Evidence for a polytopic form of the E1 envelope glycoprotein of Hepatitis C virus.
The polyprotein precursor of the Hepatitis C virus (HCV) contains multiple membrane-spanning domains that define the membrane topology and subsequent maturation of the viral structural proteins. In order to examine the biogenesis of the E1-E2 heterodimeric complex, we inserted an affinity tag (S-peptide) at specific locations within the envelope glycoproteins. In particular, and based on the prediction that the E1 glycoprotein may be able to assume a polytopic topology containing two membrane-spanning domains, we inserted the affinity tag within a putative cytoplasmic loop of the E1 glycoprotein. The HCV structural polyprotein containing this tag (at amino acids 295/296) was highly expressed and able to form a properly processed and noncovalently associated E1-E2 complex. This complex was bound by murine and conformation-dependent human monoclonal antibodies (MAbs) comparably to the native untagged complex. In addition, MAb recognition was retained upon reconstituting the tagged E1-E2 complex in lipid membrane as topologically constrained proteoliposomes. Our findings are consistent with the model of a topologically flexible E1 glycoprotein that is able to adopt a polytopic form. This form of the E1-E2 complex may be important in the HCV life cycle and in pathogenesis.
Migliaccio
Christopher T
CT
Science Complex Room 221, Montana Biotechnology Center, The University of Montana, Missoula, MT 59812, USA.
Follis
Kathryn E
KE
Matsuura
Yoshiharu
Y
Nunberg
Jack H
JH
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019281:Dimerization; D000939:Epitopes / Q000276:immunology; D016174:Hepacivirus / Q000502:physiology*; D018937:Hepatitis C Antibodies / Q000276:immunology / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011510:Proteolipids / Q000276:immunology / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; C087035:E1 protein, Hepatitis C virus; D000939:Epitopes; D018937:Hepatitis C Antibodies; D011510:Proteolipids; D014759:Viral Envelope Proteins; C020485:proteoliposomes