphatidylinositol anchor and the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial lateral sorting determinant in CHO‐K1 cells. Interestingly, the raft association of gp41 indicates a substantial cell‐to‐cell heterogeneity of the plasma membrane microdomains. In complementary fluorescence polarization microscopy, a distinct CRAC requirement was found for the oligomerization of the gp41 variants. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral sorting of viral glycoproteins for virus assembly at cellular membranes.     

Cn‐AMP2 from green coconut water is an anionic anticancer peptide

Globally, death due to cancers is likely to rise to over 20 million by 2030, which has created an urgent need for novel approaches to anticancer therapies such as the development of host defence peptides. Cn‐AMP2 (TESYFVFSVGM), an anionic host defence peptide from green coconut water of the plant Cocos nucifera, showed anti‐proliferative activity against the 1321N1 and U87MG human glioma cell lines with IC₅₀ values of 1.25 and 1.85 mM, respectively. The membrane interactive form of the peptide was found to be an extended conformation, which primarily included β‐type structures (levels > 45%) and random coil architecture (levels > 45%). On the basis of these and other data, it is suggested that the short anionic N‐terminal sequence (TES) of Cn‐AMP2 interacts with positively charged moieties in the cancer cell membrane. Concomitantly, the long hydrophobic C‐terminal sequence (YFVFSVGM) of the peptide penetrates the membrane core region, thereby driving the translocation of Cn‐AMP2 across the cancer cell membrane to attack intracellular targets and induce anti‐proliferative mechanisms. This work is the first to demonstrate that anionic host defence peptides have activity against human glioblastoma, which potentially provides an untapped source of lead compounds for development as novel agents in the treatment of these and other cancers. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.