Towards a solution for hepatitis C virus hypervariability: mimotopes of the hypervariable region 1 can induce antibodies cross-reacting with a large number of viral variants.

The hypervariable region 1 (HVR1) of the putative envelope protein E2 of hepatitis C virus (HCV) is the most variable antigenic fragment in the whole viral genome and is mainly responsible for the large inter-and intra-individual heterogeneity of the infecting virus. It contains a principal neutralization epitope and has been proposed as the major player in the mechanism of escape from host immune response. Since anti-HVR1 antibodies are the only species shown to possess protective activity up to date, developing an effective prevention therapy is a very difficult task. We have approached the problem of HVR1 variability by deriving a consensus profile from >200 HVR1 sequences from different viral isolates and used it as a template to generate a vast repertoire of synthetic HVR1 surrogates displayed on M13 bacteriophage. This library was affinity selected using many different sera from infected patients. Phages were identified which react very frequently with patients' sera and bind serum antibodies that cross-react with a large panel of HVR1 peptides derived from natural HCV variants. When injected into experimental animals, the 'mimotopes' with the highest cross-reactivity induced antibodies which recognized the same panel of natural HVR1 variants. In these mimotopes we identified a sequence pattern responsible for the observed cross-reactivity. These data may hold the key for future development of a prophylactic vaccine against HCV.     

Genetic drift in hypervariable region 1 of the viral genome in persistent hepatitis C virus infection.

The hypervariable region 1 (HVR1) of the putative second envelope glycoprotein (gp70) of hepatitis C virus (HCV) contains a sequence-specific immunological B-cell epitope that induces the production of antibodies restricted to the specific viral isolate, and anti-HVR1 antibodies are involved in the genetic drift of HVR1 driven by immunoselection (N. Kato, H. Sekiya, Y. Ootsuyama, T. Nakazawa, M. Hijikata, S. Ohkoshi, and K. Shimotohno, J. Virol. 67:3923-3930, 1993). We further investigated the sequence variability of the HCV genomic region that entirely encodes the envelope proteins (gp35 and gp70); these sequences were derived from virus isolated during the acute and chronic phases of hepatitis in one patient, and we found that HVR1 was a major site for genetic mutations in HCV after the onset of hepatitis. We carried out epitope-mapping experiments using the HVR1 sequence derived from the acute phase of hepatitis and identified two overlapping epitopes which are each composed of 11 amino acids (positions 394 to 404 and 397 to 407). The presence of two epitopes within HVR1 suggested that epitope shift happened during the course of hepatitis. Four of six amino acid substitutions detected in HVR1 were located within the two epitopes. We further examined the reactivities of anti-HVR1 antibodies to the substituted amino acid sequences within the two epitopes. HVR1 variants in both epitopes within the HVR1 escaped from anti-HVR1 antibodies that were preexisting in the patient's serum.     

Differential amplification of hypervariable region 1 of hepatitis C virus by partially mismatched primers

The quasispecies nature of hepatitis C virus (HCV) has been well documented over its whole genome and the most variable domain is located at the 5' end of the second envelope region, the so-called hypervariable region 1 (HVR1). HVR1 has therefore been extensively used as the target for characterizing HCV quasispecies profiles. In this study, we reported our finding that partially mismatched primers preferentially amplify different HVR1 sequences in a heterogeneous virus population. This finding suggests a possible mechanism of bias during the amplification of HVR1 sequences and may be responsible for some conflicting data regarding evolutionary or clinical implications of HCV quasispecies.     

丙型肝炎病毒高变区1中介导感染的关键氨基酸残基鉴定

丙型肝炎病毒( HCV)包膜E2蛋白氨基端的高变区1(HVR1)由27个氨基酸组成,是HCV蛋白中变异频率最高的肽段.HVR1含中和抗体表位,同时对HCV细胞侵入起重要作用,其结构与功能的关系目前尚不清楚.本研究对H77株包膜蛋白基因中的HVR1进行了一系列缺失突变,然后将突变体表达质粒与假病毒包装质粒共转染人胚肾(H...     

Analysis of hepatitis C virus hypervariable region 1 sequence from cryoglobulinemic patients and associated controls

Chronic hepatitis C virus (HCV) infection is frequently associated with extrahepatic manifestations, including nonmalignant and malignant B-cell lymphoproliferative disorders. It has been reported that specific changes or recurring motifs in the amino acid sequence of the HCV hypervariable region 1 (HVR1) may be associated with cryoglobulinemia. We searched for specific insertions/deletions and/or amino acid motifs within HVR1 in samples from 80 symptomatic and asymptomatic patients with and 33 patients without detectable cryoglobulins, all with chronic HCV infection. At variance with the results of a previous study which reported a high frequency of insertions at position 385 of HVR1 from cryoglobulinemic patients, we found a 6.2% prevalence of insertions in samples from patients with and a 9.1% prevalence in those without cryoglobulinemia. Moreover, statistical and bioinformatics approaches including Fisher's exact test, k-means clustering, Tree determinant-residue identification, correlation of mutations, principal component analysis, and phylogenetic analysis failed to show statistically significant differences between sequences from cryoglobulin-negative and -positive patients. Our findings suggest that cryoglobulinemia may arise by virtue of as-yet-unidentified host- rather than virus-specific factors. Specific changes in HCV envelope sequence distribution are unlikely to be directly involved in the establishment of pathological B-cell monoclonal proliferation.     

Epitope mapping of antibodies directed against hypervariable region 1 in acute self-limiting and chronic infections due to hepatitis C virus.

Epitopes of hypervariable region 1 (HVR1) were mapped by enzyme-linked immunosorbent assay using follow-up sera of patients, all of whom were infected with the same isolate of hepatitis C virus (HCV). Our results suggest that (i) an early appearance (up to month 13 postinfection) of antibodies directed to the N terminus of HVR1 is associated with acute self-limiting infections of HCV and (ii) isolate-independent antibodies which are mainly directed to the C terminus of HVR1 seem to persist in chronically infected patients. The relevance of HVR1-specific antibodies for neutralization was evaluated by characterization of a rabbit serum.     

Basic residues in hypervariable region 1 of hepatitis C virus envelope glycoprotein e2 contribute to virus entry

The N terminus of hepatitis C virus (HCV) envelope glycoprotein E2 contains a hypervariable region (HVR1) which has been proposed to play a role in viral entry. Despite strong amino acid variability, HVR1 is globally basic, with basic residues located at specific sequence positions. Here we show by analyzing a large number of HVR1 sequences that the frequency of basic residues at each position is genotype dependent. We also used retroviral pseudotyped particles (HCVpp) harboring genotype 1a envelope glycoproteins to study the role of HVR1 basic residues in entry. Interestingly, HCVpp infectivity globally increased with the number of basic residues in HVR1. However, a shift in position of some charged residues also modulated HCVpp infectivity. In the absence of basic residues, infectivity was reduced to the same level as that of a mutant deleted of HVR1. We also analyzed the effect of these mutations on interactions with some potential HCV receptors. Recognition of CD81 was not affected by changes in the number of charged residues, and we did not find a role for heparan sulfates in HCVpp entry. The involvement of the scavenger receptor class B type I (SR-BI) was indirectly analyzed by measuring the enhancement of infectivity of the mutants in the presence of the natural ligand of SR-BI, high-density lipoproteins (HDL). However, no correlation between the number of basic residues within HVR1 and HDL enhancement effect was observed. Despite the lack of evidence of the involvement of known potential receptors, our results demonstrate that the presence of basic residues in HVR1 facilitates virus entry.     

In vitro adaptation and characterization of attenuated hypervariable region 1 swap chimeras of hepatitis C virus

Hepatitis C virus (HCV) chronically infects 70 million people worldwide with an estimated annual disease-related mortality of 400,000. A vaccine could prevent spread of this pervasive human pathogen, but has proven difficult to develop, partly due to neutralizing antibody evasion mechanisms that are inherent features of the virus envelope glycoproteins, E1 and E2. A central actor is the E2 motif, hypervariable region 1 (HVR1), which protects several non-overlapping neutralization epitopes through an incompletely understood mechanism. Here, we show that introducing different HVR1-isolate sequences into cell-culture infectious JFH1-based H77 (genotype 1a) and J4 (genotype 1b) Core-NS2 recombinants can lead to severe viral attenuation. Culture adaptation of attenuated HVR1-swapped recombinants permitted us to identify E1/E2 substitutions at conserved positions both within and outside HVR1 that increased the infectivity of attenuated HVR1-swapped recombinants but were not adaptive for original recombinants. H77 recombinants with HVR1 from multiple other isolates consistently acquired substitutions at position 348 in E1 and position 385 in HVR1 of E2. Interestingly, HVR1-swapped J4 recombinants primarily acquired other substitutions: F291I (E1), F438V (E2), F447L/V/I (E2) and V710L (E2), indicating a different adaptation pathway. For H77 recombinants, the adaptive E1/E2 substitutions increased sensitivity to the neutralizing monoclonal antibodies AR3A and AR4A, whereas for J4 recombinants, they increased sensitivity to AR3A, while having no effect on sensitivity to AR4A. To evaluate effects of the substitutions on AR3A and AR4A binding, we performed ELISAs on extracted E1/E2 protein and performed immunoprecipitation of relevant viruses. However, extracted E1/E2 protein and immunoprecipitation of HCV particles only reproduced the neutralization phenotypes of the J4 recombinants. Finally, we found that the HVR1-swap E1/E2 substitutions decrease virus entry dependency on co-receptor SR-BI. Our study identifies E1/E2 positions that could be critical for intra-complex HVR1 interactions while emphasizing the need for developing novel tools for molecular studies of E1/E2 interactions.     

Monoclonal antibodies for human thrombomodulin which recognize binding sites for thrombin and protein C

Monoclonal antibodies for human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C, were prepared and their epitopes characterized. All six antibodies (MFTM-1-MFTM-6) bound to an elastase-digested active fragment of thrombomodulin, which contains six consecutive EGF domains. Binding of thrombomodulin to these antibodies did not depend on Ca2+ concentration. MFTM-4, MFTM-5, and MFTM-6 strongly inhibited protein C activation by thrombin and thrombomodulin. MFTM-4 and MFTM-5 inhibited thrombin binding to fixed thrombomodulin and bound to a recombinant mutant EGF456 protein, which contained the fourth, fifth, and sixth EGF domains of thrombomodulin. However, MFTM-6 did not inhibit thrombin binding to thrombomodulin and did not bind to EGF456 protein. Binding of thrombomodulin to fixed MFTM-4 or MFTM-5 was competitively inhibited by a recombinant mutant EGF45 protein which contained the fifth and sixth EGF-domains. These results suggest that epitopes of MFTM-4 and MFTM-5 are located in the fifth EGF domain of thrombomodulin. Thus, the binding site for thrombin is located in the fifth EGF domain. These results also suggest that an epitope for MFTM-6 is located at a region near the binding site for gamma-carboxyglutamic acid residues of protein C via Ca2+ on thrombomodulin.     

Acute hepatitis C virus structural gene sequences as predictors of persistent viremia: hypervariable region 1 as a decoy

We hypothesized that hepatitis C virus (HCV) persistence is related to the sequence variability of putative envelope genes. This hypothesis was tested by characterizing quasispecies in specimens collected every six months from a cohort of acutely HCV-infected subjects (mean duration of specimen collection, 72 months after seroconversion). We evaluated 5 individuals who spontaneously cleared viremia and 10 individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5' half of E2, including hypervariable region 1 (HVR1). To assess the quasispecies complexity and to detect variants for sequencing, the first PCR-positive sample was examined by using a previously described method that combines heteroduplex analysis and analysis of single-stranded conformational polymorphisms. The ratio of nonsynonymous to synonymous substitutions (dN/dS) within each sample was evaluated as an indicator of relative selective pressure. Amino acid sequences were analyzed for signature patterns, glycosylation signals, and charge. Quasispecies complexity was higher and E1 dN/dS ratios (selective pressure) were lower in those with persistent viremia; the association with persistence was strengthened by the presence of a combination of both characteristics. In contrast, a trend toward higher HVR1 dN/dS ratios was detected among those with persistent viremia. We did not detect any such association for factors that may affect complexity such as serum HCV RNA concentration. HVR1 had a lower positive charge in subjects with persistent viremia, although no consistent motifs were detected. Our data suggest that HCV persistence is associated with a complex quasispecies and immune response to HVR1.     

Structural basis for broad neutralization of hepatitis C virus quasispecies

Monoclonal antibodies directed against hepatitis C virus (HCV) E2 protein can neutralize cell-cultured HCV and pseudoparticles expressing envelopes derived from multiple HCV subtypes. For example, based on antibody blocking experiments and alanine scanning mutagenesis, it was proposed that the AR3B monoclonal antibody recognized a discontinuous conformational epitope comprised of amino acid residues 396–424, 436–447, and 523–540 of HCV E2 envelope protein. Intriguingly, one of these segments (436–447) overlapped with hypervariable region 3 (HVR3), a domain that exhibited significant intrahost and interhost genetic diversity. To reconcile these observations, amino-acid sequence variability was examined and homology-based structural modelling of E2 based on tick-borne encephalitis virus (TBEV) E protein was performed based on 413 HCV sequences derived from 18 subjects with chronic hepatitis C. Here we report that despite a high degree of amino-acid sequence variability, the three-dimensional structure of E2 is remarkably conserved, suggesting broad recognition of structural determinants rather than specific residues. Regions 396–424 and 523–540 were largely exposed and in close spatial proximity at the surface of E2. In contrast, region 436–447, which overlaps with HVR3, was >35 Å away, and estimates of buried surface were inconsistent with HVR3 being part of the AR3B binding interface. High-throughput structural analysis of HCV quasispecies could facilitate the development of novel vaccines that target conserved structural features of HCV envelope and elicit neutralizing antibody responses that are less vulnerable to viral escape.     

Aetiological association of virus like particles with hepatitis C

Isolation of virus-like particles from sera of anti-HCV positive patients and their ultrastructural characterization are reported. Particles were identified in sera of 5 out of 6 patients tested. Immunoelectron microscopy assay revealed small aggregates of viral particles. Size and morphological criteria suggest that these particles can be classified as Togaviridae.     

Mutational analysis of the hypervariable region of hepatitis e virus reveals its involvement in the efficiency of viral RNA replication

The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in ORF1 that tolerates small deletions with respect to infectivity. To further investigate the role of the HVR in HEV replication, we constructed a panel of mutants with overlapping deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype 1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA replication in Huh7 cells. We found that the replication levels of the HVR deletion mutants were markedly reduced in Huh7 cells, suggesting a role of the HVR in viral replication efficiency. To further verify the results, we constructed HVR deletion mutants by using a genetically divergent, nonmammalian avian HEV, and similar effects on viral replication efficiency were observed when the avian HEV mutants were tested in LMH cells. Furthermore, the impact of complete HVR deletion on virus infectivity was tested in chickens, using an avian HEV mutant with a complete HVR deletion. Although the deletion mutant was still replication competent in LMH cells, the complete HVR deletion resulted in a loss of avian HEV infectivity in chickens. Since the HVR exhibits extensive variations in sequence and length among different HEV genotypes, we further examined the interchangeability of HVRs and demonstrated that HVR sequences are functionally exchangeable between HEV genotypes with regard to viral replication and infectivity in vitro, although genotype-specific HVR differences in replication efficiency were observed. The results showed that although the HVR tolerates small deletions with regard to infectivity, it may interact with viral and host factors to modulate the efficiency of HEV replication.     

Sequence evolution of the hypervariable region in the putative envelope region E2/NS1 of hepatitis C virus is correlated with specific humoral immune responses.

Sequence evolution of the hypervariable region 1 (HVR1) in the N terminus of E2/NS1 of hepatitis C virus (HCV) was studied retrospectively in six chimpanzees inoculated with the same genotype 1b strain, containing a unique predominant HVR1 sequence. Immediately after inoculation, all animals contained the same HVR predominant sequence. Two animals developed an acute self-limiting infection. Anti-HVR1 immunoglobulin G (IgG) was produced 40 to 60 days after inoculation and rapidly disappeared after normalization of transaminases. Another chimpanzee, previously infected with human immunodeficiency virus type 1, showed a delayed response to HVR1 epitopes after superinfection with HCV. No sequence variation of HVR1 was observed in these two animals during the transient viremia in the acute phase. Three other chimpanzees developed a chronic HCV infection. During follow up, sequence evolution occurred in two animals and their anti-HVR1 response remained at varying but detectable levels. The first mutations occurred immediately after the production of anti-HVR1 during the acute phase. However, IgM anti-HVR1 was not detectable. Remarkably, HVR1 sequences remained conserved for more than 6 years in another chronically infected animal. This correlated with the complete absence of detectable anti-HVR1 during this period. Seven years after inoculation, anti-HVR1 IgG was produced and coincided with an HVR1 alteration. These results strongly suggest the involvement of neutralizing anti-HVR antibodies in sequence evolution of HVR1 through immune selection.     

Genetic heterogeneity of hypervariable region 1 of the hepatitis C virus (HCV) genome and sensitivity of HCV to alpha interferon therapy

Hepatitis C virus (HCV) populations persist in vivo as a mixture of heterogeneous viruses called quasispecies. The relationship between the genetic heterogeneity of these variants and their responses to antiviral treatment remains to be elucidated. We have studied 26 virus strains to determine the influence of hypervariable region 1 (HVR-1) of the HCV genome on the effectiveness of alpha interferon (IFN-alpha) therapy. Following PCR amplification, we cloned and sequenced HVR-1. Pretreatment serum samples from 13 individuals with chronic hepatitis C whose virus was subsequently eradicated by treatment were compared with samples from 13 nonresponders matched according to the major factors known to influence the response, i.e., sex, genotype, and pretreatment serum HCV RNA concentration. The degree of virus variation was assessed by analyzing 20 clones per sample and by calculating nucleotide sequence entropy (complexity) and genetic distances (diversity). Types of mutational changes were also determined by calculating nonsynonymous substitutions per nonsynonymous site (K(a)) and synonymous substitutions per synonymous site (K(s)). The paired-comparison analysis of the nucleotide sequence entropy and genetic distance showed no statistical differences between responders and nonresponders. By contrast, nonsynonymous substitutions were more frequent than synonymous substitutions (P 相似文献   

CD81 is required for hepatitis C virus glycoprotein-mediated viral infection

CD81 has been described as a putative receptor for hepatitis C virus (HCV); however, its role in HCV cell entry has not been characterized due to the lack of an efficient cell culture system. We have examined the role of CD81 in HCV glycoprotein-dependent entry by using a recently developed retroviral pseudotyping system. Human immunodeficiency virus (HIV) pseudotypes bearing HCV E1E2 glycoproteins show a restricted tropism for human liver cell lines. Although all of the permissive cell lines express CD81, CD81 expression alone is not sufficient to allow viral entry. CD81 is required for HIV-HCV pseudotype infection since (i) a monoclonal antibody specific for CD81 inhibited infection of susceptible target cells and (ii) silencing of CD81 expression in Huh-7.5 hepatoma cells by small interfering RNAs inhibited HIV-HCV pseudotype infection. Furthermore, expression of CD81 in human liver cells that were previously resistant to infection, HepG2 and HH29, conferred permissivity of HCV pseudotype infection. The characterization of chimeric CD9/CD81 molecules confirmed that the large extracellular loop of CD81 is a determinant for viral entry. These data suggest a functional role for CD81 as a coreceptor for HCV glycoprotein-dependent viral cell entry.