Delineation of regions important for heteromeric association of hepatitis C virus E1 and E2.
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.
Yi
M
M
Department of Molecular Biology, Cancer Research Institute, Kanazawa, Japan.
Nakamoto
Y
Y
Kaneko
S
S
Yamashita
T
T
Murakami
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004279:DNA, Viral; D006031:Glycosylation; D016174:Hepacivirus / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins / Q000378:metabolism*
D004279:DNA, Viral; C087035:E1 protein, Hepatitis C virus; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus