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Journal of Virology, June 2009, p. 5581-5591, Vol. 83, No. 11
0022-538X/09/$08.00+0     doi:10.1128/JVI.02653-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Helices 2 and 3 of West Nile Virus Capsid Protein Are Dispensable for Assembly of Infectious Virions

Petra Schlick,^1*, Christian Taucher,^2, Beate Schittl,^1, Janina L. Tran,^1, Regina M. Kofler,^2,¶ Wolfgang Schueler,¹ Alexander von Gabain,¹ Andreas Meinke,¹ and Christian W. Mandl^2,||

Intercell AG, Campus Vienna Biocenter, Vienna, Austria,¹ Clinical Institute of Virology, Medical University of Vienna, Vienna, Austria²

Received 24 December 2008/ Accepted 13 March 2009

The internal hydrophobic sequence within the flaviviral capsid protein (protein C) plays an important role in the assembly of infectious virions. Here, this sequence was analyzed in a West Nile virus lineage I isolate (crow V76/1). An infectious cDNA clone was constructed and used to introduce deletions into the internal hydrophobic domain which comprises helix 2 and part of the loop intervening helices 2 and 3. In total, nine capsid deletion mutants (4 to 14 amino acids long) were constructed and tested for virus viability. Some of the short deletions did not significantly affect growth in cell culture, whereas larger deletions removing almost the entire hydrophobic region significantly impaired viral growth. Efficient growth of the majority of mutants could, however, be restored by the acquisition of second-site mutations. In most cases, these resuscitating mutations were point mutations within protein C changing individual amino acids into more hydrophobic residues, reminiscent of what had been observed previously for another flavivirus, tick-borne encephalitis virus. However, we also identified viable spontaneous pseudorevertants with more than one-third of the capsid protein removed, i.e., 36 or 37 of a total of 105 residues, including all of helix 3 and a hydrophilic segment connecting 3 and 4. These large deletions are predicted to induce formation of large, predominantly hydrophobic fusion helices which may substitute for the loss of the internal hydrophobic domain, underlining the unrivaled structural and functional flexibility of protein C.

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* Corresponding author. Mailing address: Intercell AG, Campus Vienna Biocenter 3, A-1030 Vienna, Austria. Phone: 43-1-20620-1305. Fax: 43-1-20620-81305. E-mail: pschlick{at}intercell.com

Published ahead of print on 18 March 2009.

These authors contributed equally to the study.

Present address: Institute of Virology, Helmholtz Center Munich, Neuherberg, Germany.

Present address: Veterinary Centre Departments of the Municipal District Office, Vienna, Austria.

^¶ Present address: Lambda GmBH, Freistadt, Austria.

^|| Present address: Novartis Vaccines and Diagnostics, Inc., Cambridge, MA.

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Journal of Virology, June 2009, p. 5581-5591, Vol. 83, No. 11
0022-538X/09/$08.00+0     doi:10.1128/JVI.02653-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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