Reversible Acid-Induced Inactivation of the Membrane Fusion Protein of Semliki Forest Virus

Waarts, Barry-Lee; Smit, Jolanda M.; Aneke, Onwuchekwa J. C.; McInerney, Gerald M.; Liljeström, Peter; Bittman, Robert; Wilschut, Jan

doi:10.1128/jvi.79.12.7942-7948.2005

Cited by 13 publications

(16 citation statements)

References 42 publications

(60 reference statements)

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“…Indeed, virtually all fusion activity was lost after only 30 s of low-pH pre-incubation (Corver et al, 2000). Our result with HCVpps compared with that of SFV and Sindbis virus fusion, where 90 % of their fusion capacity is lost after 35 and 60 s, respectively, at low pH in the absence of target membranes (Smit et al, 1999;Waarts et al, 2005). When HCVpps were pre-incubated at pH 5.0 for 1 min, followed by back neutralization in the absence of liposomes, lipid mixing resumed only when reacidification was performed (Fig.…”

Section: Hcvpp Fusion Behaviour At Low Ph By Fluorimetrymentioning

confidence: 83%

Characterization of hepatitis C virus pseudoparticles by cryo-transmission electron microscopy using functionalized magnetic nanobeads

Bonnafous

Perrault

Bihan

et al. 2010

Journal of General Virology

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Cell entry and membrane fusion of the hepatitis C virus (HCV) depend on its envelope glycoproteins E1 and E2. HCV pseudotyped particles (HCVpps) are relevant and popular models to study the early steps of the HCV life cycle. However, no structural characterization of HCVpp has been available so far. Using cryo-transmission electron microscopy (cryo-TEM), providing structural information at nanometric resolution, the molecular details of HCVpps and their fusion with liposomes were studied. Cryo-TEM revealed HCVpps as regular 100 nm spherical structures containing the dense retroviral nucleocapsid surrounded by a lipid bilayer. E1-E2 glycoproteins were not readily visible on the membrane surface. Pseudoparticles bearing the E1-E2 glycoproteins of Semliki forest virus looked similar, whereas avian influenza A virus (fowl plague virus) haemagglutinin/neuraminidase-pseudotyped particles exhibited surface spikes. To further characterize HCVpp structurally, a novel method was designed based on magnetic beads covered with anti-HCV antibodies to enrich the samples with particles containing E1-E2. This strategy efficiently sorted HCVpps, which were then directly observed by cryo-TEM in the presence or absence of liposomes at low or neutral pH. After acidification, HCVpps looked the same as at neutral pH and closely contacted the liposomes. These are the first visualizations of early HCV membrane fusion events at the nanometer scale. Furthermore, fluorimetry analysis revealed a relative resistance of HCVpps regarding their fusion capacity when exposed to low pH. This study therefore brings several new molecular details to HCVpp characterization and this efficient strategy of virion immunosorting with magnetic nanobeads is direct, efficient and adaptable to extensive characterization of any virus at a nanometric resolution.

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Section: Hcvpp Fusion Behaviour At Low Ph By Fluorimetrymentioning

confidence: 83%

Characterization of hepatitis C virus pseudoparticles by cryo-transmission electron microscopy using functionalized magnetic nanobeads

Bonnafous

Perrault

Bihan

et al. 2010

Journal of General Virology

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“…Indeed HCVpp fusion became negligible only after low pH exposure longer than 10 min. This is notably not the case for TBEV (29), Alphaviruses such as SFV (60), and Sindbis (37), for example, where a complete fusion inactivation is achieved within the 1st min of preincubation at low pH. This points to a relative resistance of HCV to low pH in the absence of target membranes.…”

Section: Discussionmentioning

confidence: 95%

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

Lavillette

Bartosch

Nourrisson

et al. 2006

Journal of Biological Chemistry

126

176

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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...

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“…Labeling of WNV with the fluorescent probe pyrene was performed essentially as described previously for alphaviruses (73,81). Briefly, BHK21-15 cells cultured in the presence of 15 g/ml of 16-(1-pyrenyl)-hexadecanoic acid (Invitrogen) were infected with WNV at an multiplicity of infection of 4.…”

Section: Methodsmentioning

confidence: 99%

Human Monoclonal Antibodies against West Nile Virus Induced by Natural Infection Neutralize at a Postattachment Step

Vogt¹,

Moesker

Goudsmit

et al. 2009

J Virol

Self Cite

105

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West Nile virus (WNV) is a neurotropic flavivirus that is now a primary cause of epidemic encephalitis inWNV cycles in nature between several species of birds and Culex mosquitoes, with humans and other mammals as deadend hosts (25, 62). Infection causes syndromes ranging from a mild febrile illness to severe encephalitis and death (13, 72). WNV has spread globally and causes outbreaks with thousands of severe human cases annually in the United States. An age of greater than 55 years, a compromised immune status, and a CC5⌬32 genotype have been associated with more-severe disease (15,20). There is currently no approved vaccine or therapy for WNV infection.The mature WNV virion has a ϳ500-Å diameter and consists of a single RNA genome surrounded by the capsid protein, a lipid bilayer, and a shell of the prM/M and E proteins (31, 55). X-ray crystallography studies have elucidated the three-domain structure of the flavivirus E protein (30,48,50,58,67). Domain I (DI) is a central, eight-stranded ␤-barrel, which contains the only N-linked glycosylation site in WNV E. Domain II (DII) is a long, finger-like protrusion from DI and contains the highly conserved fusion peptide at its distal end. Domain III (DIII) adopts an immunoglobulin-like fold at the opposite end of DI and is believed to contain a site for receptor attachment (6,8,40).Within an infected cell, progeny WNV are assembled initially as immature particles. In immature virions, three pairs of E and prM interact as trimers and form 60 spiked projections with icosahedral symmetry (85,86). Exposure to mildly acidic conditions in the trans-Golgi secretory pathway promotes virus maturation through a structural rearrangement of the E proteins and cleavage of prM to M by a furin-like protease (41,83). Mature WNV virions are covered by 90 antiparallel E protein homodimers, which are arranged flat along the surface in a herringbone pattern with quasi-icosahedral symmetry (55).Upon binding to poorly characterized cell surface receptors, internalization of WNV is believed to occur through receptormediated, clathrin-dependent endocytosis (1,79,80). After trafficking to 79), the mildly acidic pH within the lumen of the endosome induces structural alterations in the flavivirus E protein (7, 49), which includes changes in its oligomeric state (7,49,77). During this process, also known as type II fusion, the hydrophobic peptide on the fusion loop of DII of the E protein inserts into the

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Reversible Acid-Induced Inactivation of the Membrane Fusion Protein of Semliki Forest Virus

Cited by 13 publications

References 42 publications

Characterization of hepatitis C virus pseudoparticles by cryo-transmission electron microscopy using functionalized magnetic nanobeads

Characterization of hepatitis C virus pseudoparticles by cryo-transmission electron microscopy using functionalized magnetic nanobeads

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

Human Monoclonal Antibodies against West Nile Virus Induced by Natural Infection Neutralize at a Postattachment Step

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