Although biological and biochemical data have been accumulated on most hepatitis C virus proteins, the structure and function of the 63-amino-acid p7 polypeptide of this virus have never been investigated. In this work, sequence analyses predicted that p7 contains two transmembrane passages connected by a short hydrophilic segment. The C-terminal transmembrane domain of p7 was predicted to function as a signal sequence, which was confirmed experimentally by analyzing the translocation of a reporter glycoprotein fused at its C terminus. The p7 polypeptide was tagged either with the ectodomain of CD4 or with a Myc epitope to study its membrane integration, its subcellular localization, and its topology. Alkaline extraction studies confirmed that p7 is an integral membrane polypeptide. The CD4-p7 chimera was detected by immunofluorescence on the surface of nonpermeabilized cells, indicating that it is exported to the plasma membrane. However, pulse-chase analyses showed that only approximately 20% of endoglycosidase H-resistant CD4-p7 was detected after long chase times, suggesting that a large proportion of p7 stays in an early compartment of the secretory pathway. Finally, by inserting a Myc epitope in several positions of p7 and analyzing the accessibility of this epitope on the plasma membrane of HepG2 cells, we showed that p7 has a double membrane-spanning topology, with both its N and C termini oriented toward the extracellular environment. Altogether, these data indicate that p7 is a polytopic membrane protein that could have a functional role in several compartments of the secretory pathway.Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma throughout the world (22). HCV is a positive-stranded RNA virus that belongs to the Hepacivirus genus. Together with the genera Flavivirus and Pestivirus, HCV belongs to the Flaviviridae family (43, 52). The HCV genome encodes a single polyprotein precursor of approximately 3,000 amino acid residues. This polyprotein precursor is co-and posttranslationally processed by cellular and viral proteases to yield the mature structural and nonstructural proteins C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (see reference 41 for a recent review) (Fig. 1A).Recently, it has been reported that an additional HCV protein can be produced by a ribosomal frameshift in the Nterminal region of the polyprotein (55). The structural proteins, i.e., capsid, E1, and E2, are released from the polyprotein by the endoplasmic reticulum (ER) signal peptidase(s) of the host cell. Further processing occurs at the C terminus of the capsid; however, the protease involved in this cleavage has not been identified (34). The nonstructural proteins (NS3, NS4A, NS4B, NS5A, and NS5B) are released from the polyprotein after cleavage by HCV proteases NS2-3 and NS3/4A. The cleavage between p7 and NS2 is supposed to be mediated by a cellular signal peptidase (30,35). Although most cleavages in the polyprotein precursor proceed to completion during or...

The hepatitis C virus genome encodes a polyprotein precursor that is co-and post-translationally processed by cellular and viral proteases to yield 10 mature protein products (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Although most cleavages in hepatitis C virus polyprotein precursor proceed to completion during or immediately after translation, the cleavages mediated by a host cell signal peptidase are partial at the E2/p7 and p7/NS2 sites, leading to the production of an E2p7NS2 precursor. The sequences located immediately N-terminally of E2/p7 and p7/NS2 cleavage sites can function as signal peptides. When fused to a reporter protein, the signal peptides of p7 and NS2 were efficiently cleaved. However, when full-length p7 was fused to the reporter protein, partial cleavage was observed, indicating that a sequence located N-terminally of the signal peptide reduces the efficiency of p7/NS2 cleavage. Sequence analyses and mutagenesis studies have also identified structural determinants responsible for the partial cleavage at both the E2/p7 and p7/NS2 sites. Finally, the short distance between the cleavage site of E2/p7 or p7/NS2 and the predicted transmembrane ␣-helix within the P region might impose additional structural constraints to the cleavage sites. The insertion of a linker polypeptide sequence between P-3 and P-4 of the cleavage site released these constraints and led to improved cleavage efficiency. Such constraints in the processing of a polyprotein precursor are likely essential for hepatitis C virus to post-translationally regulate the kinetics and/or the level of expression of p7 as well as NS2 and E2 mature proteins.

Background Mesenchymal stem cells (MSCs) are multipotent cells with broad immunosuppressive capacities. Recently, it has been reported that MSCs can transfer mitochondria to various cell types, including fibroblast, cancer, and endothelial cells. It has been suggested that mitochondrial transfer is associated with a physiological response to cues released by damaged cells to restore and regenerate damaged tissue. However, the role of mitochondrial transfer to immune competent cells has been poorly investigated. Methods and results Here, we analyzed the capacity of MSCs from the bone marrow (BM) of healthy donors (BM-MSCs) to transfer mitochondria to primary CD4 + CCR6 + CD45RO + T helper 17 (Th17) cells by confocal microscopy and fluorescent-activated cell sorting (FACS). We then evaluated the Th17 cell inflammatory phenotype and bioenergetics at 4 h and 24 h of co-culture with BM-MSCs. We found that Th17 cells can take up mitochondria from BM-MSCs already after 4 h of co-culture. Moreover, IL-17 production by Th17 cells co-cultured with BM-MSCs was significantly impaired in a contact-dependent manner. This inhibition was associated with oxygen consumption increase by Th17 cells and interconversion into T regulatory cells. Finally, by co-culturing human synovial MSCs (sMSCs) from patients with rheumatoid arthritis (RA) with Th17 cells, we found that compared with healthy BM-MSCs, mitochondrial transfer to Th17 cells was impaired in RA-sMSCs. Moreover, artificial mitochondrial transfer also significantly reduced IL-17 production by Th17 cells. Conclusions The present study brings some insights into a novel mechanism of T cell function regulation through mitochondrial transfer from stromal stem cells. The reduced mitochondrial transfer by RA-sMSCs might contribute to the persistence of chronic inflammation in RA synovitis. Electronic supplementary material The online version of this article (10.1186/s13287-019-1307-9) contains supplementary material, which is available to authorized users.