Hepatitis C virus (HCV) nonstructural protein 5A (NS5A), a phosphoprotein of unknown function, is believed to be a component of a membrane-associated viral replication complex. The determinants for membrane association of NS5A, however, have not been defined. By double label immunofluorescence analyses, NS5A was found to be associated with the endoplasmic reticulum (ER) or an ER-derived modified compartment both when expressed alone or in the context of the entire HCV polyprotein. Systematic deletion and green fluorescent protein fusion analyses allowed us to map the membrane anchor to the amino-terminal 30 amino acid residues of NS5A. Membrane association occurred by a posttranslational mechanism and resulted in properties of an integral membrane protein. Circular dichroism structural studies of a synthetic peptide corresponding to the NS5A membrane anchor, designated NS5A(1-31), demonstrated the presence of an amphipathic ␣-helix that was found to be highly conserved among 280 HCV isolates of various genotypes. The detergent-binding properties of this helical peptide together with the nature and location of its amino acids suggest a mechanism of membrane insertion via the helix hydrophobic side, yielding a topology parallel to the lipid bilayer in the cytoplasmic leaflet of the ER membrane. These findings have important implications for the structural and functional organization of the HCV replication complex and may define novel targets for antiviral intervention. Hepatitis C virus (HCV)1 infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide (1). A protective vaccine does not exist to date, and therapeutic options are still limited (2, 3). HCV has been classified in the Hepacivirus genus within the Flaviviridae family which includes the classical flaviviruses, such as yellow fever virus, and the animal pestiviruses, such as bovine viral diarrhea virus (BVDV) (4). The structure and replication cycle of HCV are incompletely understood due to the low viral titers found in sera and livers of HCV-infected individuals and the lack of an efficient cell culture system or small animal model permissive for HCV infection. Nevertheless, considerable progress has been made using heterologous expression systems, functional cDNA clones, and more recently, selectable subgenomic replicons (see Refs. 5 and 6 for recent reviews).HCV contains a single-stranded RNA genome of positive polarity and ϳ9600 nucleotides (nt) length that encodes a polyprotein precursor of about 3000 amino acids (aa) (Fig. 1A). The polyprotein precursor is co-and posttranslationally processed by cellular and viral proteases to yield the mature structural and nonstructural proteins. The structural proteins include the core protein, which forms the viral nucleocapsid, and the envelope glycoproteins E1 and E2. The non-structural proteins NS2 through NS5B include the NS2-3 autoprotease and the NS3 serine protease, an RNA helicase located in the carboxyl-terminal region of NS3, the NS4A polypeptide, the NS4B ...

Next to a profound T cell immunodeficiency, HIV-1 infection induces activation and dysfunction of B cells, resulting in hypergammaglobulinemia. Whereas T cell immune reconstitution with potent antiretroviral therapy has been extensively documented, limited data are available on B cell immune reconstitution. We studied the effect of potent antiretroviral therapy on antibody titers to the viral proteins gp120 and p24 and on total IgG concentrations. Three retrospectively chosen groups were studied: a successfully treated group, untreated controls, and subjects with virological failure after several months of successful therapy. In the successfully treated group, the median total IgG concentrations normalized, whereas they remained elevated in the untreated group and rebounded after an initial decline in the therapy failure group. The HIV-1-specific antibody titers declined in the successfully treated group and followed the rebound of the HIV RNA levels in the therapy failure group. With potent antiretroviral therapy the hypergammaglobulinemia normalized whereas HIV-1-specific immune responses were weakened. The weakening of antiviral immunity with therapy may be relevant for current attempts to gain immunological control over the virus through structured treatment interruptions or therapeutic vaccinations.

Liposomes of sufficiently different surface charge can be separated on a DEAE-cellulose column. This made possible the present study on the transfer of phosphatidylcholine between liposomes consisting of phosphatidylcholine and various amounts of phosphatidic acid. It was demonstrated that the redistribution of ['4C]phosphatidylcholine between liposomes as catalyzed by the phosphatidylcholine exchange protein, followed the kinetics of an isotope-exchange reaction. Furthermore, it was shown that the phospholipid composition of the liposomes affected the activity of the exchange protein.It has been demonstrated for a number of tissues that soluble proteins are present which catalyze the transfer of phospholipids between membrane structures [l -41. Recently, one of these proteins denoted as phosphatidylcholine exchange protein, has been purified from beef liver and partially characterized [5]. It was shown that one mole of phosphatidylcholine was bound per mole of protein. The exchange protein exerted its action as a carrier of phosphatidylcholine between membranes by the exchange of its bound lipid molecule for one present in the membrane [6]. Recently, Ehnholm and Zilversmit [7] provided evidence for the catalyzed transfer of phosphatidylcholine between antigen-sensitized and non-sensitized liposomes after separation of the liposomes by immunoprecipitation. A drawback of this technique according to the authors was the relatively long time required to separate the liposomes.The present paper demonstrates the catalyzed transfer of phosphatidylcholine between two populations of liposomes one of which contains [14C]phosphatidylcholine. Differences of surface charge made possible the rapid separation of these liposomes on an ion-exchange column. It is shown that the redistribution of [14C]phosphatidylcholine between the liposomes as catalyzed by the exchange protein follows the kinetics of an isotope-exchange reaction. It is also shown that particularly the incorporation of negatively charged phospholipids into the liposomes has a pronounced inhibitory effect on the transfer. MATERIALS AND METHODSPhosphatidylcholine from egg yolk, phosphatidylinositol from rat liver and sphingomyelin from bovine erythrocytes were isolated according to established procedures [8 -101. Phosphatidic acid was prepared from phosphatidylcholine by degradation with phospholipase D [ll]. A [14C]methyl group was introduced into the polar head group of phosphatidylcholine by the method of Stoffel et al. [12]. [7cl-3H]Cholesteryloleate was purchased from New England Nuclear. Phosphatidylcholine exchange protein was isolated from beef liver according to the procedure of Kamp et al. [5].Liposomes were prepared in 0.05 M KCl-0.01 M glycine-KOH (pH 8.6) by sonication of a phospholipid dispersion for 30 min following the procedure of Huang [13]. Chromatography on a Sepharose-4B column indicated that more than 95 o/, of the phospholipid dispersed was present in single bilayer liposomes.Exchange of phosphatidylcholine between liposomes was determined b...

We use a randomised experiment to study the effect of offering half of 556 freshman students a learning analytics dashboard and a weekly email with a link to their dashboard, on student behaviour in the online environment and final exam performance. The dashboard shows their online progress in the learning management systems, their predicted chance of passing, their predicted grade and their online intermediate performance compared with the total cohort. The email with dashboard access, as well as dashboard use, has positive effects on student behaviour in the online environment, but no effects are found on student performance in the final exam of the programming course. However, we do find differential effects by specialisation and student characteristics.

An enzyme-linked immunosorbent assay (ELISA) was developed based on sera from patients convalescent from non-A, non-B hepatitis and haemophilia A patients who had been frequently treated with commercial blood products. Using this ELISA, an antigen was detected which appears to be related to non-A, non-B hepatitis. The antigen is provisionally designated as DS-antigen (DS-Ag). The serum of another patient with haemophilia A, which was strongly positive for the DS-Ag, caused a typical case of non-A, non-B hepatitis in a chimpanzee. DS-Ag could be detected in the serum of the chimpanzee during the acute phase of the infection. The ELISA for DS-Ag reacted with neither hepatitis A or B virus antigens, nor Epstein-Barr virus or cytomegalovirus. The assay was provisionally evaluated using sera from different groups of patients. Out of 17 patients with posttransfusion hepatitis non-A, non-B, 11 were found positive in the ELISA for DS-Ag (65%). As expected, a relatively high prevalence of DS-Ag (9%) was found in patients with haemophilia, who are regularly treated with blood-clotting factor-concentrates. Antibodies to DS-Ag were found in 48% of these patients. The DS-Ag was found in 8 of 1400 (0.6%) volunteer blood donors, and antibody to DS-Ag in 3% of the sera. Remarkably, a high incidence (41%) of antibodies to DS-Ag was found in prostitutes, suggesting that this antigen may be transmitted by a sexual route. The DS-Ag was pelleted by ultracentrifugation for four hours at 100,000g and was found to have a buoyant density of 1.32 g/cm3 in a CsCl gradient.