Syntenin regulates hepatitis C virus sensitivity to neutralizing antibody by promoting E2 secretion through exosomes.

BACKGROUND & AIMS: Assembly of infectious hepatitis C virus (HCV) particles is known to involve host lipoproteins, giving rise to unique lipo-viro-particles (LVPs), but proteome studies now suggest that additional cellular proteins are associated with HCV virions or other particles containing the viral envelope glycoprotein E2. Many of these host cell proteins are common markers of exosomes, most notably the intracellular adaptor protein syntenin, which is required for exosome biogenesis. We aimed to elucidate the role of syntenin/E2 in HCV infection. METHODS: Using cell culture-derived HCV, we studied the biogenesis and function of E2-coated exosomes in both hepatoma cells and primary human hepatocytes (PHHs). RESULTS: Knockout of syntenin had a negligible impact on HCV replication and virus production, whereas ectopic expression of syntenin at physiological levels reduced intracellular E2 abundance, while concomitantly increasing the secretion of E2-coated exosomes. Importantly, cells expressing syntenin and HCV structural proteins efficiently released exosomes containing E2 but lacking the core protein. Furthermore, infectivity of HCV released from syntenin-expressing hepatoma cells and PHHs was more resistant to neutralization by E2-specific antibodies and chronic-phase patient serum. We also found that high E2/syntenin levels in sera correlate with lower serum neutralization capability. CONCLUSIONS: E2- and syntenin-containing exosomes are a major type of particle released from cells expressing high levels of syntenin. Efficient production of E2-coated exosomes renders HCV infectivity less susceptible to antibody neutralization in hepatoma cells and PHHs. LAY SUMMARY: This study identifies a key role for syntenin in the regulation of E2 secretion via exosomes. Efficient production of E2-coated exosomes was shown to make hepatitis C virus less sensitive to antibody neutralization. These results may have implications for the development of a hepatitis C virus vaccine.

Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication.

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.

Biochemical and morphological properties of hepatitis C virus particles and determination of their lipidome.

A hallmark of hepatitis C virus (HCV) particles is their association with host cell lipids, most notably lipoprotein components. It is thought that this property accounts for the low density of virus particles and their large heterogeneity. However, the composition of infectious virions and their biochemical and morphological properties are largely unknown. We developed a system in which the envelope glycoprotein E2 was N-terminally tagged with a FLAG epitope. This virus, designated Jc1E2(FLAG), produced infectivity titers to wild type levels and allowed affinity purification of virus particles that were analyzed for their protein and lipid composition. By using mass spectrometry, we found the lipid composition of Jc1E2(FLAG) particles to resemble the one very low- and low density-lipoprotein with cholesteryl esters accounting for almost half of the total HCV lipids. Thus, HCV particles possess a unique lipid composition that is very distinct from all other viruses analyzed so far and from the human liver cells in which HCV was produced. By electron microscopy (EM), we found purified Jc1E2(FLAG) particles to be heterogeneous, mostly spherical structures, with an average diameter of about 73 nm. Importantly, the majority of E2-containing particles also contained apoE on their surface as assessed by immuno-EM. Taken together, we describe a rapid and efficient system for the production of large quantities of affinity-purified HCV allowing a comprehensive analysis of the infectious virion, including the determination of its lipid composition.

High rate of infection control with one-stage revision of septic knee prostheses excluding MRSA and MRSE.

BACKGROUND: The rate of infection control for one-stage revision of infected knee arthroplasties is unclear as are the factors influencing infection control. Such factors include duration of infection and the type of infected prosthesis. QUESTIONS/PURPOSES: We therefore determined: (1) the rate of infection control with one-stage revision of septic knee prostheses, (2) the clinical knee scores that can be achieved, (3) whether the duration of infection or the type of prosthesis influence the level of infection control, and (4) whether different types of prostheses influence the knee scores. METHODS: We retrospectively reviewed prospectively collected data from 63 patients who underwent one-stage revisions of septic knee endoprostheses (six unicondylar, 37 primary total knee replacement prostheses, and 20 hinged knee endoprostheses) between 2004 and 2006. All were treated locally and systemically with microorganism-specific antibiotics. For this study we excluded patients with Methicillin-resistant Staphylococcus aureus and Methicillin-resistant Staphylococcus epidermidis or unknown microorganisms. The patients were examined for infection every 3 months and Oxford and Knee Society scores were assessed at the same time. The minimum followup was 24 months (mean followup, 36 months; range, 24-70 months). RESULTS: None of the patients with replacement unicondylar and primary total knee replacement prostheses had recurrence of infection. Three of the 20 patients with the hinged infected knee prostheses had recurrences; these three patients had chronic infections and had undergone two to three revision operations during at least a 5-year period. The likelihood of infection control was influenced by the duration of infection. The mean Knee Society knee score 24 months after surgery was 72 points (range, 20-98 points), the Knee Society function score was 71 points (range, 10-100 points), and the Oxford-12 knee score was 27 points (range, 13-44 points). CONCLUSIONS: One-stage revision of septic knee prostheses achieved an infection control rate of 95% and higher knee scores than reported for two-stage revisions. Higher rates of recurrent infection appeared to be associated with long-term chronic infections of hinged prostheses.

Radical Dimerization of 5,5'-Diphenyl-3,3',4,4'-tetramethoxy-2,2'-bipyrrole: π Dimer in the Crystal, σ Dimer in Solution.

The crystalline, spinless π dimer (1.+ ⋅PF6 )2 was obtained by the oxidation of bipyrrole 1 with ferrocenium hexafluorophosphate. The diamagnetic species generated electrochemically in solution was identified as the σ dimer by NMR spectroscopy.

Composition and three-dimensional architecture of the dengue virus replication and assembly sites.

Positive-strand RNA viruses are known to rearrange cellular membranes to facilitate viral genome replication. The biogenesis and three-dimensional organization of these membranes and the link between replication and virus assembly sites is not fully clear. Using electron microscopy, we find Dengue virus (DENV)-induced vesicles, convoluted membranes, and virus particles to be endoplasmic reticulum (ER)-derived, and we detect double-stranded RNA, a presumed marker of RNA replication, inside virus-induced vesicles. Electron tomography (ET) shows DENV-induced membrane structures to be part of one ER-derived network. Furthermore, ET reveals vesicle pores that could enable release of newly synthesized viral RNA and reveals budding of DENV particles on ER membranes directly apposed to vesicle pores. Thus, DENV modifies ER membrane structure to promote replication and efficient encapsidation of the genome into progeny virus. This architecture of DENV replication and assembly sites could explain the coordination of distinct steps of the flavivirus replication cycle.

3,3'- and 4,4'-Dimethoxy-2,2'-bipyrroles: highly electron-rich model compounds for polypyrrole formation.

3,3'-Dimethoxy-2,2'-bipyrrole (1) and 4,4'-dimethoxy-2,2'-bipyrrole (2) were obtained in short sequences and good yields from N-benzyl-3-hydroxypyrrole-2,4-dicarboxylic acid. The key intermediate leading to 1 is an N-benzyl-3-methoxypyrrole, which is dimerized by lithiation and oxidation with NiCl(2). The formation of 2 is achieved by a classical Ullmann coupling of diethyl 1-benzyl-2-bromo-4-methoxypyrrole-3,5-dicarboxylate. The N-benzyl protection groups of 1 and 2 are cleaved under reducing conditions with sodium in liquid ammonia. Both isomeric bipyrroles are extremely sensitive toward air. Compound 1 has a very low oxidation potential of 0.09 V against AgCl but film formation hardly occurs. On the other hand, compound 2 with a potential of 0.35 V readily forms stable polypyrrole films with anodic waves at -0.51 and -0.35 V and a cathodic wave at -0.77 V, the lowest potential ever observed for a p-doped polymer.