Luc Snyers

Association of stomatin with lipid-protein complexes in the plasma membrane and the endocytic compartment

Membrane protein - microvilli - lipid raft - GPI-anchored protein - epithelial cell The 31 kDa integral membrane protein stomatin (protein 7.2b) has a monotopic structure and a cytofacial orientation. We have shown previously that stomatin is located in plasma membrane protruding structures and forms high-order homo-oligomers in the human epithelial cell line UAC, suggesting that this protein has a structural function in the cortical morphogenesis of the cells. It is also present in a pool of juxtanuclear vesicles. In this study, we show that stomatin colocalizes with the GPI-anchored proteins placental alkaline phosphatase (PLAP) and membrane folate receptor alpha (MFRalpha) endogenously expressed in UAC cells. This observation enabled us to demonstrate two different aspects of stomatin. First, using anti-PLAP antibody internalization, we show that the peri-centrosomal vesicles containing stomatin correspond to a subset of endosomes, which can also be labeled with the late endosomal/lysosomal marker LAMP-2. Secondly, we found that stomatin is partially present in detergent-insoluble membrane domains and co-patches with PLAP on the plasma membrane, after cross-linking of PLAP by antibodies. These data indicate that stomatin and GPI-anchored proteins are linked through lipid rafts and undergo the same sorting events. We propose that stomatin, through its affinity for lipid rafts, functions in concentrating GPI-anchored proteins in membrane microvillar structures. Consistent with this hypothesis, we found that stomatin is expressed exclusively in microvilli of the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells.

Human Rhinovirus Type 2 Is Internalized by Clathrin-Mediated Endocytosis

ABSTRACT Using several approaches, we investigated the importance of clathrin-mediated endocytosis in the uptake of human rhinovirus serotype 2 (HRV2). By means of confocal immunofluorescence microscopy, we show that K+ depletion strongly reduces HRV2 internalization. Viral uptake was also substantially reduced by extraction of cholesterol from the plasma membrane with methyl-β-cyclodextrin, which can inhibit clathrin-mediated endocytosis. In accordance with these data, overexpression of dynamin K44A in HeLa cells prevented HRV2 internalization, as judged by confocal immunofluorescence microscopy, and strongly reduced infection. We also demonstrate that HRV2 bound to the surface of HeLa cells is localized in coated pits but not in caveolae. Finally, transient overexpression of the specific dominant-negative inhibitors of clathrin-mediated endocytosis, the SH3 domain of amphiphysin and the C-terminal domain of AP180, potently inhibited internalization of HRV2. Taken together, these results indicate that HRV2 uses clathrin-mediated endocytosis to infect cells.

The Minor Receptor Group of Human Rhinovirus (HRV) Includes HRV23 and HRV25, but the Presence of a Lysine in the VP1 HI Loop Is Not Sufficient for Receptor Binding

ABSTRACT Like all 10 minor receptor group human rhinoviruses (HRVs), HRV23 and HRV25, previously classified as major group viruses, are neutralized by maltose binding protein (MBP)-V33333 (a soluble recombinant concatemer of five copies of repeat 3 of the very-low-density lipoprotein receptor fused to MBP), bind to low-density lipoprotein receptor in virus overlay blots, and replicate in intercellular adhesion molecule 1 (ICAM-1)-negative COS-7 cells. From phylogenetic analysis of capsid protein VP1-coding sequences, they are also known to cluster together with other minor group strains. Therefore, they belong to the minor group; there are now 12 minor group and 87 major group HRV serotypes. Sequence comparison of the VP1 capsid proteins of all HRVs revealed that the lysine in the HI loop, strictly conserved in the 12 minor group HRVs, is also present in 9 major group serotypes that are neutralized by soluble ICAM-1. Despite the presence of this lysine, they are not neutralized by MBP-V33333 and fail to replicate in COS-7 cells and in HeLa cells in the presence of an ICAM-1-blocking antibody. These nine serotypes are therefore “true” major group viruses.

Cysteine 29 is the major palmitoylation site on stomatin

The 31 kDa membrane protein stomatin was metabolically labeled with tritiated palmitic acid in the human amniotic cell line UAC and immunoprecipitated. We show that the incorporated palmitate is sensitive to hydroxylamine, indicating the binding to cysteine residues. Stomatin contains three cysteines. By expressing a myc‐tagged stomatin and substituting the three cysteines by serine, individually or in combination, we demonstrate that Cys‐29 is the predominant site of palmitoylation and that Cys‐86 accounts for the remaining palmitate labeling. Disruption of Cys‐52 alone does not show any detectable reduction of palmitic acid incorporation. Given the organization of stomatin into homo‐oligomers, the presence of multiple palmitate chains is likely to increase greatly the affinity of these oligomers for the membrane and perhaps particular lipid domains within it.

A Cellular Receptor of Human Rhinovirus Type 2, the Very-Low-Density Lipoprotein Receptor, Binds to Two Neighboring Proteins of the Viral Capsid

ABSTRACT The very-low-density lipoprotein receptor (VLDL-R) is a receptor for the minor-group human rhinoviruses (HRVs). Only two of the eight binding repeats of the VLDL-R bind to HRV2, and their footprints describe an annulus on the dome at each fivefold axis. By studying the complex formed between a selection of soluble fragments of the VLDL-R and HRV2, we demonstrate that it is the second and third repeats that bind. We also show that artificial concatemers of the same repeat can bind to HRV2 with the same footprint as that for the native receptor. In a 16-Å-resolution cryoelectron microscopy map of HRV2 in complex with the VLDL-R, the individual repeats are defined. The third repeat is strongly bound to charged and polar residues of the HI and BC loops of viral protein 1 (VP1), while the second repeat is more weakly bound to the neighboring VP1. The footprint of the strongly bound third repeat extends down the north side of the canyon. Since the receptor molecule can bind to two adjacent copies of VP1, we suggest that the bound receptor “staples” the VP1s together and must be detached before release of the RNA can occur. When the receptor is bound to neighboring sites on HRV2, steric hindrance prevents binding of the second repeat.

Species-Specific Receptor Recognition by a Minor-Group Human Rhinovirus (HRV): HRV Serotype 1A Distinguishes between the Murine and the Human Low-Density Lipoprotein Receptor

ABSTRACT Human rhinoviruses (HRV) of the minor receptor group use several members of the low-density lipoprotein receptor superfamily for cell entry. These proteins are evolutionarily highly conserved throughout species and are almost ubiquitously expressed. Their common building blocks, cysteine-rich ligand binding repeats about 40 amino acids in length, exhibit considerable sequence similarity. Various numbers of these repeats are present in the different receptors. We here demonstrate that HRV type 1A (HRV1A) replicates in mouse cells without adaptation. Furthermore, although closely related to HRV2, it fails to bind to the human low-density lipoprotein receptor but recognizes the murine protein, whereas HRV2 binds equally well to both homologues. This difference went unnoticed due to the presence of other receptors, such as the low-density lipoprotein receptor-related protein, which allow species-independent attachment. The species specificity of HRV1A reported here will aid in defining amino acid residues establishing the contact between the viral surface and the receptor.

Human Rhinovirus Type 2-Antibody Complexes Enter and Infect Cells via Fc-γ Receptor IIB1

ABSTRACT HeLa cells were stably transfected with a cDNA clone encoding the B1 isoform of the mouse FcγRII receptor (hereafter referred to as HeLa-FcRII cells). The receptor was expressed at high level at the plasma membrane in about 90% of the cells. These cells bound and internalized mouse monoclonal virus-neutralizing antibodies 8F5 and 3B10 of the subtype immunoglobulin G2a (IgG2a) and IgG1, respectively. Binding of the minor-group human rhinovirus type 2 (HRV2) to its natural receptors, members of the low-density lipoprotein receptor family, is dependent on the presence of Ca2+ ions. Thus, chelating Ca2+ ions with EDTA prevented HRV2 binding, entry, and infection. However, upon complex formation of 35S-labeled HRV2 with 8F5 or 3B10, virus was bound, internalized, and degraded in HeLa-FcRII cells. Furthermore, challenge of these cells with HRV2-8F5 or HRV2-3B10 complexes resulted in de novo synthesis of viral proteins, as shown by indirect immunofluorescence microscopy. These data demonstrate that minor-group receptors can be replaced by surrogate receptors to mediate HRV2 cell entry, delivery into endosomal compartments, and productive uncoating. Consequently, the conformational change and uncoating of HRV2 appears to be solely triggered by the low-pH (pH ≤ 5.6) environment in these compartments.

Identification of the Human Rhinovirus Serotype 1A Binding Site on the Murine Low-Density Lipoprotein Receptor by Using Human-Mouse Receptor Chimeras

ABSTRACT Human rhinovirus serotype 1A (HRV1A) binds more strongly to the mouse low-density lipoprotein receptor (LDLR) than to the human homologue (M. Reithmayer, A. Reischl, L. Snyers, and D. Blaas, J. Virol. 76:6957-6965, 2002). Here, we used this fact to determine the binding site of HRV1A by replacing selected ligand binding modules of the human receptor with the corresponding ligand binding modules of the mouse receptor. The chimeric proteins were expressed in mouse fibroblasts deficient in endogenous LDLR and LDLR-related protein, both used by minor group HRVs for cell entry. Binding was assessed by virus overlay blots, by immunofluorescence microscopy, and by measuring cell attachment of radiolabeled virus. Replacement of ligand binding repeat 5 of the human LDLR with the corresponding mouse sequence resulted in a substantial increase in HRV1A binding, whereas substitution of repeats 3 and 4 was without effect. Replacement of human receptor repeats 1 and 2 with the murine homologues also increased virus binding. Finally, murine receptor modules 1, 2, and 5 simultaneously introduced into the human receptor resulted in HRV1A binding indistinguishable from mouse wild-type receptor. Thus, repeats 1 and/or 2 and repeat 5 are involved in HRV1A attachment. Changing CDGGPD in the acidic cluster of module 5 in the human receptor to CDGEAD present in the mouse receptor led to substantially increased binding of HRV1A, indicating an important role of the glutamate residue in HRV1A recognition.

Distinct chromatin signature of histone H3 variant H3.3 in human cells.

Actively transcribed regions of the genome have been found enriched for the histone H3 variant H3.3. This variant is incorporated into nucleosomes throughout the cell cycle whereas the canonical isoforms are predominately deposited in association with replication. In order to obtain a global picture of the deposition pattern at the single cell level we expressed H3.3 in both normal and malignant human cells and analyzed nuclei using conventional and structured illumination imaging (SIM). We found that the distribution pattern of H3.3 in interphase differs from that of the canonical histone H3 variants and this difference is conveyed to mitotic chromosomes which display a distinct H3.3 banding pattern. Histone H3.3 localization positively correlated with markers for transcriptionally active chromatin and, notably, H3.3 was almost completely absent from the inactive X chromosome. Collectively, our data show that histone variant H3.3 occupies distinct intranuclear chromatin domains and that these genomic loci are associated with gene expression.

A Mutation in the First Ligand-Binding Repeat of the Human Very-Low-Density Lipoprotein Receptor Results in High-Affinity Binding of the Single V1 Module to Human Rhinovirus 2

ABSTRACT Minor group human rhinoviruses (HRVs) bind members of the low-density lipoprotein receptor family for cell entry. The ligand-binding domains of these membrane proteins are composed of various numbers of direct repeats of about 40 amino acids in length. Residues involved in binding of module 3 (V3) of the very-low-density lipoprotein receptor (VLDLR) to HRV2 have been identified by X-ray crystallography (N. Verdaguer, I. Fita, M. Reithmayer, R. Moser, and D. Blaas, Nat. Struct. Mol. Biol. 11:429-434, 2004). Sequence comparisons of the eight repeats of VLDLR with respect to the residues implicated in the interaction between V3 and HRV2 suggested that (in addition to V3) V1, V2, V5, and V6 also fulfill the requirements for interacting with the virus. Using a highly sensitive binding assay employing phage display, we demonstrate that single modules V2, V3, and V5 indeed bind HRV2. However, V1 does not. A single mutation from threonine 17 to proline converted the nonbinding wild-type form of V1 into a very strong binder. We interpret the dramatic increase in affinity by the generation of a hydrophobic patch between virus and receptor; in the presence of threonine, the contact area might be disturbed. This demonstrates that the interaction between virus and its natural receptors can be strongly enhanced by mutation.