A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). C9orf72 encodes two C9orf72 protein isoforms of unclear function. Reduced levels of C9orf72 expression have been reported in C9ALS/FTD patients, and although C9orf72 haploinsufficiency has been proposed to contribute to C9ALS/FTD, its significance is not yet clear. Here, we report that C9orf72 interacts with Rab1a and the Unc‐51‐like kinase 1 (ULK1) autophagy initiation complex. As a Rab1a effector, C9orf72 controls initiation of autophagy by regulating the Rab1a‐dependent trafficking of the ULK1 autophagy initiation complex to the phagophore. Accordingly, reduction of C9orf72 expression in cell lines and primary neurons attenuated autophagy and caused accumulation of p62‐positive puncta reminiscent of the p62 pathology observed in C9ALS/FTD patients. Finally, basal levels of autophagy were markedly reduced in C9ALS/FTD patient‐derived iNeurons. Thus, our data identify C9orf72 as a novel Rab1a effector in the regulation of autophagy and indicate that C9orf72 haploinsufficiency and associated reductions in autophagy might be the underlying cause of C9ALS/FTD‐associated p62 pathology.

Expansion of GGGGCC repeats in C9orf72 causes familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but the underlying mechanism is unclear. Using RNA pulldown and immunohistochemistry in ALS biosamples, Cooper-Knock et al. identify proteins that bind to the repeat expansions. Disrupted RNA splicing and/or nuclear export may underlie C9orf72-ALS pathogenesis.

CD81 has been described as a putative receptor for hepatitis C virus (HCV); however, its role in HCV cell entry has not been characterized due to the lack of an efficient cell culture system. We have examined the role of CD81 in HCV glycoprotein-dependent entry by using a recently developed retroviral pseudotyping system. Human immunodeficiency virus (HIV) pseudotypes bearing HCV E1E2 glycoproteins show a restricted tropism for human liver cell lines. Although all of the permissive cell lines express CD81, CD81 expression alone is not sufficient to allow viral entry. CD81 is required for HIV-HCV pseudotype infection since (i) a monoclonal antibody specific for CD81 inhibited infection of susceptible target cells and (ii) silencing of CD81 expression in Huh-7.5 hepatoma cells by small interfering RNAs inhibited HIV-HCV pseudotype infection. Furthermore, expression of CD81 in human liver cells that were previously resistant to infection, HepG2 and HH29, conferred permissivity of HCV pseudotype infection. The characterization of chimeric CD9/CD81 molecules confirmed that the large extracellular loop of CD81 is a determinant for viral entry. These data suggest a functional role for CD81 as a coreceptor for HCV glycoprotein-dependent viral cell entry. Hepatitis C virus (HCV) is an enveloped, positive-stranded RNA virus classified in the familyFlaviviridae. An estimated 170 million individuals worldwide are infected with HCV. Infection is associated with the development of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The principal site of virus replication is thought to be the liver; however, the specific cell types targeted by HCV remain unclear. Recent reports of HCV antigen detection in the chronically infected liver (43) and serum-derived virus infection of primary liver cell cultures (7) suggest that hepatocytes are the primary target cells in vivo. However, authors from several laboratories have suggested that HCV may infect a wider range of cell types, including B cells and cells of the monocyte/macrophage lineage within the central nervous system (14,18,32,40). Since attachment of a virus to a target cell is determined by specific interactions between the viral glycoproteins (gp) and cell surface receptors, this suggests that liver-specific molecules may act as receptors for HCV.HCV encodes two putative envelope gp's, E1 and E2, which are believed to be type I integral transmembrane proteins. Our understanding of gp maturation and virus assembly is limited by the lack of a tissue culture system supporting virus assembly and release. Hence, the mechanism(s) by which HCV enters target cells is currently unknown. In the absence of a cell culture system, surrogate assays have been developed to study HCV entry, including the expression of a truncated version(s) of the E2 gp (10, 29), E1E2-liposomes (15), vesicular stomatitis virus (VSV)-HCV pseudotypes (6,22,24), and viruslike particles expressed in insect cell systems (4,42,44). Truncated versions of E2 bind specifically to human cells and were used...

Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).CD81 is a member of the tetraspanin family. Tetraspanins are membrane proteins containing four transmembrane domains, short intracellular domains and two extracellular loops (8). Tetraspanins have been shown to be involved in cell activation, proliferation, motility, and metastasis, as well as in cell fusion (14). Although CD81 is widely expressed, its level of expression varies in specific cell lineages and during differentiation. Moreover, its association with cell surface proteins differs in cell types of various lineages. In B cells CD81 is a component of the CD19-CD21-CD81-Leu-13 molecular complex, which plays a role in B-cell activation (2), whereas in T cells the molecule is associated with T-cell-specific molecules, including CD4 and CD8 (5, 15). In addition to its association with lineage-specific proteins, CD81 is associated with integrins and other tetraspanins (8). As a consequence of such protein associations, it is possible to activate multiple adhesion/signaling pathways in different cell types by engaging CD81 at their surface. For example, treatment of B-cell lines with a monoclonal antibody (MAb) specific for CD81 induces changes in cell adhesion and inhibits proliferation, whereas treatment of T-cell lines affects cell adhesion but not proliferation (10).CD81 was recently reported to interact with the hepatitis C virus (HCV) envelope glycoprotein (gp) E2 and hypothesized to act as a putative viral receptor (12). We have confirmed this observation and have shown that cell surface-expressed human CD81, but not murine or monkey (Chlorocebus aethiops, African green monkey [AGM]) CD81, binds E2 661 , a truncated soluble version of the E2 gp. Furthermore, interaction of E2 661 with the B-cell line Daudi had effects on cell adhesion and proliferation similar to the effects induced by anti-CD81 antibodies (3). This observation is of particular interest since in addition to causing acute and chronic liver disease, HCV is the major cause for mixed cryoglobulinemia...

IntroductionPlasma membrane contains small organized microdomains (lipid rafts) in which restricted repertoires of proteins are arranged together. 1,2 In resting cells, lipid rafts are estimated to be around 100 nm in diameter, including a few dozen proteins, and are distributed randomly on the cell surface, covering up to 50% of the plasma membrane. Upon cell activation, raft domains coalesce, recruiting and excluding different receptors, and allowing the proper organization of signaling complexes for efficient signal transduction. 1,2 Tetraspanins comprise a large number of small palmitoylated polypeptides that span the plasma membrane 4 times, [3][4][5][6] and form microdomains that contain a restricted repertoire of proteins. Biochemically, they share some properties with lipid rafts, but tetraspanin microdomains are based on protein-protein interactions. [7][8][9][10] Tetraspanins have a highly conserved structure with a short and a large extracellular loop (LEL) where 2 or 3 disulfide bonds can be formed. 11 This large loop interacts noncovalently with other tetraspanins and transmembrane proteins, including integrins and adhesion receptors of the immunoglobulin (Ig) superfamily. Although all mammalian cells express different tetraspanins, genetic approaches have been elusive and their function has not yet been fully elucidated. However, their role in antigen presentation and sperm-egg binding has been recently underscored. [12][13][14][15][16][17][18][19][20] The association of certain plasma membrane proteins to the cortical actin cytoskeleton is critical for their proper localization and function. Thus, the concentration of selectins and their ligands on the tip of microvilli 21,22 both at the leukocyte and the apical surface of endothelial cells favors their interaction during the rolling phase of leukocyte extravasation. Likewise, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), which are relevant in the subsequent leukocyte firm adhesion step, are also displayed anchored to actin through ezrin-radixin-moesin proteins (ERMs) 23,24 at the apical surface on endothelial cells. Upon leukocyte firm adhesion, the engagement of VCAM-1 and ICAM-1 triggers the reorganization of the endothelial cortical actin cytoskeleton, building up a 3-dimensional docking structure that prevents the detachment of leukocytes by shear stress. 22,23 Here, we show that ICAM-1 and VCAM-1 are included in tetraspanin microdomains that regulate their membrane expression and the efficient adhesive function necessary for proper leukocyte transendothelial migration under flow conditions. Materials and methods Cells and cell culturesHuman umbilical vein endothelial cells (HUVECs) were obtained and cultured as previously described. 25 Cells were used up to the third passage in all assays. To activate HUVECs, tumor necrosis factor-␣ (TNF-␣; 20 ng/mL)(R&D Systems, Minneapolis, MN) was added to the culture media 20 hours before the assays were performed. T lymphoblasts were derived The online vers...