Critical interaction between E1 and E2 glycoproteins determines binding and fusion properties of hepatitis C virus during cell entry

Douam, Florian; Thi, Viet Loan Dao; Maurin, Guillemette; Fresquet, Judith; Mompelat, Dimitri; Zeisel, Mirjam B.; Baumert, Thomas F.; Cosset, François‐Loïc; Lavillette, Dimitri

doi:10.1002/hep.26733

Cited by 87 publications

(99 citation statements)

References 30 publications

(69 reference statements)

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“…HCV pseudoparticles (HCVpp) were produced in 293T cells and used to infect cell lines as previously described (29,30). Infected cells were quantified by FACSCanto II (BD Biosciences) to measure percentages of green fluorescent protein (GFP) expression.…”

Section: Methodsmentioning

confidence: 99%

“…Pseudoparticles were purified and concentrated from the cell culture medium by ultracentrifugation at 82,000 ϫ g for 1 h 45 min through a 20% sucrose cushion. Cell lysates and viral pellets were subjected to Western blot analysis using antibody 3/11 and an anti-MLV-CA antibody as described previously (29).…”

Section: Methodsmentioning

confidence: 99%

“…L1a and L3k E1E2-encoding sequences (without core-encoding sequences) were inserted into the H77/JFH1/HQL genome to generate, respectively, the L1a/JFH1 and L3k/JFH1 recombinant genomes. Derived HCV RNAs were electroporated in Huh-7.5 cells as described previously (7,29). Huh-7.5 cells were infected with different dilutions of cell culture supernatants harvested at 48 h and 72 h postelectroporation or with intracellular viral particles obtained 72 h postelectroporation after 3 freeze-thaw cycles of Huh-7.5.…”

Section: Methodsmentioning

confidence: 99%

“…To quantify viral particle production, viral RNAs were isolated from cell supernatant using Tri Reagent solution (Sigma-Aldrich) as recommended by the manufacturer. Viral RNAs were then reverse transcribed using random primers (Bio-Rad) and quantified by quantitative PCR (qPCR) using a light cycler LC480 (Roche Applied Science) as described previously (29).…”

Section: Methodsmentioning

confidence: 99%

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Specialization of Hepatitis C Virus Envelope Glycoproteins for B Lymphocytes in Chronically Infected Patients

Douam

Bobay

Maurin

et al. 2016

J Virol

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Hepatitis C virus (HCV) productively infects hepatocytes. Virion surface glycoproteins E1 and E2 play a major role in this restricted cell tropism by mediating virus entry into particular cell types. However, several pieces of evidence have suggested the ability of patientderived HCV particles to infect peripheral blood mononuclear cells. The viral determinants and mechanisms mediating such events remain poorly understood. Here, we aimed at isolating viral determinants of HCV entry into B lymphocytes. For this purpose, we constructed a library of full E1E2 sequences isolated from serum and B lymphocytes of four chronically infected patients. We observed a strong phylogenetic compartmentalization of E1E2 sequences isolated from B lymphocytes in one patient, indicating that E1E2 glycoproteins can represent important mediators of the strong segregation of two specialized populations in some patients. Most of the E1E2 envelope glycoproteins were functional and allowed transduction of hepatocyte cell lines using HCV-derived pseudoparticles. Strikingly, introduction of envelope glycoproteins isolated from B lymphocytes into the HCV JFH-1 replicating virus switched the entry tropism of this nonlymphotropic virus from hepatotropism to lymphotropism. Significant detection of viral RNA and viral proteins within B cells was restricted to infections with JFH-1 harboring E1E2 from lymphocytes and depended on an endocytic, pH-dependent entry pathway. Here, we achieved for the first time the isolation of HCV viral proteins carrying entry-related lymphotropism determinants. The identification of genetic determinants within E1E2 represents a first step for a better understanding of the complex relationship between HCV infection, viral persistence, and extrahepatic disorders. IMPORTANCEHepatitis C virus (HCV) mainly replicates within the liver. However, it has been shown that patient-derived HCV particles can slightly infect lymphocytes in vitro and in vivo, highlighting the existence of lymphotropism determinants within HCV viral proteins. We isolated HCV envelope glycoproteins from patient B lymphocytes that conferred to a nonlymphotropic HCV the ability to enter B cells, thus providing a platform for characterization of HCV entry into lymphocytes. This unusual tropism was accompanied by a loss of entry function into hepatocytes, suggesting that HCV lymphotropic variants likely constitute a distinct but parallel source for viral persistence and immune escape within chronically infected patients. Moreover, the level of genetic divergence of B-cell-derived envelopes correlated with their degree of lymphotropism, underlining a long-term specialization of some viral populations for B-lymphocytes. Consequently, the clearance of both hepatotropic and nonhepatotropic HCV populations may be important for effective treatment of chronically infected patients.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Specialization of Hepatitis C Virus Envelope Glycoproteins for B Lymphocytes in Chronically Infected Patients

Douam

Bobay

Maurin

et al. 2016

J Virol

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“…It has been proposed that a conserved hydrophobic sequence, CSALYVGDLC (residues 272 to 281), in E1, which was not included in the structural analysis of nE1, may represent a truncated class II fusion peptide (27). The role of this sequence in virus entry and membrane fusion was supported by data from several studies (27)(28)(29)(30)(31)(32). The potential role of this putative fusion peptide in E1 in other aspects of the HCV life cycle has not been explored.…”

mentioning

confidence: 99%

Functional Analysis of Hepatitis C Virus (HCV) Envelope Protein E1 Using a trans -Complementation System Reveals a Dual Role of a Putative Fusion Peptide of E1 in both HCV Entry and Morphogenesis

Tong

Chi

Yang

et al. 2017

J Virol

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Hepatitis C virus (HCV) is an enveloped RNA virus belonging to the Flaviviridae family. It infects mainly human hepatocytes and causes chronic liver diseases, including cirrhosis and cancer. HCV encodes two envelope proteins, E1 and E2, that form a heterodimer and mediate virus entry. While E2 has been extensively studied, less has been done so for E1, and its role in the HCV life cycle still needs to be elucidated. Here we developed a new cell culture model for HCV infection based on the trans-complementation of E1. Virus production of the HCV genome lacking the E1-encoding sequence can be efficiently rescued by the ectopic expression of E1 in trans. The resulting virus, designated HCVΔE1, can propagate in packaging cells expressing E1 but results in only single-cycle infection in naive cells. By using the HCVΔE1 system, we explored the role of a putative fusion peptide (FP) of E1 in HCV infection. Interestingly, we found that the FP not only contributes to HCV entry, as previously reported, but also may be involved in virus morphogenesis. Finally, we identified amino acid residues in FP that are critical for biological functions of E1. In summary, our work not only provides a new cell culture model for studying HCV but also provides some insights into understanding the role of E1 in the HCV life cycle. IMPORTANCE Hepatitis C virus (HCV), an enveloped RNA virus, encodes two envelope proteins, E1 and E2, that form a heterodimeric complex to mediate virus entry. Compared to E2, the biological functions of E1 in the virus life cycle are not adequately investigated. Here we developed a new cell culture model for single-cycle HCV infection based on the trans-complementation of E1. The HCV genome lacking the E1-encoding sequence can be efficiently rescued for virus production by the ectopic expression of E1 in trans. This new model renders a unique system to dissect functional domains and motifs in E1. Using this system, we found that a putative fusion peptide in E1 is a multifunctional structural element contributing to both HCV entry and morphogenesis. Our work has provided a new cell culture model to study HCV and provides insights into understanding the biological roles of E1 in the HCV life cycle. KEYWORDS E1, envelope proteins, fusion peptide, hepatitis C virus, transcomplementation, virus entry, virus morphogenesis H epatitis C virus (HCV) is a major human pathogen that infects about 170 million people worldwide and can lead to chronic hepatitis and more severe liver diseases such as cirrhosis and hepatocellular carcinoma (1-3). For a long time, standard therapy for chronic HCV infection consisted of the administration of pegylated interferon alpha Functional analysis of hepatitis C virus (HCV) envelope protein E1 using a transcomplementation system reveals a dual role of a putative fusion peptide of E1 in both HCV entry and morphogenesis. J Virol 91:e02468-16.

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Selection of a hepatitis C virus with altered entry factor requirements reveals a genetic interaction between the E1 glycoprotein and claudins

Hopcraft

Evans

2015

Hepatology

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Hepatitis C virus (HCV) cell entry is a complex, multistep process requiring numerous host cell factors, including the tight junction protein claudin-1 (CLDN1). It is not known whether CLDN1 and the HCV glycoproteins physically interact. Therefore, the focus of this work was to study genetic interactions between CLDN1 and HCV. We used CRISPR technology to generate CLDN1 knockout (KO) Huh-7.5 cells, which could not be infected by genotype 2a Jc1 HCV unless CLDN1 expression was restored. Passage of Jc1-transfected CLDN1 KO cells resulted in the selection of a virus that could infect these cells. This virus encoded a single mutation, H316N (numbered relative to the HCV polyprotein), in the E1 glycoprotein. Whereas Jc1 H316N efficiently infected cells lacking CLDN1, such infection was blocked by an antibody targeting CLDN6, another member of the claudin family that is expressed in these cells. Furthermore, HuH6 cells, which express CLDN6, but not CLDN1, were infectable only with the mutant virus. Thus, this mutant virus adapted to the loss of CLDN1 by developing the capacity to utilize other CLDNs. Indeed, CLDN1/CLDN6 double-KO Huh-7.5 cells supported infection by the mutant virus only when CLDN1, CLDN6, or CLDN9 was expressed. Finally, this phenotype was not genotype dependent, given that the H316N mutation rendered a Japanese fulminant hepatitis 1 chimeric HCV genome encoding the genotype 5a glycoproteins able to utilize CLDN6 for host cell entry. Conclusion These data demonstrate plasticity of HCV virus-host interactions, where a previously CLDN1-dependent virus was capable of evolving to use CLDN6. They also reveal a role for E1 in determining entry factor usage and imply a direct, physical interaction between E1 and CLDNs.

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Critical interaction between E1 and E2 glycoproteins determines binding and fusion properties of hepatitis C virus during cell entry

Cited by 87 publications

References 30 publications

Specialization of Hepatitis C Virus Envelope Glycoproteins for B Lymphocytes in Chronically Infected Patients

Specialization of Hepatitis C Virus Envelope Glycoproteins for B Lymphocytes in Chronically Infected Patients

Functional Analysis of Hepatitis C Virus (HCV) Envelope Protein E1 Using a trans -Complementation System Reveals a Dual Role of a Putative Fusion Peptide of E1 in both HCV Entry and Morphogenesis

Selection of a hepatitis C virus with altered entry factor requirements reveals a genetic interaction between the E1 glycoprotein and claudins

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