Abstract
Oligomerization of viral envelope proteins is essential to control virus assembly and fusion. The transmembrane domains (TMDs) of hepatitis C virus envelope glycoproteins E1 and E2 have been shown to play multiple functions during the biogenesis of E1E2 heterodimer. This makes them very unique among known transmembrane sequences. In this report, we used alanine scanning insertion mutagenesis in the TMDs of E1 and E2 to examine their role in the assembly of E1E2 heterodimer. Alanine insertion within the center of the TMDs of E1 or E2 or in the N-terminal part of the TMD of E1 dramatically reduced heterodimerization, demonstrating the essential role played by these domains in the assembly of hepatitis C virus envelope glycoproteins. To better understand the alanine scanning data obtained for the TMD of E1 which contains GXXXG motifs, we analyzed by circular dichroism and nuclear magnetic resonance the three-dimensional structure of the E1-(350-370) peptide encompassing the N-terminal sequence of the TMD of E1 involved in heterodimerization. Alanine scanning results and the three-dimensional molecular model we obtained provide the first framework for a molecular level understanding of the mechanism of hepatitis C virus envelope glycoprotein heterodimerization.
Highlights
After their synthesis and integration into the membrane, a large number of membrane proteins associate and form homoor hetero-oligomeric complexes with new functions
To better understand the alanine scanning data obtained for the transmembrane domains (TMDs) of E1 which contains GXXXG motifs, we analyzed by circular dichroism and nuclear magnetic resonance the three-dimensional structure of the E1-(350 –370) peptide encompassing the N-terminal sequence of the TMD of E1 involved in heterodimerization
To confirm the role played by the TMDs of hepatitis C virus (HCV) envelope glycoproteins in heterodimerization, we used alanine scanning insertion mutagenesis, a technique which has been shown to disrupt helix-helix interaction in a membrane environment [19, 20]
Summary
Cell Culture—The HepG2, CV-1, and 143B (thymidine kinase-deficient) cell lines were obtained from the American Type Culture Collection, Rockville, MD. A plasmid expressing the chimeric protein CD4-E1A358Ј was constructed as described [16] This plasmid contains the sequence of the signal peptide of CD4, followed by the sequence of the ectodomain of CD4 in fusion with the sequence encoding the C-terminal 37 amino acids of E1 with an alanine inserted at position 358Ј. For in vivo labeling of glycan moieties, HepG2 cells were infected with the appropriate vaccinia virus recombinants and pulse-labeled for 30 min with [2-3H]mannose (3.7 ϫ 106 Bq/ml; Amersham Pharmacia Biotech) in ␣-minimum essential medium containing 0.5 mM glucose and 10% dialyzed fetal bovine serum. The numbering of this synthetic peptide (denoted E1-(350 –370)) refers to the genotype 1a and its sequence (GAHWGVLAGIAYFSMVGNWAK-NH2) is identical to that of an infectious HCV cDNA clone recently published [33] (EMBL access number: AF009606). The secondary structure elements and Ramachandran plots were analyzed according to the Kabsch-Sander definition rules, as incorporated in the program PROCHEK-NMR [41]
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