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Journal of Virology, August 2009, p. 7375-7383, Vol. 83, No. 15
0022-538X/09/$08.00+0     doi:10.1128/JVI.00331-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cryo-Electron Microscopy Structure of Adenovirus Type 2 Temperature-Sensitive Mutant 1 Reveals Insight into the Cell Entry Defect ^,

Mariena Silvestry,¹ Steffen Lindert,^1,2 Jason G. Smith,³ Oana Maier,⁴ Christopher M. Wiethoff,⁴ Glen R. Nemerow,³ and Phoebe L. Stewart^1*

Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 2215 Garland Avenue, Nashville, Tennessee 37232,¹ Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37212,² Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, IMM-19, La Jolla, California 92037,³ Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Loyola University Chicago, Building 105, 2160 South First Avenue, Maywood, Illinois 60153⁴

Received 13 February 2009/ Accepted 11 May 2009

The structure of the adenovirus type 2 temperature-sensitive mutant 1 (Ad2ts1) was determined to a resolution of 10 Å by cryo-electron microscopy single-particle reconstruction. Ad2ts1 was prepared at a nonpermissive temperature and contains the precursor forms of the capsid proteins IIIa, VI, and VIII; the core proteins VII, X (mu), and terminal protein (TP); and the L1-52K protein. Cell entry studies have shown that although Ad2ts1 can bind the coxsackievirus and Ad receptor and undergo internalization via v integrins, this mutant does not escape from the early endosome and is targeted for degradation. Comparison of the Ad2ts1 structure to that of mature Ad indicates that Ad2ts1 has a different core architecture. The Ad2ts1 core is closely associated with the icosahedral capsid, a connection which may be mediated by preproteins IIIa and VI. Density within hexon cavities is assigned to preprotein VI, and membrane disruption assays show that hexon shields the lytic activity of both the mature and precursor forms of protein VI. The internal surface of the penton base in Ad2ts1 appears to be anchored to the core by interactions with preprotein IIIa. Our structural analyses suggest that these connections to the core inhibit the release of the vertex proteins and lead to the cell entry defect of Ad2ts1.

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* Corresponding author. Mailing address: Vanderbilt University Medical Center, Department of Molecular Physiology and Biophysics, 710 Light Hall, 2215 Garland Avenue, Nashville, TN 37232. Phone: (615) 322-7908. Fax: (615) 322-7236. E-mail: phoebe.stewart{at}vanderbilt.edu

Published ahead of print on 20 May 2009.

Supplemental material for this article may be found at http://jvi.asm.org/.

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Journal of Virology, August 2009, p. 7375-7383, Vol. 83, No. 15
0022-538X/09/$08.00+0     doi:10.1128/JVI.00331-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.