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A critical step in the folding of influenza virus HA determined with a novel folding assay

Nature Structural & Molecular Biology volume 12, pages 258–263 (2005)Cite this article

Abstract

Most principles of protein folding emerged from refolding studies in vitro on small, soluble proteins, because large ones tend to misfold and aggregate. We developed a folding assay allowing the study of large proteins in detergent such that the extent of cellular assistance required for proper folding can be determined. We identified a critical step in the in vivo folding pathway of influenza virus hemagglutinin (HA). Only the formation of the first few disulfides in the top domain of HA required the intact endoplasmic reticulum. After that, HA proceeded to fold efficiently in a very dilute solution, despite its size and complexity. This study paves the way for detailed structural analyses during the folding of complex proteins.

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Figure 1: HA folds in a diluted detergent cell lysate.
Figure 2: In vitro folded HA acquires the correct epitopes.
Figure 3: Reduced HA cannot fold in a diluted lysate.
Figure 5: The bottleneck of HA folding: formation of the first few disulfides.
Figure 4: Reduced HA can fold in vivo in the absence of CNX or CRT binding.
Figure 6: Rescue of reduced HA in the absence of calnexin binding.

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Acknowledgements

We thank H. Tabak, A. Ora and A. Azuaga-Fortes for comments, discussions and critical reading of the manuscript, and B. Kleizen, M. Molinari, A. Helenius and members of the Braakman lab for fruitful discussions. We thank J. Smit for preparing the cartoon of HA (Fig. 2a). The work was supported by a grant from Telethon Foundation, Italy (M.C.M.), a European Union Marie Curie Research Training grant (M.C.M.), and Netherlands Organization for Scientific Research/Chemical Sciences (I.M.L. and I.B.).

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  1. M Claudia Maggioni and I Marije Liscaljet: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Chemistry, Utrecht University, Padualaan 8, Utrecht, 3584 CH, The Netherlands

    M Claudia Maggioni, I Marije Liscaljet & Ineke Braakman

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  1. M Claudia Maggioni
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Correspondence to Ineke Braakman.

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Maggioni, M., Liscaljet, I. & Braakman, I. A critical step in the folding of influenza virus HA determined with a novel folding assay. Nat Struct Mol Biol 12, 258–263 (2005). https://doi.org/10.1038/nsmb897

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