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J. Virol. doi:10.1128/JVI.01060-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Conserved Salt-bridge between the N- and C-Terminal Heptad Repeat Regions of HIV-1 gp41 Core Structure Is Critical for Virus Entry and Inhibition

From the Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York10021, USA; Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China

^* To whom correspondence should be addressed. Email: yhe{at}nybloodcenter.org.

	Abstract

The fusogenic HIV-1 gp41 core structure is a stable six-helix bundle formed by its N- and C-terminal heptad-repeat sequences (NHR and CHR). Notably, the negatively charged residue Asp⁶³² located at the pocket-binding motif in the CHR interacts with the positively charged residue Lys⁵⁷⁴ in the pocket-formation region of the NHR to form a salt-bridge. We previously demonstrated that the residue Lys⁵⁷⁴ plays an essential role for the six-helix bundle formation and virus infectivity and is a key determinant of the target for anti-HIV fusion inhibitors. In this report, the functionality of the residue Asp⁶³² has been specifically characterized by mutational analysis and biophysical approaches. We show that substitutions of Asp⁶³² with positively charged residues (D632K and D632R) or hydrophobic residue (D632V) could completely abolish Env-mediated viral entry while conserved substitution (D632E) retained its activity. Similar to the Lys⁵⁷⁴ mutations, non-conserved 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, non-conserved 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.