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J. Biol. Chem., Vol. 280, Issue 8, 6792-6801, February 25, 2005

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Structural and Dynamics Studies of the D54A Mutant of Human T Cell Leukemia Virus-1 Capsid Protein^*

From the ^aHoward Hughes Medical Institute, New York, New York, the ^dLaboratory of Biophysical Chemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, the ^fDepartment of Biochemistry and Molecular Biophysics and Department of Microbiology, Columbia University, College of Physicians and Surgeons, and the ⁱDepartment of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York 11794-5222

The human T cell leukemia virus and the human immunodeficiency virus share a highly conserved, predominantly helical two-domain mature capsid (CA) protein structure with an N-terminal -hairpin. Despite overall structural similarity, differences exist in the backbone dynamic properties of the CA N-terminal domain. Since studies with other retroviruses suggest that the -hairpin is critical for formation of a CA-CA interface, we investigated the functional role of the human T cell leukemia virus -hairpin by disrupting the salt bridge between Pro¹ and Asp⁵⁴ that stabilizes the -hairpin. NMR ¹⁵N relaxation data were used to characterize the backbone dynamics of the D54A mutant in the context of the N-terminal domains, compared with the wild-type counterpart. Moreover, the effect of the mutation on proteolytic processing and release of virus-like particles (VLPs) from human cells in culture was determined. Conformational and dynamic changes resulting from the mutation were detected by NMR spectroscopy. The mutation also altered the conformation of mature CA in cells and VLPs, as reflected by differential antibody recognition of the wild-type and mutated CA proteins. In contrast, the mutation did not detectably affect antibody recognition of the CA protein precursor or release of VLPs assembled by the precursor, consistent with the fact that the hairpin cannot form in the precursor molecule. The particle morphology and size were not detectably affected. The results indicate that the -hairpin contributes to the overall structure of the mature CA protein and suggest that differences in the backbone dynamics of the -hairpin contribute to mature CA structure, possibly introducing flexibility into interface formation during proteolytic maturation.

Received for publication, July 19, 2004 , and in revised form, November 23, 2004.

^* This work was supported in part by National Institutes of Health (NIH) Grant GM 48294 (to C. A. C.) and facilitated by the infrastructure and resources provided by the NIH-funded center for AIDS research program P30 AI 42848. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The ¹H^N, ¹⁵N, ¹³C, ¹H, ¹³C', ¹³C, and ¹H chemical shifts for the D54A mutant have been deposited in the BioMagnetic Resonance Bank (www.bmrb.wisc.edu) under BMRB accession code 5334.

^b A Howard Hughes Medical Institute (HHMI) associate.

^c These authors contributed equally to this work.

^e Present address: Center for Eukaryotic Structural Genomics, Dept. of Biochemistry, University of Wisconsin, Madison, WI 53706-1544.

^g An HHMI principal investigator.

^h To whom correspondence may be addressed. Tel.: 301-402-3029; Fax: 301-402-3405; E-mail: nico{at}helix.nih.gov. j To whom correspondence may be addressed. Tel.: 631-632-8801; Fax: 631-632-9797; E-mail: ccarter{at}ms.cc.sunysb.edu.

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