Hepatitis C Virus Glycoproteins Mediate Low pH-dependent Membrane Fusion with Liposomes

Abstract

It has been suggested that the hepatitis C virus (HCV) infects host cells through a pH-dependent internalization mechanism, but the steps leading from virus attachment to the fusion of viral and cellular membranes remain uncharacterized. Here we studied the mechanism underlying the HCV fusion process in vitro using liposomes and our recently described HCV pseudoparticles (pp) bearing functional E1E2 envelope glycoproteins. The fusion of HCVpp with liposomes was monitored with fluorescent probes incorporated into either the HCVpp or the liposomes. To validate these assays, pseudoparticles bearing either the hemagglutinin of the influenza virus or the amphotropic glycoprotein of murine leukemia virus were used as models for pH-dependent and pH-independent entry, respectively. The use of assays based either on fusion-induced dequenching of fluorescent probes or on reporter systems, which produce fluorescence when the virus and liposome contents are mixed, allowed us to demonstrate that HCVpp mediated a complete fusion process, leading to the merging of both membrane leaflets and to the mixing of the internal contents of pseudoparticle and liposome. This HCVpp-mediated fusion was dependent on low pH, with a threshold of 6.3 and an optimum at about 5.5. Fusion was temperature-dependent and did not require any protein or receptor at the surface of the target liposomes. Most interestingly, fusion was facilitated by the presence of cholesterol in the target membrane. These findings clearly indicate that HCV infection is mediated by a pH-dependent membrane fusion process. This paves the way for future studies of the mechanisms underlying HCV membrane fusion. Hepatitis C virus (HCV)2 belongs to the genus Hepacivirus of the Flaviviridae family (1) and has infected some 170 million people worldwide (2). In the majority of HCV-infected patients, the progression to chronic disease is associated with an increased risk of liver diseases and hepatocellular carcinoma. No vaccine is presently available, and the current treatment for chronic hepatitis C (a combination of pegylated interferon and ribavirin) has a limited efficacy (3). A more detailed understanding of the molecular mechanisms of virus entry would be beneficial to the development of novel therapeutic strategies.The HCV genome is a positive-stranded RNA encoding a precursor polyprotein of about 3,000 amino acids. This polyprotein is cleaved coand post-translationally to generate 10 viral proteins, classified into structural (capsid protein and the envelope glycoproteins E1 and E2) and nonstructural proteins (NS2 to NS5B), separated by the membrane ion channel polypeptide p7 (1, 4). The type I transmembrane envelope glycoproteins E1 and E2 form noncovalently linked heterodimers in the endoplasmic reticulum and are highly glycosylated, containing up to 6 and 11 potential glycosylation sites, respectively (5-8). Studies of the HCV life cycle have been hampered by the lack of an efficient and reliable cell culture system to produce and isolate the functional virus. Most re...

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Bafilomycin A1 or Concanamycin A) so as to prevent the natural HCV entry pathway. Last, fusion can be tested between virus particles and liposomes ( Figure 6C) [175,176]. By labelling one of the protagonists (virions or liposomes) with a fluorescence dye at a self-quenching concentration, it is possible to assess fusion by monitoring fluorescence dequenching and emission.…”

“…This system, as opposed to the two assays described above, is an indicator for hemifusion, as dequenching occurs as soon as the outer hemilayers of virions and liposomes merge. Another liposome-based assay was developed to monitor complete fusion [175]. This "content mixing assay" however depends on a retroviral core and can therefore only be applied to the HCVpp system.…”

“…Defining inhibitors that would act on HCV entry without affecting fusion in these setups would consolidate their usefulness. On the opposite, the liposome-based assays are temperature-dependent but receptor-independent [175,176]. Therefore these systems are simpler than cell-based experiments and more amenable to perform perturbation studies and define the true determinants of fusion.…”

HCV and lipoproteins: Is oxLDL an Achilles' heel of the Trojan horse?

“…Furthermore, cell entry of HCV has been shown to occur via endocytosis and membrane fusion to be dependent on acidic pH and cholesterol. 10,14,16,26 How do oxidized lipoproteins interfere with HCV entry and what is the significance of this event in vivo? First, it is important to point out that lipoproteins are completely dispensable in infection assays in vitro with HCVpp or HCVcc.…”

“…[11][12][13] HCVpp and HCVcc allow a robust investigation of most functions mediated by the HCV glycoproteins and, hence, may reproduce some cell entry features of native HCV. [11][12][13][14][15][16][17][18] Of note, these different HCV model systems are generated in vitro from transformed cell lines, which could preclude some feature of HCV association with lipoprotein metabolism. However, recent results suggest that there may be more similarities than anticipated between current in vitro HCV models and virus purified from patients' plasma.…”