Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly

Abstract

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. IMPORTANCE Hepatitis 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.

The acidic domain of the hepatitis C virus NS4A protein is required for viral assembly and envelopment through interactions with the viral E1 glycoprotein

“…The first hydrophobic region of E1 has previously been implicated in viral particle production, and several amino acids within this region are important for viral infectivity [47, 48]. Additionally, one specific E1 mutation, D263A (E1 aa71), attenuated viral infectivity and resulted in secreted Core protein but not HCV RNA [48]. Taken together with our findings that NS4A binds to the E1 the hydrophobic region, which contains aaD263, we hypothesized that Y45F may have a similar phenotype as the E1 D263A mutant.…”

“…Importantly, we found that NS4A binds to E1 and that antagonizing this interaction with one amino acid change in NS4A prevents viral envelopment. We mapped the binding site of NS4A on E1 and found that it interacts with the first hydrophobic region, a region that is known to be important for viral particle production [47, 48]. Finally, we found that the Y45F mutation in NS4A, which prevents envelopment, also results in secretion of noninfectious, incompletely formed virions that are composed of low density Core protein oligomers that lack HCV RNA.…”

New insights into the ORF2 capsid protein, a key player of the hepatitis E virus lifecycle

“…Western blotting analyses were performed as described previously (31). Briefly, supernatants and lysates of wt and mt PLC3/HEV-p6 cells were heated for 20 min at 80°C in the presence of Laemmli buffer (reducing or non-reducing).…”

Development and characterization of a human monoclonal antibody targeting the N-terminal region of hepatitis C virus envelope glycoprotein E1

“…It is also the main target for the host's adaptive immune system. Recently the E1 protein was also shown to be involved in viral entry and virion assembly (Haddad et al, 2017). …”

“…Despite the availability of multiple in vitro systems as well as experimental animal models, little is still known about the structure and actual function of the E1 glycoprotein (Douam et al, 2014; Haddad et al, 2017; Lavillette et al, 2007; Wahid et al, 2013). Evidence for the immunogenicity and the induction of neutralizing antibodies by E1 has been reported.…”