Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus

Due to the recent development of a cell culture model, hepatitis C virus (HCV) can be efficiently propagated in cell culture. This allowed us to reinvestigate the subcellular localization of HCV structural proteins in the context of an infectious cycle. In agreement with previous reports, confocal immunofluorescence analysis of the subcellular localization of HCV structural proteins indicated that, in infected cells, the glycoprotein heterodimer is retained in the endoplasmic reticulum. However, in contrast to other studies, the glycoprotein heterodimer did not accumulate in other intracellular compartments or at the plasma membrane. As previously reported, an association between the capsid protein and lipid droplets was also observed. In addition, a fraction of labeling was consistent with the capsid protein being localized in a membranous compartment that is associated with the lipid droplets. However, in contrast to previous reports, the capsid protein was not found in the nucleus or in association with mitochondria or other well-defined intracellular compartments. Surprisingly, no colocalization was observed between the glycoprotein heterodimer and the capsid protein in infected cells. Electron microscopy analyses allowed us to identify a membrane alteration similar to the previously reported "membranous web." However, no virus-like particles were found in this type of structure. In addition, dense elements compatible with the size and shape of a viral particle were seldom observed in infected cells. In conclusion, the cell culture system for HCV allowed us for the first time to characterize the subcellular localization of HCV structural proteins in the context an infectious cycle.     

Hepatitis C patient-derived glycoproteins exhibit marked differences in susceptibility to serum neutralizing antibodies: genetic subtype defines antigenic but not neutralization serotype

Neutralizing antibodies have a role in controlling hepatitis C virus (HCV) infection. A successful vaccine will need to elicit potently neutralizing antibodies that are capable of preventing the infection of genetically diverse viral isolates. However, the specificity of the neutralizing antibody response in natural HCV infection still is poorly understood. To address this, we examined the reactivity of polyclonal antibodies isolated from chronic HCV infection to the diverse patient-isolated HCV envelope glycoproteins E1 and E2 (E1E2), and we also examined the potential to neutralize the entry of pseudoparticles bearing these diverse E1E2 proteins. The genetic type of the infection was found to determine the pattern of the antibody recognition of these E1E2 proteins, with the greatest reactivity to homologous E1E2 proteins. This relationship was strongest when the component of the antibody response directed only to linear epitopes was analyzed. In contrast, the neutralization serotype did not correlate with genotype. Instead, serum-derived antibodies displayed a range of neutralization breadth and potency, while different E1E2 glycoproteins displayed different sensitivities to neutralization, such that these could be divided broadly into neutralization-sensitive and -resistant phenotypes. An important additional observation was that entry mediated by some E1E2 proteins was enhanced in the presence of some of the polyclonal antibody fractions isolated during chronic infection. These data highlight the need to use diverse E1E2 isolates, which represent extremes of neutralization sensitivity, when screening antibodies for therapeutic potential and for testing antibodies generated following immunization as part of vaccine development.     

The Tol proteins of Escherichia coli and their involvement in the translocation of group A colicins

The Tol proteins are involved in outer membrane stability of Gram-negative bacteria. The TolQRA proteins form a complex in the inner membrane while TolB and Pal interact near the outer membrane. These two complexes are transiently connected by an energy-dependent interaction between Pal and TolA. The Tol proteins have been parasitized by group A colicins for their translocation through the cell envelope. Recent advances in the structure and energetics of the Tol system, as well as the interactions between the N-terminal translocation domain of colicins and the Tol proteins are presented.     

Regulation of hepatitis C virus polyprotein processing by signal peptidase involves structural determinants at the p7 sequence junctions

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 alpha-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.     

Genetic ablation of caveolin-2 sensitizes mice to bleomycin-induced injury

Caveolar domains act as platforms for the organization of molecular complexes involved in signal transduction. Caveolin proteins, the principal structural components of caveolae, have been involved in many cellular processes. Caveolin-1 (Cav-1) and caveolin-2 (Cav-2) are highly expressed in the lung. Cav-1-deficient mice (Cav-1^−/−) and Cav-2-deficient mice (Cav-2^−/−) exhibit severe lung dysfunction attributed to a lack of Cav-2 expression. Recently, Cav-1 has been shown to regulate lung fibrosis in different models. Here, we show that Cav-2 is also involved in modulation of the fibrotic response, but through distinct mechanisms. Treatment of wild-type mice with the pulmonary fibrosis-inducer bleomycin reduced the expression of Cav-2 and its phosphorylation at tyrosine 19. Importantly, Cav-2^−/− mice, but not Cav-1^−/− mice, were more sensitive to bleomycin-induced lung injury in comparison to wild-type mice. Bleomycin-induced lung injury was characterized by alveolar thickening, increase in cell density, and extracellular matrix deposition. The lung injury observed in bleomycin-treated Cav-2^−/− mice was not associated with alterations in the TGF-β signaling pathway and/or in the ability to produce collagen. However, apoptosis and proliferation were more prominent in lungs of bleomycin-treated Cav-2^−/− mice. Since Cav-1^−/− mice also lack Cav-2 expression and show a different outcome after bleomycin treatment, we conclude that Cav-1 and Cav-2 have distinct roles in bleomycin induced-lung fibrosis, and that the balance of both proteins determines the development of the fibrotic process.     

Two distinct conformational states define the interaction of human RAD51‐ATP with single‐stranded DNA

An essential mechanism for repairing DNA double‐strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single‐stranded DNA, promoting DNA‐strand exchange. Here, we study the interaction of hRAD51 with single‐stranded DNA using a single‐molecule approach. We show that ATP‐bound hRAD51 filaments can exist in two different states with different contour lengths and with a free‐energy difference of ~4 k_BT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly‐competent ADP‐bound configuration. In agreement with the single‐molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51‐ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51‐ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.     

DNA divergence in and around the alcohol dehydrogenase locus in five closely related species of Hawaiian Drosophila

The alcohol dehydrogenase (Adh) region from five planitibia subgroup species of Hawaiian picture-wing Drosophila has been cloned. A total of 15 kb of DNA in and around the Adh gene has been compared among the five species. Genetic distances were calculated to determine evolutionary relationships. These distances agree with previous distances determined by protein polymorphism and DNA hybridization techniques and can be interpreted in terms of specific island colonization and speciation (founder) events over the past 5 Myr. Examination of the restriction maps of the cloned Adh region from the five species shows many instances of small deletions, insertion of a transposable element in D. heteroneura, and the existence of a highly variable region on the 3' side of the Adh gene. Clustering relationships and rates of DNA change are calculated and compared with the relationship found for other species of Drosophila.      

Serological survey of herpesvirus infections in wild ruminants of France and Belgium

The presence of antibodies against bovine herpesvirus 1 (BHV-1), bovid herpesvirus 6 (BHV-6), herpesvirus of Cervidae type 1 (HVC-1), reindeer herpesvirus, bovine herpesvirus 2 (BHV-2) and bovid herpesvirus 4 (BHV-4) was investigated in wild ruminants of France and Belgium between 1981 and 1986. There were no animals serologically positive for BHV-4. Antibodies against BHV-2 were demonstrated in roe deer (Cervus capreolus) (less than 1%) and chamois (Rupicapra rupicapra) (1%) in France. Animals seropositive to the four related viruses (BHV-1, BHV-6, HVC-1, reindeer herpesvirus) were detected in red deer (Cervus elaphus) in France and Belgium (1% and 11%, respectively), in roe deer (less than 1%) from France, in chamois (4%) in France and in ibex (Capra ibex) (4%) from France. The presence of antibodies against HVC-1, especially in red deer from Belgium, may suggest that wild ruminants in continental Europe are now infected with this virus, which previously has been isolated only in Scotland.     

Ultrastructure of impuberal vaginas cultivated under various hormonal conditions]

Differentiation of rat vaginal epithelium has been studied in organotypic culture in vitro under different hormonal conditions [evolution in association with ovary or testis, and after injection to rats of progesterone 5 mg. and estradiol 0.1 lambda). Whatever the hormonal preconditioning before the vaginal culture the same, complet cornification occurs.     

Assembly of a functional HCV glycoprotein heterodimer

The two HCV envelope glycoproteins E1 and E2 are released from HCV polyprotein by signal peptidase cleavages. These glycoproteins are type I transmembrane proteins with a highly glycosylated N-terminal ectodomain and a C-terminal hydrophobic anchor. After their synthesis, HCV glycoproteins E1 and E2 associate as a noncovalent heterodimer. The transmembrane domains of HCV envelope glycoproteins play a major role in E1E2 heterodimer assembly and subcellular localization. The envelope glycoprotein complex E1E2 has been proposed to be essential for HCV entry. However, for a long time, HCV entry studies have remained limited because of the lack of a robust cell culture system to amplify this virus. A few years ago, a model mimicking the entry process of HCV lifecycle has been developed by pseudotyping retroviral particles with native HCV envelope glycoproteins. This model allowed the characterization of functional E1E2 envelope glycoproteins. The data obtained can now be confirmed with the help of a newly developed cell-culture system that allows efficient amplification of HCV (HCVcc). Here, we present the recent data that have been accumulated on the assembly of the functional HCV glycoprotein heterodimer.