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Journal of Virology, November 2008, p. 11129-11139, Vol. 82, No. 22
0022-538X/08/$08.00+0     doi:10.1128/JVI.01060-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Conserved Salt Bridge between the N- and C-Terminal Heptad Repeat Regions of the Human Immunodeficiency Virus Type 1 gp41 Core Structure Is Critical for Virus Entry and Inhibition

Yuxian He,^1,2* Shuwen Liu,³ Jingjing Li,¹ Hong Lu,¹ Zhi Qi,¹ Zhonghua Liu,² Asim K. Debnath,¹ and Shibo Jiang^1,3

Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York 10021,¹ Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China,² School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China³

Received 20 May 2008/ Accepted 28 August 2008

The fusogenic human immunodeficiency virus type 1 (HIV-1) gp41 core structure is a stable six-helix bundle formed by its N- and C-terminal heptad repeat sequences. Notably, the negatively charged residue Asp⁶³² located at the pocket-binding motif in the C-terminal heptad repeat interacts with the positively charged residue Lys⁵⁷⁴ in the pocket formation region of the N-terminal heptad repeat to form a salt bridge. We previously demonstrated that the residue Lys⁵⁷⁴ plays an essential role in six-helix bundle formation and virus infectivity and is a key determinant of the target for anti-HIV fusion inhibitors. In this study, the functionality of residue Asp⁶³² has been specifically characterized by mutational analysis and biophysical approaches. We show that Asp⁶³² substitutions with positively charged residues (D632K and D632R) or a hydrophobic residue (D632V) could completely abolish Env-mediated viral entry, while a protein with a conserved substitution (D632E) retained its activity. Similar to the Lys⁵⁷⁴ mutations, nonconserved substitutions of Asp⁶³² also severely impaired the -helicity, stability, and conformation of six-helix bundles as shown by N36 and C34 peptides as a model system. Furthermore, nonconserved substitutions of Asp⁶³² significantly reduced the potency of C34 to sequestrate six-helix bundle formation and to inhibit HIV-1-mediated cell-cell fusion and infection, suggesting its importance for designing antiviral fusion inhibitors. Taken together, these data suggest that the salt bridge between the N- and C-terminal heptad repeat regions of the fusion-active HIV-1 gp41 core structure is critical for viral entry and inhibition.

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* Corresponding author. Mailing address: Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67th Street, New York, NY 10021. Phone: (212) 570-3366. Fax: (212) 570-3099. E-mail: yhe{at}nybloodcenter.org

Published ahead of print on 3 September 2008.

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Journal of Virology, November 2008, p. 11129-11139, Vol. 82, No. 22
0022-538X/08/$08.00+0     doi:10.1128/JVI.01060-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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