Anne Op De Beeck

Characterization of Functional Hepatitis C Virus Envelope Glycoproteins

ABSTRACT Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2, that assemble as a noncovalent heterodimer which is mainly retained in the endoplasmic reticulum. Because assembly into particles and secretion from the cell lead to structural changes in viral envelope proteins, characterization of the proteins associated with the virion is necessary in order to better understand how they mature to be functional in virus entry. There is currently no efficient and reliable cell culture system to amplify HCV, and the envelope glycoproteins associated with the virion have therefore not been characterized yet. Recently, infectious pseudotype particles that are assembled by displaying unmodified HCV envelope glycoproteins on retroviral core particles have been successfully generated. Because HCV pseudotype particles contain fully functional envelope glycoproteins, these envelope proteins, or at least a fraction of them, should be in a mature conformation similar to that on the native HCV particles. In this study, we used conformation-dependent monoclonal antibodies to characterize the envelope glycoproteins associated with HCV pseudotype particles. We showed that the functional unit is a noncovalent E1E2 heterodimer containing complex or hybrid type glycans. We did not observe any evidence of maturation by a cellular endoprotease during the transport of these envelope glycoproteins through the secretory pathway. These envelope glycoproteins were recognized by a panel of conformation-dependent monoclonal antibodies as well as by CD81, a molecule involved in HCV entry. The functional envelope glycoproteins associated with HCV pseudotype particles were also shown to be sensitive to low-pH treatment. Such conformational changes are likely necessary to initiate fusion.

Biogenesis of hepatitis C virus envelope glycoproteins.

IP: 54.191.40.80 On: Sat, 09 Sep 2017 23:23:00 Journal of General Virology (2001), 82, 2589–2595. Printed in Great Britain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Hepatitis C Virus E2 Has Three Immunogenic Domains Containing Conformational Epitopes with Distinct Properties and Biological Functions

ABSTRACT Mechanisms of virion attachment, interaction with its receptor, and cell entry are poorly understood for hepatitis C virus (HCV) because of a lack of an efficient and reliable in vitro system for virus propagation. Infectious HCV retroviral pseudotype particles (HCVpp) were recently shown to express native E1E2 glycoproteins, as defined in part by HCV human monoclonal antibodies (HMAbs) to conformational epitopes on E2, and some of these antibodies block HCVpp infection (A. Op De Beeck, C. Voisset, B. Bartosch, Y. Ciczora, L. Cocquerel, Z. Y. Keck, S. Foung, F. L. Cosset, and J. Dubuisson, J. Virol. 78:2994-3002, 2004). Why some HMAbs are neutralizing and others are nonneutralizing is looked at in this report by a series of studies to determine the expression of their epitopes on E2 associated with HCVpp and the role of antibody binding affinity. Antibody cross-competition defined three E2 immunogenic domains with neutralizing HMAbs restricted to two domains that were also able to block E2 interaction with CD81, a putative receptor for HCV. HCVpp immunoprecipitation showed that neutralizing and nonneutralizing domains are expressed on E2 associated with HCVpp, and affinity studies found moderate-to-high-affinity antibodies in all domains. These findings support the perspective that HCV-specific epitopes are responsible for functional steps in virus infection, with specific antibodies blocking distinct steps of virus attachment and entry, rather than the perspective that virus neutralization correlates with increased antibody binding to any virion surface site, independent of the epitope recognized by the antibody. Segregation of virus neutralization and sensitivity to low pH to specific regions supports a model of HCV E2 immunogenic domains similar to the antigenic structural and functional domains of other flavivirus envelope E glycoproteins.

The Transmembrane Domains of Hepatitis C Virus Envelope Glycoproteins E1 and E2 Play a Major Role in Heterodimerization

Oligomerization of viral envelope proteins is essential to control virus assembly and fusion. The transmembrane domains (TMDs) of hepatitis C virus envelope glycoproteins E1 and E2 have been shown to play multiple functions during the biogenesis of E1E2 heterodimer. This makes them very unique among known transmembrane sequences. In this report, we used alanine scanning insertion mutagenesis in the TMDs of E1 and E2 to examine their role in the assembly of E1E2 heterodimer. Alanine insertion within the center of the TMDs of E1 or E2 or in the N-terminal part of the TMD of E1 dramatically reduced heterodimerization, demonstrating the essential role played by these domains in the assembly of hepatitis C virus envelope glycoproteins. To better understand the alanine scanning data obtained for the TMD of E1 which contains GXXXG motifs, we analyzed by circular dichroism and nuclear magnetic resonance the three-dimensional structure of the E1-(350–370) peptide encompassing the N-terminal sequence of the TMD of E1 involved in heterodimerization. Alanine scanning results and the three-dimensional molecular model we obtained provide the first framework for a molecular level understanding of the mechanism of hepatitis C virus envelope glycoprotein heterodimerization.

Role of low-density lipoprotein receptor in the hepatitis C virus life cycle.

Hepatitis C virus (HCV) particles are known to be in complex with lipoproteins. As a result of this interaction, the low‐density lipoprotein (LDL) receptor (LDLR) has been proposed as a potential entry factor for HCV; however, its implication in virus entry remains unclear. Here, we reinvestigated the role of the LDLR in the HCV life cycle by comparing virus entry to the mechanism of lipoprotein uptake. A small interfering RNA targeting the LDLR in Huh‐7 cells reduced HCV infectivity, confirming that this receptor plays a role in the life cycle of HCV generated in cell culture. However, kinetics of internalization were much faster for lipoproteins than for infectious HCV particles. Furthermore, a decrease in HCV RNA replication was observed by blocking the LDLR with a specific antibody, and this was associated with an increase in the ratio of phosphatidylethanolamine to phosphatidylcholine in host cells. Nevertheless, a soluble form of the LDLR inhibited both HCV entry into the hepatocytes and its binding to the LDLR expressed on Chinese hamster ovary cells, suggesting a direct interaction between the HCV particle and the LDLR. Finally, we showed that modification of HCV particles by lipoprotein lipase (LPL) reduces HCV infectivity and increases HCV binding to LDLR. Importantly, LPL treatment also induced an increase in RNA internalization, suggesting that LDLR, at least in some conditions, leads to nonproductive internalization of HCV. Conclusion: The LDLR is not essential for infectious HCV particle entry, whereas the physiological function of this receptor is important for optimal replication of the HCV genome. (HEPATOLOGY 2012)

Topological changes in the transmembrane domains of hepatitis C virus envelope glycoproteins.

Hepatitis C virus proteins are synthesized as a polyprotein cleaved by a signal peptidase and viral proteases. The behaviour of internal signal sequences at the C‐terminus of the transmembrane domains of hepatitis C virus envelope proteins E1 and E2 is essential for the topology of downstream polypeptides. We determined the topology of these transmembrane domains before and after signal sequence cleavage by tagging E1 and E2 with epitopes and by analysing their accessibility in selectively permeabilized cells. We showed that, after cleavage by signal peptidase in the endoplasmic reticulum, the C‐terminal orientation of these transmembrane domains changed from luminal to cytosolic. The dynamic behaviour of these transmembrane domains is unique and it is linked to their multifunctionality. By reorienting their C‐terminus toward the cytosol and being part of a transmembrane domain, the signal sequences at the C‐terminus of E1 and E2 contribute to new functions: (i) membrane anchoring; (ii) E1E2 heterodimerization; and (iii) endoplasmic reticulum retention.

Statins potentiate the in vitro anti‐hepatitis C virus activity of selective hepatitis C virus inhibitors and delay or prevent resistance development

Statins are 3‐hydroxyl‐3‐methylglutaryl coenzyme A (HMG CoA) reductase inhibitors used for the treatment of hypercholesterolemia. It was recently reported that statins inhibit in vitro hepatitis C virus (HCV) RNA replication. We here report that, of five statins studied, mevastatin and simvastatin exhibit the strongest in vitro anti‐HCV activity, lovastatin and fluvastatin have moderate inhibitory effects, and pravastatin is devoid of an antiviral effect. A combination of statins with interferon‐alpha (IFN‐α) or HCV nonstructural (NS)5B polymerase or NS3 protease inhibitors results in an additive antiviral activity in short‐term (3 days) antiviral assays. Neither statins, at a concentration of five‐fold their median effective concentration (EC50) value, nor polymerase, protease inhibitors, or IFN‐α, at concentrations 10‐ or 20‐fold their EC50 value, were able to clear cells from their replicon following four or six consecutive passages of antiviral pressure. However, the combination of HCV polymerase or protease inhibitors with mevastatin or simvastatin resulted in an efficient clearance of the cultures from their replicon. In colony formation experiments, mevastatin reduced the frequency or prevented the selection of HCV replicons resistant to the nonnucleoside inhibitor HCV‐796. Conclusion: A combination of specific HCV inhibitors with statins may result in a more profound antiviral effect and may delay or prevent the development of resistance to such inhibitors. (HEPATOLOGY 2009.)

Viruses and the cell cycle

Viruses depend on the hosts machineries to replicate and express their genome. Actively replicating cells have large pools of deoxynucleotides and high levels of key enzyme activities that viruses exploit to their own needs. Some viruses have developed strategies for driving quiescent cells into the S phase of the cell cycle, e.g. adenovirus, others, such as parvovirus, wait until the host itself begins to replicate. Viruses may also force the host cell to stay in a favourable phase, e.g. Epstein-Barr virus, or, if necessary, they may inhibit apoptotic cell death, e.g. human cytomegalovirus. In this review, we focus on the different strategies that viruses use to create in infected cells an environment favourable to the accomplishment of the viral life cycle through acting on cell cycle regulators.

Ceramide enrichment of the plasma membrane induces CD81 internalization and inhibits hepatitis C virus entry

Virus entry is a major step in which host‐cell lipids can play an essential role. In this report, we investigated the importance of sphingolipids in hepatitis C virus (HCV) entry. For this purpose, sphingomyelin present in the plasma membrane of target cells was hydrolysed into ceramide by sphingomyelinase treatment. Interestingly, ceramide enrichment of the plasma membrane strongly inhibited HCV entry. To understand how ceramide affected HCV entry, we analysed the effect of ceramide enrichment of the plasma membrane on three cell‐surface molecules identified as entry factors for HCV: CD81 tetraspanin, scavenger receptor BI and Claudin‐1. These proteins, which we found to be mainly associated with detergent‐soluble membranes in Huh‐7 cells, were not relocated in detergent‐resistant microdomains after sphingomyelin hydrolysis into ceramide. Importantly, ceramide enrichment of the plasma membrane led to a 50% decrease in cell‐surface CD81, which was due to its ATP‐independent internalization. Our results strongly suggest that the ceramide‐induced internalization of CD81 is responsible for the inhibitory effect of ceramide on HCV entry. Together, these data indicate that some specific lipids of the plasma membrane are essential for HCV entry and highlight plasma membrane lipids as potential targets to block HCV entry.

NS1- and Minute Virus of Mice-Induced Cell Cycle Arrest: Involvement of p53 and p21 cip1

ABSTRACT The nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVMp) is cytolytic when expressed in transformed cells. Before causing extensive cell lysis, NS1 induces a multistep cell cycle arrest in G1, S, and G2, well reproducing the arrest in S and G2 observed upon MVMp infection. In this work we investigated the molecular mechanisms of growth inhibition mediated by NS1 and MVMp. We show that NS1-mediated cell cycle arrest correlates with the accumulation of the cyclin-dependent kinase (Cdk) inhibitor p21 cip1 associated with both the cyclin A/Cdk and cyclin E/Cdk2 complexes but in the absence of accumulation of p53, a potent transcriptional activator of p21 cip1 . By comparison, MVMp infection induced the accumulation of both p53 and p21 cip1 . We demonstrate that p53 plays an essential role in the MVMp-induced cell cycle arrest in both S and G2 by using p53 wild-type (+/+) and null (−/−) cells. Furthermore, only the G2 arrest was abrogated in p21 cip1 null (−/−) cells. Together these results show that the MVMp-induced cell cycle arrest in S is p53 dependent but p21 cip1 independent, whereas the arrest in G2 depends on both p53 and its downstream effector p21 cip1 . They also suggest that induction of p21 cip1 by the viral protein NS1 arrests cells in G2 through inhibition of cyclin A-dependent kinase activity.