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Delineation of regions important for heteromeric association of hepatitis C virus E1 and E2
1Department of Molecular Biology, Cancer Research Institute, Kanazawa, Japan.
Insights
Hepatitis C virus envelope proteins E1 and E2 form a complex, crucial for viral function. Key interactions involve specific residues in E2
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Hepatitis C virus (HCV) causes blood-borne non-A, non-B hepatitis.
- HCV persistence is linked to mutations in the hypervariable region 1 (HVR1) of envelope protein 2 (E2), aiding immune evasion.
- The precise organization of HCV envelope proteins E1 and E2 in their heteromeric complex remains unclear.
Purpose of the Study:
- To investigate the interaction between Hepatitis C virus envelope proteins E1 and E2.
- To map the specific regions and amino acid residues involved in the E1-E2 complex formation.
- To determine the role of glycosylation in the heteromeric complex formation.
Main Methods:
- Far-Western blotting using bacterial recombinant proteins.
- Pull-down assays using mammalian-expressed proteins.
- Site-directed mutagenesis to analyze critical amino acid residues.
Main Results:
- The major E1-interacting site on E2 was mapped to its N-terminal domain (NCD1, aa 415-500).
- HVR1 and HVR2 on E2 are not essential for E1 interaction; conserved sequences flanking HVR2 are significant.
- Specific residues (e.g., 'WHY' at aa 489-491) in E2 are crucial for E1 binding.
- The N-terminal region of E1 is important for binding E2.
- Glycosylation of E1 and E2 is not required for heteromeric complex formation.
Conclusions:
- The E1-E2 heteromeric complex formation is mediated by specific interactions within the N-terminal regions of both proteins.
- Conserved residues in E2's NCD1, particularly 'WHY' (aa 489-491), play a critical role in stabilizing the E1-E2 interaction.
- The study clarifies the molecular basis of HCV envelope protein complexation, independent of glycosylation.
Abstract:
Hepatitis C virus (HCV) is the major causative agent of blood-borne non-A non-B hepatitis. The persistence of HCV infection is believed to reflect escape from the host immunosurveillance system by mutations in hypervariable region 1 (HVR1) of the envelope protein 2 (E2). Two envelope proteins of HCV, E1 and E2, have been reported to form a heteromeric complex but the exact organization of the viral envelope proteins remains uncertain. We examined the interaction of E1 and E2 by far- Western blotting using the bacterial recombinant proteins and also by pull-down assay using mammalian expressed proteins. The major E1-interacting site of E2 was mapped within the N-terminal part of E2 (NCD1) (aa 415 to 500 of the polyprotein). Both HVR1 and HVR2, located at the N-terminal part of E2, were dispensable for the interaction. Although several discontinuous regions within NCD1 seemed to contribute to the strong binding to E1, the highly conserved amino acid sequences flanking HVR2 had the most significant effect. The amino acid residues "WHY" from 489 to 491 of E2 played an especially crucial role since the constructs with the internal deletion or substitution of the residues showed severely impaired E1-binding. The N-terminal part of E1 is important for the E2-binding as determined by far-Western blotting using the mammalian- and bacterial-expressed E2 proteins as probes. The mammalian-expressed, glycosylated forms of the E1 and E2 proteins exhibited E1-E2 binding activities similar to those of the bacterial-expressed, nonglycosylated forms in pull-down assays, suggesting that glycosylation is not prerequisite for the heteromeric complex formation of E1 and E2.