A single point mutation in E2 enhances hepatitis C virus infectivity and alters lipoprotein association of viral particles

c Hypervariable region 1 (HVR1) of envelope protein 2 (E2) of hepatitis C virus (HCV) serves important yet undefined roles in the viral life cycle. We previously showed that the viability of HVR1-deleted JFH1-based recombinants with Core-NS2 of H77 (H77 ⌬HVR1 , genotype 1a) and S52 (S52 ⌬HVR1 , genotype 3a) in Huh7.5 cells was rescued by E2 substitutions N476D/S733F and an E1 substitution, A369V, respectively; HVR1-deleted J6 (J6 ⌬HVR1 , genotype 2a) was fully viable. In single-cycle production assays, where HCV RNA was transfected into entry-deficient Huh7-derived S29 cells with low CD81 expression, we found no effect of HVR1 deletion on replication or particle release for H77 and S52. HCV pseudoparticle assays in Huh7.5 cells showed that HVR1 deletion decreased entry by 20-to 100-fold for H77, J6, and S52; N476D/S733F restored entry for H77 ⌬HVR1 , while A369V further impaired S52 ⌬HVR1 entry. We investigated receptor usage by antibody blocking and receptor silencing in Huh7.5 cells, followed by inoculation of parental and HVR1-deleted HCV recombinants. Compared to parental viruses, scavenger receptor class B type I (SR-BI) dependency was decreased for H77 ⌬HVR1/N476D/S733F , H77 N476D/S733F , S52 ⌬HVR1/A369V , and S52 A369V , but not for J6 ⌬HVR1 . Low-density lipoprotein receptor (LDLr) dependency was decreased for HVR1-deleted viruses, but not for H77 N476D/S733F and S52 A369V . Soluble LDLr neutralization revealed strong inhibition of parental HCV but limited effect against HVR1-deleted viruses. Apolipoprotein E (ApoE)-specific HCV neutralization was similar for H77, J6, and S52 viruses with and without HVR1. In conclusion, HVR1 and HVR1-related adaptive envelope mutations appeared to be involved in LDLr and SR-BI dependency, respectively. Also, LDLr served ApoE-independent but HVR1-dependent functions in HCV entry.A pproximately 180 million people worldwide are chronically infected with hepatitis C virus (HCV) with an increased risk of developing liver cirrhosis and hepatocellular carcinoma (1). HCV is an enveloped positive-strand RNA virus of the family Flaviviridae with a 9.6-kb genome consisting of 5= and 3= untranslated regions (UTRs) flanking an open reading frame (ORF) that encodes a single polyprotein. This polyprotein is processed into structural proteins (Core and envelope proteins E1 and E2), p7, and six nonstructural proteins (NS2 to NS5B) (2). HCV is a highly diverse virus, and isolates are divided into seven major genotypes, most containing multiple subtypes and differing by ϳ30% and ϳ20%, respectively, at the nucleotide and amino acid levels (2). Previous studies have shown genotype or isolate differences when analyzing HCV neutralization and in reverse genetics studies of HCV proteins (3-5). This highlights the importance of including several isolates, preferably of diverse genotypes, in functional studies.While the process of HCV entry into the human hepatocyte remains incompletely understood, it is known to be a complex multistep process involving several receptors acting at (i) initial a...

Section: Discussionsupporting

confidence: 74%

Section: S29 Cell Transfectionmentioning

confidence: 97%

Hypervariable Region 1 Deletion and Required Adaptive Envelope Mutations Confer Decreased Dependency on Scavenger Receptor Class B Type I and Low-Density Lipoprotein Receptor for Hepatitis C Virus

Prentoe

Serre

Ramírez

et al. 2014

“…HCVcc (JFH1 strain, genotype 2a) (GenBank accession number AB047639) (46,47) was used in these studies. The protocols for the generation of HCVcc or HCVΔE1, including in vitro RNA transcription and electroporation, were described previously (18,33,47).…”

Section: Methodsmentioning

confidence: 99%

“…Through their C-terminal transmembrane domains (TMDs), E1 and E2 form a stable noncovalent heterodimeric complex that mediates virus entry and determines viral pathogenicity and the host immune response (13)(14)(15). The HCV envelope proteins may also mediate the association of viral particles with host low-density lipoprotein (LDL) or very-low-density lipoprotein (VLDL), which plays important roles in virus entry, egress, and evasion of the host immune response (12,(16)(17)(18). E2 contains the major viral structural elements that are responsible for binding to host receptors, including CD81 and scavenger receptor class B type I (SR-BI) (13)(14)(15)(16).…”

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

Self Cite

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.

“…These proposed mechanisms include genetic escape (61), the occlusion of neutralizing epitopes through glycan shielding (18,26) and lipid associations (23,54), infection enhancement via serum components such as high-density lipoprotein (HDL) and apolipoproteins (13,14,60), and cell-to-cell transmission (7,63). An additional mechanism was recently proposed by Zhang and colleagues, who reported that a linear region of E2 encompassing amino acids 434 to 446 (the so-called epitope II) elicits nonneutralizing antibodies in humans and chimpanzees that can inhibit the neutralizing activity of antibodies targeting aa 412 to 423 (65,66).…”

mentioning

confidence: 99%

Naturally Occurring Antibodies That Recognize Linear Epitopes in the Amino Terminus of the Hepatitis C Virus E2 Protein Confer Noninterfering, Additive Neutralization

Tarr

Urbanowicz

Jayaraj

et al. 2012

Chronic hepatitis C virus (HCV) infection can persist even in the presence of a broadly neutralizing antibody response. Various mechanisms that underpin viral persistence have been proposed, and one of the most recently proposed mechanisms is the presence of interfering antibodies that negate neutralizing responses. Specifically, it has been proposed that antibodies targeting broadly neutralizing epitopes located within a region of E2 encompassing residues 412 to 423 can be inhibited by nonneutralizing antibodies binding to a less conserved region encompassing residues 434 to 446. To investigate this phenomenon, we characterized the neutralizing and inhibitory effects of human-derived affinity-purified immunoglobulin fractions and murine monoclonal antibodies and show that antibodies to both regions neutralize HCV pseudoparticle (HCVpp) and cell culture-infectious virus (HCVcc) infection albeit with different breadths and potencies. Epitope mapping revealed the presence of overlapping but distinct epitopes in both regions, which may explain the observed differences in neutralizing phenotypes. Crucially, we failed to demonstrate any inhibition between these two groups of antibodies, suggesting that interference by nonneutralizing antibodies, at least for the region encompassing residues 434 to 446, does not provide a mechanism for HCV persistence in chronically infected individuals. H epatitis C virus (HCV) has infected approximately 180 million people worldwide (2). Following infection, most people fail to clear the virus, and a chronic infection, often with serious sequelae, ensues (1, 38). HCV-related end-stage liver disease is the leading indication for liver transplantation, and reinfection of the grafted liver occurs rapidly (32). A systematic review of the research literature recently suggested that there is little, if any, benefit gained by the treatment of liver transplant recipients with standard antiviral regimens (24), and possible adverse effects associated with newly emerging direct-acting antivirals may limit their usefulness in this clinical setting. Antibodies are usually well tolerated, and the successful administration of anti-hepatitis B virus immunoglobulin (Ig) (HBIG) (50, 59) sets an important precedent for HCV. The administration of HCV-neutralizing antibodies during the anhepatic phase and following transplantation could likewise prevent the reinfection of the grafted liver; the reduced incidence of HCV in individuals receiving HBIG containing anti-HCV antibodies (20) supports this notion. However, to date, the therapeutic administration of serum immunoglobulin or monoclonal antibodies targeting HCV has been disappointing (10, 51), indicating that further studies of the polyclonal response are needed, if we are to harness the opportunity that antibody therapy offers.There is also an urgent need for the development of safe and effective HCV vaccines to prevent infection. Significant progress has been made toward T-cell-based vaccines (22), but these vaccines will not be sufficient to elici...