Three Different Functional Microdomains In The Hepatitis C Virus Hypervariable Region1(HVR1)Mediate Entry And Immune Evasion

Hepatitis C virus (HCV), an enveloped single-stranded positive-sense RNA virus in the family Flaviviridae, infects more than180million individuals worldwide and in most cases, patients with acute HCV infection will progress towards chronic infection, and some of them process to liver cirrhosis and hepatocellular carcinoma. HCV genome has a high mutation rate resulting from an error-prone RNA polymerase. These sequence variations are concentrated in some special regions of the genome and the best-characterized hypervariable region of HCV is the27amino acid residues located at the N-terminal part of the envelope protein E2(hypervariable region1, HVR1). HVR1exposes on the surface of viral particles, and contains dominant neutralizing epitopes. Its variation may lead to virus escape from pre-existing neutralizing antibodies. Mutation of HVR1has been proposed to be driven by immune pressure and play an important role in the establishment of persistent infection and disease progression. HVR1is used to be a target for the development of prophylactic vaccine. But from HCV was named in1989, a vaccine is not available presently.HCV infection in hepatic cell disturbs cellular biochemistry, including lipid metabolism and apolipoprotein synthesis. Therefore, moderate to severe hepatic steatosis symptoms, including lipid droplet aggregation in plasma, are typical histopathology features distinctive in HCV chronic infection. HCV is an enveloped RNA virus, which is usually wrapped in vivo with a lipoprotein covering. HCV particles purified from patient serum subjected todensity gradient centrifugation show up in different layers, with each layer exhibiting different levels of infectivity. It is reported that deletion of HVR1reduces the number of low-density virus particles, that is, impaired the virus binding with lipoproteins. Hence, HVR1has important functions also in the association between viral particles and lipoproteins. HVR1is notably responsible for the interaction between HCV envelope glycoproteins and SR-BI and plays an important role in HCV cell entry. Although HVR1is highly variable, its importance in virus entry suggests that structural constraints might be present within this region. In this context, it has been suggested that the variability of HVR1is no random, as its chemicophysical properties and conformation are conserved. Despite these observations, the relevance between the structure and function of HVR1remains to be defined.I. Identification of key residues mediating HCV cell entry within HVR1We used HCVpp model, and investigated27residues within HVR1of H77isolate. Using deletion mutagenesis and function analyses, we found that five residues (A14, G15, K25, Q26and N27) within HVR1of H77isolate play a key role in HCVpp cell entry. Deletion of any a single residue in these five amino acids led to significant loss of pseudoparticle infectivity, while deletion of any a single residue in other regions only led to slight effect on infectivity of pseudoparticles. In this study, binding of envelope mutants to SR-BI directly correlated with the entry efficiency of the corresponding pseudoparticles, suggesting that the five key residues mediate entry predominantly through regulation of interaction between envelope proteins and SR-BI. The importance of these five residues is further supported by the findings that deletion of aa1-13(△1-13) attenuated HCVpp infectivity slightly, whereas deletion of aa16-24(△16-24) did not decrease infectivity of the resulting HCVpp. Furthermore, the amino terminal region of HVR1may regulate the infectivity of the virus and in turn to protect the virus from neutralizing antibodies targeting to the epitope within the carboxyl terminus of HVR1.Ⅱ. Identification of antigenic epitopes within HVR1To identify antigenic epitopes within HVR1, HVR1-specific antibodies were raised by immunizing rabbits with thioredoxin-HVR1peptide fusion proteins. It seems that, the N-terminal13amino acids and middle13amino acids do not contain an epitope. Our data provide evidence that neutralizing epitope in H77HVR1is located in region across aa16-24. This9residues exist in HVR1is dispensable for HCV entry, but play an important role in HCV envelope-heparin binding, and this region is necessary for enhancement of HCV infection by HDL and conferring HCV resistant to neutralizing antibodies targeted to epitope outside of HVR1. The result was further supported by antibody binding assay using sera from mice that received DNA vaccines carrying HBsAg and HVR1fusion gene. Ⅲ. Identification of key region mediating lipoprotein binding within HVR1The concentrated HCVcc were separated by overnight centrifugation through a iodixanol gradients, compared with fractions with higher density, fractions with lower density contained more infective particles, and enhancement by HDL was only restricted to infectivity of HCV viral particles with higher density, but no enhancement to viral particles with low density. We have identified a sole neutralizing epitope comprising aa16-24in HVR1of H77isolate of genotype la, and this region is necessary for enhancement of HCV infection by HDL. We found the populations of△16-24HCVcc were characterized by decrease of particles with low buoyant density ranging and increase of high density particles, and△HVR1exhibited the similar tendency. It demonstrated this neutralizing epitope acts as a key microdomain to modulate the association between viral particles and lipoprotein, and we found HDL could not enhance infectivity of△16-24particles of each density fraction.SummaryOur data showed that HCV HVR1contains three different functional microdomains that cooperate to confer HCV cell entry and immune evasion:The antigenic determinant region (aal6-25) shape a "Lipo-claw" on the surface of viral particle to modulate the association with lipoprotein, and this "Lipo-claw" can bind lipoprotein and promote HCV cell entry by more prone to be recognized by SR-BI receptor. Furthermore, it also can conceal other conserved neutralizing epitopes on the surface of envelope to evade immune response. This "Lipo-claw" rears on the surface of viral particles, and acts as an "immune decoy" to constantly evade immune clearance by its own highly heterogeneity. People are used to targeting HVR1as a whole region. These findings provide novel insights into understanding the exact role of HVR1in mediating HCV cell entry, immune evasion and antibody-mediated neutralization, and will thus contribute to the development of entry inhibitors or a protective vaccine that targets the interaction between HVR1and cell entry factors.