Little is known about the assembly pathway and structure of hepatitis C virus (HCV) since insufficient quantities of purified virus are available for detailed biophysical and structural studies. Here, we show that bacterially expressed HCV core proteins can efficiently self-assemble in vitro into nucleocapsid-like particles. These particles have a regular, spherical morphology with a modal distribution of diameters of approximately 60 nm. Self-assembly of nucleocapsid-like particles requires structured RNA molecules. The 124 N-terminal residues of the core protein are sufficient for self-assembly into nucleocapsid-like particles. Inclusion of the carboxy-terminal domain of the core protein modifies the core assembly pathway such that the resultant particles have an irregular outline. However, these particles are similar in size and shape to those assembled from the 124 N-terminal residues of the core protein. These results provide novel opportunities to delineate protein-protein and protein-RNA interactions critical for HCV assembly, to study the molecular details of HCV assembly, and for performing high-throughput screening of assembly inhibitors.
Lysosomal degradation of the receptor-tyrosine kinase cMet requires receptor ubiquitination by the E3 ubiquitin ligase Cbl followed by clathrin-dependent internalization. A role for Cbl as an adaptor for cMet internalization has been previously reported. However, the requirement for Cbl ubiquitin ligase activity in this process and its mode of recruitment to cMet has yet to be determined. Cbl can directly bind cMet at phosphotyrosine 1003 or indirectly via Grb2 to phosphotyrosine 1356 in the multisubstrate binding domain of cMet. The direct binding of Cbl with cMet is critical for receptor degradation and not receptor internalization. Here we show a strict requirement for Grb2 and the ubiquitin ligase activity of Cbl for cMet endocytosis. Receptor internalization was impaired by small interfering RNA depletion of Grb2, overexpression of dominant negative Grb2 mutants, and point mutations in the cMet multisubstrate docking site that inhibits the direct association of Grb2 with cMet. The requirement for Grb2 was specific and did not involve the multiadaptor Gab1. cMet internalization was impaired in cells expressing an ubiquitin ligase-deficient Cbl mutant or conjugation-deficient ubiquitin but was unaffected in cells expressing a Cbl mutant that is unable to bind cMet directly. Expression of a Cbl-Grb2 chimera rescued impaired cMet endocytosis in cells depleted of endogenous Grb2. These results indicate that the ubiquitin ligase activity of Cbl is critical for clathrin-dependent cMet internalization and suggest a role for Grb2 as an intermediary linking Cbl ubiquitin ligase activity to this process.
For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2-mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2-mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by facilitating mCAT-1 trafficking.A s obligatory parasites, viruses have evolved to exploit host cellular mechanisms to facilitate viral replication and infection. Cell entry is the first step in viral infection. Viral entry involves receptor binding and movement, either into the cell or across the cell membrane, followed by the penetration of the cell membrane. In the case of enveloped viruses, this step involves membrane fusion between the virus and cell membranes (15). For many retroviruses, active receptor recruitment and trafficking occur during entry. For example, receptor trafficking is indispensable for HIV infection. The binding of HIV to CD4, which resides in lipid rafts (membrane microdomains enriched in cholesterol, glycosphingolipids, and signaling phospholipids), results in the subsequent recruitment of the coreceptors CXCR4 and CCR5 to the lipid raft (44). For ecotropic murine leukemia virus (MLV) (eMLV), a distantly related retrovirus receptor, trafficking is also important. Soon after cell contact, eMLV appears to "surf" along cell filopodia toward ...
Increased signaling from cMet, the receptor for Hepatocyte Growth Factor (HGF) has been implicated in the progression of many cancers, particularly pancreatic cancer, correlating with cellular transformation, tumor invasion and poor patient prognosis. In contrast to other cancers, receptor mutations that result in increased cMet signaling have yet to be detected in pancreatic cancers. This led us to hypothesize that post translational mechanisms such as impaired cMet down regulation, a process that normally functions to attenuate cMet signaling in vivo, could be a factor for increased cMet signaling in pancreatic tumors. To test this, we examined the lysosomal down regulation of cMet in a panel of pancreatic adenocarcinoma cell lines. Here we report that uncoupled cMet down regulation in pancreatic tumor cells correlates with increased cMet stability and signaling in response to HGF. In pancreatic Suit-2 cells, impaired binding of the E3 ubiquitin ligase Cbl to cMet, results in reduced receptor ubiquitination and increased cMet recycling to the cell surface. Conversely, transient HGF induced cMet ubiquitination in pancreatic BxPC-3 cells correlates with prolonged cMet stability and signaling, suggesting that the kinetics of cMet ubiquitination rather than receptor ubiquitination per se is critical for efficient cMet down regulation. These data mark the first evidence that disrupted cMet down regulation could be a contributing factor in the aggressiveness of certain pancreatic tumor cells and highlights the importance of the endocytic machinery as a novel therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 277.
Entry of the bacterial pathogen Listeria monocytogenes into host epithelial cells is critical for infection and virulence. One major pathway for
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