Sandrine Belouzard

Hepatitis C Virus Entry Depends on Clathrin-Mediated Endocytosis

ABSTRACT Due to difficulties in cell culture propagation, the mechanisms of hepatitis C virus (HCV) entry are poorly understood. Here, postbinding cellular mechanisms of HCV entry were studied using both retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the HCV clone JFH-1 propagated in cell culture (HCVcc). HCVpp entry was measured by quantitative real-time PCR after 3 h of contact with target cells, and HCVcc infection was quantified by immunoblot analysis and immunofluorescence detection of HCV proteins expressed in infected cells. The functional role of clathrin-mediated endocytosis in HCV entry was assessed by small interfering RNA-mediated clathrin heavy chain depletion and with chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. In both conditions, HCVpp entry and HCVcc infection were inhibited. HCVcc infection was also inhibited by pretreating target cells with bafilomycin A1 or chloroquine, two drugs known to interfere with endosome acidification. These data indicate that HCV enters target cells by clathrin-mediated endocytosis, followed by a fusion step from within an acidic endosomal compartment.

Subcellular Localization of Hepatitis C Virus Structural Proteins in a Cell Culture System That Efficiently Replicates the Virus

ABSTRACT 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.

(−)‐Epigallocatechin‐3‐gallate is a new inhibitor of hepatitis C virus entry

Here, we identify (−)‐epigallocatechin‐3‐gallate (EGCG) as a new inhibitor of hepatitis C virus (HCV) entry. EGCG is a flavonoid present in green tea extract belonging to the subclass of catechins, which has many properties. Particularly, EGCG possesses antiviral activity and impairs cellular lipid metabolism. Because of close links between HCV life cycle and lipid metabolism, we postulated that EGCG may interfere with HCV infection. We demonstrate that a concentration of 50 μM of EGCG inhibits HCV infectivity by more than 90% at an early step of the viral life cycle, most likely the entry step. This inhibition was not observed with other members of the Flaviviridae family tested. The antiviral activity of EGCG on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition, using binding assays at 4°C, we demonstrate that EGCG prevents attachment of the virus to the cell surface, probably by acting directly on the particle. We also show that EGCG has no effect on viral replication and virion secretion. By inhibiting cell‐free virus transmission using agarose or neutralizing antibodies, we show that EGCG inhibits HCV cell‐to‐cell spread. Finally, by successive inoculation of naïve cells with supernatant of HCV‐infected cells in the presence of EGCG, we observed that EGCG leads to undetectable levels of infection after four passages. Conclusion: EGCG is a new, interesting anti‐HCV molecule that could be used in combination with other direct‐acting antivirals. Furthermore, it is a novel tool to further dissect the mechanisms of HCV entry into the hepatocyte. (HEPATOLOGY 2012;)

Mechanisms of Coronavirus Cell Entry Mediated by the Viral Spike Protein

Coronaviruses are enveloped positive-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S) mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes—A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.

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)

Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites

The coronavirus spike protein (S) plays a key role in the early steps of viral infection, with the S1 domain responsible for receptor binding and the S2 domain mediating membrane fusion. In some cases, the S protein is proteolytically cleaved at the S1–S2 boundary. In the case of the severe acute respiratory syndrome coronavirus (SARS-CoV), it has been shown that virus entry requires the endosomal protease cathepsin L; however, it was also found that infection of SARS-CoV could be strongly induced by trypsin treatment. Overall, in terms of how cleavage might activate membrane fusion, proteolytic processing of the SARS-CoV S protein remains unclear. Here, we identify a proteolytic cleavage site within the SARS-CoV S2 domain (S2′, R797). Mutation of R797 specifically inhibited trypsin-dependent fusion in both cell–cell fusion and pseudovirion entry assays. We also introduced a furin cleavage site at both the S2′ cleavage site within S2 793-KPTKR-797 (S2′), as well as at the junction of S1 and S2. Introduction of a furin cleavage site at the S2′ position allowed trypsin-independent cell–cell fusion, which was strongly increased by the presence of a second furin cleavage site at the S1–S2 position. Taken together, these data suggest a novel priming mechanism for a viral fusion protein, with a critical proteolytic cleavage event on the SARS-CoV S protein at position 797 (S2′), acting in concert with the S1–S2 cleavage site to mediate membrane fusion and virus infectivity.

Griffithsin has antiviral activity against hepatitis C virus

ABSTRACT Hepatitis C virus (HCV)-infected patients undergoing liver transplantation universally experience rapid reinfection of their new liver graft. Current treatment protocols do not prevent graft reinfection and, in addition, an accelerated disease progression is observed. In the present study, we have evaluated a novel strategy to prevent HCV infection using a lectin, griffithsin (GRFT) that specifically binds N-linked high-mannose oligosaccharides that are present on the viral envelope. The antiviral effect of GRFT was evaluated in vitro using the HCV pseudoparticle (HCVpp) and HCV cell culture (HCVcc) systems. We show here that preincubation of HCVpp and HCVcc with GRFT prevents infection of Huh-7 hepatoma cells. Furthermore, GRFT interferes with direct cell-to-cell transmission of HCV. GRFT acts at an early phase of the viral life cycle by interfering in a genotype-independent fashion with the interaction between the viral envelope proteins and the viral receptor CD81. The capacity of GRFT to prevent infection in vivo was evaluated using uPA+/+-SCID mice (uPA stands for urokinase-type plasminogen activator) that harbor human primary hepatocytes in their liver (chimeric mice). In this proof-of-concept trial, we demonstrated that GRFT can mitigate HCV infection of chimeric mice. Treated animals that did become infected demonstrated a considerable delay in the kinetics of the viral infection. Our data demonstrate that GRFT can prevent HCV infection in vitro and mitigate HCV infection in vivo. GRFT treatment of chronically infected HCV patients undergoing liver transplantation may be a suitable strategy to prevent infection of the liver allograft.

Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity

Obesity is a major public health problem and is often associated with type 2 diabetes mellitus, cardiovascular disease, and metabolic syndrome. Leptin is the crucial adipostatic hormone that controls food intake and body weight through the activation of specific leptin receptors (OB-R) in the hypothalamic arcuate nucleus (ARC). However, in most obese patients, high circulating levels of leptin fail to bring about weight loss. The prevention of this “leptin resistance” is a major goal for obesity research. We report here a successful prevention of diet-induced obesity (DIO) by silencing a negative regulator of OB-R function, the OB-R gene-related protein (OB-RGRP), whose transcript is genetically linked to the OB-R transcript. We provide in vitro evidence that OB-RGRP controls OB-R function by negatively regulating its cell surface expression. In the DIO mouse model, obesity was prevented by silencing OB-RGRP through stereotactic injection of a lentiviral vector encoding a shRNA directed against OB-RGRP in the ARC. This work demonstrates that OB-RGRP is a potential target for obesity treatment. Indeed, regulators of the receptor could be more appropriate targets than the receptor itself. This finding could serve as the basis for an approach to identifying potential new therapeutic targets for a variety of diseases, including obesity.

Low Levels of Expression of Leptin Receptor at the Cell Surface Result from Constitutive Endocytosis and Intracellular Retention in the Biosynthetic Pathway

The leptin receptor is mainly localized in intracellular compartments in target tissues. To study the mechanisms leading to this intracellular localization, two main isoforms of leptin receptors, OB-Ra and OB-Rb, were expressed in HeLa cells. Both isoforms were localized at steady state in the trans-Golgi network, in endosomes, and to a lesser extent, at the cell surface. They turned over with a half-life of less than 2 h. Both isoforms of leptin receptors were constitutively endocytosed in a ligand-independent manner and degraded in lysosomes with no evidence of recycling to the cell surface or to the trans-Golgi network. The endocytosis was inhibited by the deletion of the cytoplasmic domain. Newly synthesized leptin receptors were partially retained in the Golgi complex or in a post-Golgi intracellular compartment. The transmembrane domain was found to be important for this intracellular retention in the biosynthetic pathway, whereas the cytoplasmic domain was not involved. The data suggest that the low levels of expression of leptin receptors at the cell surface results from partial retention in the biosynthetic pathway, coupled to constitutive removal from the plasma membrane via ligand-independent, constitutive endocytosis.

Bovine Viral Diarrhea Virus Entry Is Dependent on Clathrin-Mediated Endocytosis

ABSTRACT Cellular mechanisms of bovine viral diarrhea virus (BVDV) entry in MDBK cells were investigated. Chloroquine, bafilomycin A1, or ammonium chloride inhibited BVDV infection, indicating that an acidic endosomal pH is required for BVDV entry. The tyrosine kinase inhibitor genistein partially inhibited BVDV infection at a postentry step, whereas BVDV entry was strongly inhibited by chlorpromazine or by the overexpression of a dominant-negative form of EPS15, a protein essential for the formation of clathrin-coated vesicles at the plasma membrane. Together, these data indicate that BVDV infection requires an active clathrin-dependent endocytic pathway.