The core (C) protein of hepatitis C virus (HCV) appears to be a multifunctional protein that is involved in many viral and cellular processes. Although its effects on host cells have been extensively discussed in the literature, little is known about its main function, the assembly and packaging of the viral genome. We have studied the in vitro assembly of several deleted versions of recombinant HCV C protein expressed in E. coli. We demonstrated that the 75 N-terminal residues of the C protein were sufficient to assemble and generate nucleocapsid-like particles (NLPs) in vitro. However, homogeneous particles of regular size and shape were observed only when NLPs were produced from at least the first 79 N-terminal amino acids of the C protein. This small protein unit fused to the endoplasmic reticulum-anchoring domain also generated NLPs in yeast cells. These data suggest that the N-terminal half of the C protein is important for formation of NLPs. Similarities between the HCV C protein and C proteins of other members of the Flaviviridae are discussed.

IntroductionMonosodium urate crystals (MSU), the etiological agent of gout, are one of the most potent proinflammatory stimuli for neutrophils. The modulation of MSU-induced neutrophil activation by inhibitory receptors remains poorly characterized. The expression of the myeloid inhibitory C-type lectin-like receptor (MICL) in neutrophils is downregulated by several proinflammatory stimuli, suggestive of a role for this receptor in neutrophil function. We thus investigated the potential role of MICL in MSU-induced neutrophil activation.MethodsThe expression of MICL was monitored in human neutrophils by flow cytometry and Western blot analysis after stimulation with MSU. Protein tyrosine phosphorylation was also assessed by Western blot analysis and the production of IL-1 and IL-8 by enzyme-linked immunosorbent assay. Changes in the concentration of cytoplasmic free calcium were monitored with the Fura-2-acetoxymethyl ester calcium indicator. MICL expression was modulated with an anti-MICL antibody in neutrophils and siRNA in the PLB-985 neutrophil-like cell line.ResultsMSU induced the downregulation of MICL expression in neutrophils. A diminution in the expression of MICL induced by antibody cross-linking or siRNA enhanced the MSU-dependent increase in cytoplasmic calcium levels, protein tyrosine phosphorylation and IL-8 but not IL-1 production. Pretreatment of neutrophils with colchicine inhibited the MSU-induced downregulation of MICL expression.ConclusionsOur findings strongly suggest that MICL acts as an inhibitory receptor in human neutrophils since the downregulation of MICL expression enhances MSU-induced neutrophil activation. Since MSU downregulates the expression of MICL, MICL may play a pathogenic role in gout by enhancing neutrophil effector functions. In support of this notion, colchicine counteracts the MSU-induced loss of MICL expression. Our findings thus also provide further insight into the potential molecular mechanisms behind the anti-inflammatory properties of this drug.

Angiostatin is a potent inhibitor of angiogenesis. One mechanism through which angiostatin inhibits angiogenesis is by binding to the cell surface protein p80-angiomotin. The p80-angiomotin protein promotes angiogenesis, in part, by conferring a hypermigratory phenotype to endothelial cells. Although p80-angiomotin is extensively characterized, less is known about the related protein angiomotin-like 1. We report that angiomotin-like 1 forms part of a protein complex containing p80-angiomotin. Structure-function studies revealed that angiomotin-like 1 associates with this p80-angiomotin-containing complex via its coiled-coil domain. Since p80-angiomotin plays a role in cell migration, a process that involves the remodeling of the actin cytoskeleton, we then addressed the hypothesis that angiomotin-like 1 may interact with the cytoskeleton. Immunofluorescence studies reveal that angiomotin-like 1 not only co-localizes with filamentous actin but also significantly modifies the architecture of the actin cytoskeleton. Regarding migration, angiomotin-like 1 increases the velocity of migration and decreases the persistence of migration directionality. Together these observations strongly suggest that angiomotin-like 1 is involved in actin-cytoskeleton-based processes, in part, via its interaction with a p80-angiomotin-containing complex and the actin cytoskeleton. These findings have important implications for angiogenesis-driven disease since angiomotin and angiomotin-like 1 are both expressed in capillaries.

We previously described a non-classical mechanism that arrests Fc␥RIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. The engagement of Fc␥RIIa leads to its ubiquitination by the ubiquitin ligase c-Cbl and degradation by the proteasome. Herein, we further examined some of the events regulating this novel pathway. The adaptor protein CIN85 was described in other systems to be involved in the regulation of the c-Cbl-dependent pathway. We found that CIN85 is expressed in human neutrophils and that it translocates like c-Cbl from the cytosol to the plasma membrane following receptor cross-linking. CIN85 was also recruited to the same subset of high density detergent-resistant membrane fractions in which stimulated Fc␥RIIa partitioned with c-Cbl. The integrity of these microdomains is essential to the Fc␥RIIa degradation process because the cholesterol-depleting agent methyl-␤-cyclodextrin inhibits this event. Silencing the expression of CIN85 by siRNA in dibutyryl cyclic AMP-differentiated PLB 985 cells prevented Fc␥RIIa degradation and increased IgG-mediated phagocytosis. Confocal microscopy revealed that the presence of CIN85 is essential to the proper sorting of Fc␥RIIa during endocytosis. We also provide direct evidence that CIN85 is a substrate of serine/threonine kinase PKCs. Classical PKCs positively regulate Fc␥RIIa ubiquitination and degradation because these events were inhibited by Gö6976, a classical PKC inhibitor. We conclude that the ubiquitination and degradation of stimulated Fc␥RIIa mediated by c-Cbl are positively regulated by the adaptor protein CIN85 in a PKC-dependent manner and that these events contribute to the termination of Fc␥RIIa signaling. Fc␥ receptors (Fc␥Rs)3 represent a family of membrane proteins involved in the recognition of the Fc portion of immunoglobulin G. They act as sensors for opsonized pathogens or immune complexes, and their engagement initiates intracellular signals that lead to multiple cell functions, such as degranulation, activation of the respiratory burst, and phagocytosis (1-3). Fc␥R isoforms either express an immunoreceptor tyrosine-based activating motif (ITAM) in their intracellular portion or associate with ITAM-containing accessory proteins with the exception of Fc␥RIIb, the unique inhibitory member of the Fc␥R family that transmits inhibitory signals through an immunoreceptor tyrosine-based inhibition motif (ITIM) (4, 5). In the case of antibody-mediated responses, such as IgG-dependent phagocytosis, neutrophils and other immune effectors, such as macrophages, have the capacity to trigger strong proinflammatory responses, which must be tightly controlled. On human neutrophils, only two Fc␥Rs, namely Fc␥RIIa (CD32a) and Fc␥RIIIb (CD16b), are constitutively expressed, neither of which possess an ITIM. The available data indicate that in contrast to other human phagocytes, including macrophages, human neutrophils express very little (6) or no (7) Fc␥RIIb.Several lines of evidence indicate that Fc␥RIIa is directly involve...