Summary auto-generated
This study investigates the N-linked glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and their role in forming the E1E2 glycoprotein complex. HCV encodes two envelope glycoproteins, E1 and E2, which are heavily modified by N-linked glycosylation and interact to form a noncovalent heterodimeric complex in the endoplasmic reticulum. The researchers used site-directed mutagenesis to create E1 glycosylation mutants and expressed them in HepG2 cells. They identified that E1 contains five potential glycosylation sites at amino acid positions 196, 209, 234, 305, and 325, but only four are actually utilized. The fifth site (position 325) remains unglycosylated because a proline residue immediately following the glycosylation consensus sequence prevents core glycosylation. Through analysis of complex formation using conformation-sensitive antibodies, the researchers found that glycosylation at position 4 (amino acid 305) is critical for proper E1E2 complex assembly. Mutations at positions 1 and 4 reduced complex formation efficiency, while mutations at positions 2 and 3 had minimal effects. In vitro translation studies revealed that position 4 is only partially glycosylated at 66% efficiency, compared to higher efficiency at other sites.
Key findings
- HCV glycoprotein E1 has five potential N-linked glycosylation sites, but only four are functionally used; the fifth site is not glycosylated due to a proline residue following the consensus sequence
- The glycosylation site at position 4 (amino acid 305) is critical for efficient formation of noncovalent E1E2 heterodimeric complexes
- Position 4 glycosylation occurs at only 66% efficiency compared to positions 1, 2, and 3, which are efficiently glycosylated
- Mutations at glycosylation positions 1 and 4 significantly impair E1E2 complex formation, while mutations at positions 2 and 3 have minimal effects on complex assembly
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Abstract
The hepatitis C virus (HCV) genome encodes two membrane-associated envelope glycoproteins (E1 and E2), which are released from the viral polyprotein precursor by host signal peptidase cleavages. These glycoproteins interact to form a noncovalent heterodimeric complex, which is retained in the endoplasmic reticulum. HCV glycoproteins, E1 and E2, are heavily modified by N-linked glycosylation. A recent study has revealed that upon partial deglycosylation with endoglycosidase H only four of the five potential glycosylation sites of HCV glycoprotein E1 are utilized. In this work, the unused glycosylation site on the E1 glycoprotein was identified and the influence of N-linked glycosylation on the formation of the HCV glycoprotein complex was studied by expressing a panel of E1 glycosylation mutants in HepG2 cells. Each of the five potential N-linked glycosylation sites, located at amino acid positions 196, 209, 234, 305 and 325, respectively, on the HCV polyprotein, was mutated separately as well as in combination with the other sites. Expression of the mutated E1 proteins in HepG2 cells indicated that the fifth glycosylation site is not used for the addition of N-linked oligosaccharides and the Pro immediately following the sequon (Asn-Trp-Ser) precludes core glycosylation. The effect of each mutation on the formation of noncovalent E1E2 complexes was also analysed. As determined with the use of a conformation-sensitive monoclonal antibody, mutations at positions N2 and N3 had no, or only minor, effects on the assembly of the E1E2 complex, whereas a mutation at position N1 and predominantly at position N4 dramatically reduced the efficiency of the formation of noncovalent E1E2 complexes.