Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: Putative CD81 binding region 1 is not involved in CD81 binding

Little is known about the structure of the envelope glycoproteins of hepatitis C virus (HCV). To identify new regions essential for the function of these glycoproteins, we generated HCV pseudoparticles (HCVpp) containing HCV envelope glycoproteins, E1 and E2, from different genotypes in order to detect intergenotypic incompatibilities between these two proteins. Several genotype combinations were nonfunctional for HCV entry. Of interest, a combination of E1 from genotype 2a and E2 from genotype 1a was nonfunctional in the HCVpp system. We therefore used this nonfunctional complex and the recently described structural model of E2 to identify new functional regions in E2 by exchanging protein regions between these two genotypes. The functionality of these chimeric envelope proteins in the HCVpp system and/or the cell-cultured infectious virus (HCVcc) was analyzed. We showed that the intergenotypic variable region (IgVR), hypervariable region 2 (HVR2), and another segment in domain II play a role in E1E2 assembly. We also demonstrated intradomain interactions within domain I. Importantly, we also identified a segment (amino acids [aa] 705 to 715 [segment 705-715]) in the stem region of E2, which is essential for HCVcc entry. Circular dichroism and nuclear magnetic resonance structural analyses of the synthetic peptide E2-SC containing this segment revealed the presence of a central amphipathic helix, which likely folds upon membrane binding. Due to its location in the stem region, segment 705-715 is likely involved in the reorganization of the glycoprotein complexes taking place during the fusion process. In conclusion, our study highlights new functional and structural regions in HCV envelope glycoprotein E2.Hepatitis C virus (HCV) infects approximately 3% of the world population (72) and is currently the major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (43). A vaccine is not yet available, and the treatment fails in around 50% of the cases, depending on the virus genotype (43). Although the cloning of the HCV genome more than 20 years ago (4) allowed for a rapid analysis of the genomic organization and a biochemical characterization of its proteins (reviewed in reference 57), the lack of a cell culture system to efficiently amplify this virus has long been a major obstacle for the study of the HCV life cycle. Fortunately, in 2005, the development of a cell culture system that allowed for a relatively efficient amplification of HCV (HCVcc) was finally reported (42,71,78).HCV is an enveloped, positive-stranded RNA virus that belongs to the Flaviviridae family (41). Its genome encodes a single polyprotein of about 3,000 amino acids, which is cleaved co-and posttranslationally by cellular and viral proteases to yield at least 10 mature products (reviewed in reference 57). Cleavage of the viral polyprotein by a cellular signal peptidase gives rise to the envelope glycoproteins E1 and E2 (reviewed in reference 17). HCV envelope glycoproteins are type I transmembrane (TM) proteins containing a h...

Section: Discussionsupporting

confidence: 77%

Identification of New Functional Regions in Hepatitis C Virus Envelope Glycoprotein E2

Albecka

Montserret

Krey

et al. 2011

“…Alanine scanning mutagenesis revealed that two conserved E2 residues at G530 and D535 are required for binding by all domain B HMAbs, while G523 or W529 is required for some, but not all, of these antibodies (21,22,29). Importantly, W529, G530, and D535 have been shown to participate in the interaction of E2 with CD81 (30,33). The data thus suggest that domain B HMAbs exert potent and potentially broad neutralizing effects on HCV by competing with CD81 for binding to conserved residues on E2 that are important for viral entry.…”

mentioning

confidence: 99%

Mapping a Region of Hepatitis C Virus E2 That Is Responsible for Escape from Neutralizing Antibodies and a Core CD81-Binding Region That Does Not Tolerate Neutralization Escape Mutations

Keck

Saha

Xia

et al. 2011

147

Understanding the interaction between broadly neutralizing antibodies and their epitopes provides a basis for the rational design of a preventive hepatitis C virus (HCV) vaccine. CBH-2, HC-11, and HC-1 are representatives of antibodies to overlapping epitopes on E2 that mediate neutralization by blocking virus binding to CD81. To obtain insights into escape mechanisms, infectious cell culture virus, 2a HCVcc, was propagated under increasing concentrations of a neutralizing antibody to isolate escape mutants. Three escape patterns were observed with these antibodies. First, CBH-2 escape mutants that contained mutations at D431G or A439E, which did not compromise viral fitness, were isolated. Second, under the selective pressure of HC-11, escape mutations progressed from a single L438F substitution at a low antibody concentration to double substitutions, L438F and N434D or L438F and T435A, at higher antibody concentrations. Escape from HC-11 was associated with a loss of viral fitness. An HCV pseudoparticle (HCVpp) containing the L438F mutation bound to CD81 half as efficiently as did wild-type (wt) HCVpp. Third, for HC-1, the antibody at a critical concentration completely suppressed viral replication and generated no escape mutants. Epitope mapping revealed contact residues for CBH-2 and HC-11 in two regions of the E2 glycoprotein, amino acids (aa) 425 to 443 and aa 529 to 535. Interestingly, contact residues for HC-1 were identified only in the region encompassing aa 529 to 535 and not in aa 425 to 443. Taken together, these findings point to a region of variability, aa 425 to 443, that is responsible primarily for viral escape from neutralization, with or without compromising viral fitness. Moreover, the region aa 529 to 535 is a core CD81 binding region that does not tolerate neutralization escape mutations.Up to 170 million people worldwide are chronically infected with hepatitis C virus (HCV), with many at significant risk for liver failure and hepatocellular carcinoma (http://www.who.int /vaccine_research/diseases/viral_cancers/en/index2.html). The virus is transmitted primarily by parenteral routes and injection drug use in developed countries, whereas contaminated injection equipment appears to be the major risk factor for HCV infection in developing countries. From unsafe needle injections alone, the World Health Organization estimates an annual increase in the global burden by 2 million new infections (35). Current therapy with combined pegylated interferon and ribavirin has led to clinical improvement for some patients, but treatment is associated with adverse side effects and a high relapse rate off therapy. Clearly, additional approaches are needed for treatment and prevention of infection. However, an effective HCV vaccine has yet to be achieved, despite considerable effort. A major impediment is the genetic diversity of the virus. The phylogenetic tree of HCV contains seven major genotypes with more than 30% divergence between genotypes, and each genotype contains a large number of related subtype...

“…Furthermore, we assessed their relationships with other HCV HMAbs that are directed at two distinct clusters of overlapping epitopes, designated antigenic domains B and D (30)(31)(32)(33)(34). Antibodies to antigenic domain B share two contact residues at G530 and D535 (30,33,35), which are universally conserved and have been shown to participate in the interaction of E2 with CD81 (19,36). Some of the antigenic domain B epitopes also contain residues located within aa 434 to 446, e.g., an epitope identified by an antibody designated HC-11 (35,37 425 ) were synthesized using a C-terminal biotin residue with a Gly-Ser-Gly linker (American Peptide, Sunnyvale, CA).…”

mentioning

confidence: 99%

Cooperativity in Virus Neutralization by Human Monoclonal Antibodies to Two Adjacent Regions Located at the Amino Terminus of Hepatitis C Virus E2 Glycoprotein

Keck

Wang

et al. 2013

110

176

A challenge for hepatitis C virus (HCV) vaccine development is defining conserved epitopes that induce protective antibodies against this highly diverse virus. An envelope glycoprotein (E2) segment located at amino acids (aa) 412 to 423 contains highly conserved neutralizing epitopes. While polyclonal antibodies to aa 412 to 423 from HCV-infected individuals confirmed broad neutralization, conflicting findings have been reported on polyclonal antibodies to an adjacent region, aa 434 to 446, that may or may not interfere with neutralization by antibodies to aa 412 to 423. To define the interplay between these antibodies, we isolated human monoclonal antibodies (HMAbs) to aa 412 to 423, designated HC33-related HMAbs (HC33 HMAbs), and characterized their interactions with other HMAbs to aa 434 to 446. A subset of the HC33 HMAbs neutralized genotype 1 to 6 infectious cell culture-derived HCV virions (HCVcc) with various activities. Although nonneutralizing HC33 HMAbs were isolated, they had lower binding affinities than neutralizing HC33 HMAbs. These antibodies could be converted to neutralizing antibodies by affinity maturation. Unidirectional competition for binding to E2 was observed between HC33 HMAbs and HMAbs to aa 434 to 446. When HMAbs to aa 434 to 446, which mediated neutralization, were combined with neutralizing HC33 HMAbs, biphasic patterns in neutralization were observed. A modest degree of antagonism was observed at lower concentrations, and a modest degree of synergism was observed at higher concentrations. However, the overall effect was additive neutralization. A similar pattern was observed when these antibodies were combined to block E2 binding to the HCV coreceptor, CD81. These findings demonstrate that both of these E2 regions participate in epitopes mediating virus neutralization and that the antibodies to aa 412 to 423 and aa 434 to 446 do not hinder their respective virus-neutralizing activities.