Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly.
Hepatitis C virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis.IMPORTANCEHepatitis C virus (HCV) infection is a major public health problem worldwide. It encodes two envelope proteins, E1 and E2, which play a major role in the life cycle of this virus. E2 has been extensively characterized, whereas E1 remains poorly understood. Here, we investigated E1 functions by using site-directed mutagenesis in the context of the viral life cycle. Our results identify unique phenotypes. Unexpectedly, two mutants clearly showed a shift in receptor dependence for cell entry, highlighting a role for E1 in modulating HCV particle interaction with a cellular receptor(s). More importantly, another mutant led to the assembly and release of viral particles devoid of genomic RNA. This unique phenotype was further characterized, and we observed a change in subcellular colocalization between HCV RNA and E1. This unique observation highlights for the first time cross talk between a viral envelope protein and genomic RNA during morphogenesis.
University of Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019, UMR 8204, Centre d'Infection et d'Immunité de Lille, Lille, France.|Laboratoire Microbiologie Santé et Environnement, Ecole Doctorale en Sciences et Technologie, Faculté de Santé Publique, Université Libanaise, Tripoli, Liban.
Juliano G
JG
Haddad
University of Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019, UMR 8204, Centre d'Infection et d'Immunité de Lille, Lille, France.
Yves
Y
Rouillé
University of Lille, CNRS, UMR 8576, Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France.
Xavier
X
Hanoulle
Laboratoire de Virologie EA4294, Centre Hospitalier Universitaire d'Amiens, Université de Picardie Jules Verne, Amiens, France.
Véronique
V
Descamps
Laboratoire Microbiologie Santé et Environnement, Ecole Doctorale en Sciences et Technologie, Faculté de Santé Publique, Université Libanaise, Tripoli, Liban.
Monzer
M
Hamze
Laboratoire Microbiologie Santé et Environnement, Ecole Doctorale en Sciences et Technologie, Faculté de Santé Publique, Université Libanaise, Tripoli, Liban.
Fouad
F
Dabboussi
Inserm, U1110, University of Strasbourg, Pôle Hépato-digestif-Hôpitaux Universitaires de Strasbourg, Strasbourg, France.
Thomas F
TF
Baumert
Laboratoire de Virologie EA4294, Centre Hospitalier Universitaire d'Amiens, Université de Picardie Jules Verne, Amiens, France.
Gilles
G
Duverlie
University of Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019, UMR 8204, Centre d'Infection et d'Immunité de Lille, Lille, France muriel.lavie@ibl.cnrs.fr jean.dubuisson@ibl.cnrs.fr.
Muriel
M
Lavie
University of Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019, UMR 8204, Centre d'Infection et d'Immunité de Lille, Lille, France muriel.lavie@ibl.cnrs.fr jean.dubuisson@ibl.cnrs.fr.
Jean
http://orcid.org/0000-0003-1626-7693
J
Dubuisson
D002460:Cell Line; D004252:DNA Mutational Analysis; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D020125:Mutation, Missense; D017510:Protein Folding; D055503:Protein Multimerization; D011991:Receptors, Virus / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly; D053586:Virus Internalization
C087035:E1 protein, Hepatitis C virus; D011991:Receptors, Virus; D014759:Viral Envelope Proteins
envelope proteins; glycoprotein; glycoproteins; hepatitis C virus; viral assembly; viral entry; viral envelope