Efficient replication of hepatitis C virus (HCV) subgenomic RNA in cell culture requires the introduction of adaptive mutations. In this report we describe a system which enables efficient replication of the Con1 subgenomic replicon in Huh7 cells without the introduction of adaptive mutations. The starting hypothesis was that high amounts of the NS5A hyperphosphorylated form, p58, inhibit replication and that reduction of p58 by inhibition of specific kinase(s) below a certain threshold enables HCV replication. Upon screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro and the formation of NS5A p58 in cell culture. Cells, transfected with the HCV Con1 wild-type sequence, support HCV RNA replication upon addition of any of the three compounds. The effect of the kinase inhibitors was found to be synergistic with coadaptive mutations in NS3. This is the first direct demonstration that the presence of high amounts of NS5A-p58 causes inhibition of HCV RNA replication in cell culture and that this inhibition can be relieved by kinase inhibitors.
Fibro/Adipogenic Progenitors (FAPs) are muscle-interstitial progenitors mediating pro-myogenic signals that are critical for muscle homeostasis and regeneration. In myopathies, the autocrine/paracrine constraints controlling FAP adipogenesis are released causing fat infiltrates. Here, by combining pharmacological screening, high-dimensional mass cytometry and in silico network modeling with the integration of single-cell/bulk RNA sequencing data, we highlighted the canonical WNT/ GSK/β-catenin signaling as a crucial pathway modulating FAP adipogenesis triggered by insulin signaling. Consistently, pharmacological blockade of GSK3, by the LY2090314 inhibitor, stabilizes β-catenin and represses PPARγ expression abrogating FAP adipogenesis ex vivo while limiting fatty degeneration in vivo. Furthermore, GSK3 inhibition improves the FAP pro-myogenic role by efficiently stimulating, via follistatin secretion, muscle satellite cell (MuSC) differentiation into mature myotubes. Combining, publicly available single-cell RNAseq datasets, we characterize FAPs as the main source of WNT ligands inferring their potential in mediating autocrine/paracrine responses in the muscle niche. Lastly, we identify WNT5a, whose expression is impaired in dystrophic FAPs, as a crucial WNT ligand able to restrain the detrimental adipogenic differentiation drift of these cells through the positive modulation of the β-catenin signaling.
Hepatitis C virus (HCV) glycoprotein E2 binds to human cells by interacting with the CD81 molecule, which has been proposed to be the viral receptor. A correlation between binding to CD81 and species permissiveness to HCV infection has also been reported. We have determined the sequence of CD81 from the tamarin, a primate species known to be refractory to HCV infection. Tamarin CD81 (t-CD81) differs from the human molecule at 5 amino acid positions (155, 163, 169, 180, and 196) within the large extracellular loop (LEL), where the binding site for E2 has been located. Soluble recombinant forms of human CD81 (h-CD81), t-CD81, and African green monkey CD81 (agm-CD81) LEL molecules were analyzed by enzyme-linked immunosorbent assay for binding to E2 glycoprotein. Both h-CD81 and t-CD81 molecules were able to bind E2. Competition experiments showed that the two receptors cross-compete and that the t-CD81 binds with stronger affinity than the human molecule. Recently, h-CD81 residue 186 has been characterized as the critical residue involved in the interaction with E2. Recombinant CD81 mutant proteins were expressed to test whether human and tamarin receptors interacted with E2 in a comparable manner. Mutation of residue 186 (F186L) dramatically reduced the binding capability of t-CD81, a result that has already been demonstrated for the human receptor, whereas the opposite mutation (L186F) in agm-CD81 resulted in a neat gain of binding activity. Finally, the in vitro data were confirmed by detection of E2 binding to cotton-top tamarin (Saguinus oedipus) cell line B95-8 expressing endogenous CD81. These results indicate that the binding of E2 to CD81 is not predictive of an infection-producing interaction between HCV and host cells.Hepatitis C virus (HCV) is the major etiologic agent of non-A non-B hepatitis, infecting an estimated 400 million people worldwide (3). A striking feature of HCV infection is the tendency towards a chronic status leading to liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma (24). Moreover, HCV infection is also associated with mixed cryoglobulinemia, a B-lymphocyte proliferative disorder (2). HCV is an enveloped virus classified as a Flavivirus (20); its genome is a positive-strand RNA of 9.5 kb, containing a single open reading frame encoding a large polyprotein precursor (5, 6), whose cleavage results in mature structural and nonstructural viral proteins (18). A basic polypeptide (core) and two envelope glycoproteins (E1 and E2) are the putative HCV structural proteins (30). Because of the lack of a cell culture system for virus growth, most of the studies aimed at understanding the biological activity of viral envelope proteins have been performed on recombinant proteins (15,16,27,28). A truncated soluble recombinant form of HCV E2 glycoprotein has been reported to bind the surface of human cells (22) by interacting with the CD81 molecule (9, 19).CD81 is a member of the tetraspanin family, membrane proteins containing four transmembrane domains, a short int...
Interleukin‐6 (IL‐6) triggers the formation of a high affinity receptor complex with the ligand binding subunit IL‐6Ralpha and the signal transducing chain gp130. Since the intracytoplasmic region of the IL‐6Ralpha does not contribute to signaling, soluble forms of the extracytoplasmic domain (sIL‐6Ralpha), potentiate IL‐6 bioactivity and induce a cytokine‐responsive status in cells expressing gp130 only. This observation, together with the detection of high levels of circulating soluble human IL‐6Ralpha (shIL‐6Ralpha) in sera, suggests that the hIL‐6‐shIL‐6Ralpha complex is an alternative form of the cytokine. Here we describe the generation of human IL‐6 (hIL‐6) variants with strongly enhanced shIL‐6Ralpha binding activity and bioactivity. Homology modeling and site‐directed mutagenesis of hIL‐6 suggested that the binding interface for hIL‐6Ralpha is constituted by the C‐terminal portion of the D‐helix and residues contained in the AB loop. Four libraries of hIL‐6 mutants were generated by each time fully randomizing four different amino acids in the predicted AB loop. These libraries were displayed monovalently on filamentous phage surface and sorted separately for binding to immobilized shIL‐6Ralpha. Mutants were selected which, when expressed as soluble proteins, showed a 10‐ to 40‐fold improvement in shIL‐6Ralpha binding; a further increase (up to 70‐fold) was achieved by combining variants isolated from different libraries. Interestingly, high affinity hIL‐6 variants show strongly enhanced bioactivity on cells expressing gp13O in the presence of shIL‐6Ralpha at concentrations similar to those normally found in human sera.
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