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J Mol Biol. 2013 Jun 12;425(11):1899-1914. doi: 10.1016/j.jmb.2013.02.025. Epub 2013 Feb 28.

Glycan shifting on hepatitis C virus (HCV) E2 glycoprotein is a mechanism for escape from broadly neutralizing antibodies.

Author information

1
Department of Infectious Diseases, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
2
Department of Structural Biology, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
3
Department of Antibody Engineering, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
4
Department of Protein Chemistry, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
5
Department of Bioinformatics, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
6
Therapeutic Antibody Group, MRC Technology, London NW7 1AD, UK.
7
MRC-University of Glasgow Centre for Virus Research, Glasgow G11 5JR, UK.
8
Department of Structural Biology, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA; Department of Antibody Engineering, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address: eigenbrot.c@gene.com.
9
Department of Infectious Diseases, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address: kapadia.sharookh@gene.com.

Abstract

Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma. Glycan shielding has been proposed to be a mechanism by which HCV masks broadly neutralizing epitopes on its viral glycoproteins. However, the role of altered glycosylation in HCV resistance to broadly neutralizing antibodies is not fully understood. Here, we have generated potent HCV neutralizing antibodies hu5B3.v3 and MRCT10.v362 that, similar to the previously described AP33 and HCV1, bind to a highly conserved linear epitope on E2. We utilize a combination of in vitro resistance selections using the cell culture infectious HCV and structural analyses to identify mechanisms of HCV resistance to hu5B3.v3 and MRCT10.v362. Ultra deep sequencing from in vitro HCV resistance selection studies identified resistance mutations at asparagine N417 (N417S, N417T and N417G) as early as 5days post treatment. Comparison of the glycosylation status of soluble versions of the E2 glycoprotein containing the respective resistance mutations revealed a glycosylation shift from N417 to N415 in the N417S and N417T E2 proteins. The N417G E2 variant was glycosylated neither at residue 415 nor at residue 417 and remained sensitive to MRCT10.v362. Structural analyses of the E2 epitope bound to hu5B3.v3 Fab and MRCT10.v362 Fab using X-ray crystallography confirmed that residue N415 is buried within the antibody-peptide interface. Thus, in addition to previously described mutations at N415 that abrogate the β-hairpin structure of this E2 linear epitope, we identify a second escape mechanism, termed glycan shifting, that decreases the efficacy of broadly neutralizing HCV antibodies.

PMID:
23458406
DOI:
10.1016/j.jmb.2013.02.025
[Indexed for MEDLINE]
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