The Fusion Activity of HIV-1 gp41 Depends on Interhelical Interactions

Infection by human immunodeficiency virus type I requires the fusogenic activity of gp41, the transmembrane subunit of the viral envelope protein. Crystallographic studies have revealed that fusion-active gp41 is a "trimer-of-hairpins" in which three central N-terminal helices form a trimeric coiled coil surrounded by three antiparallel C-terminal helices. This structure is stabilized primarily by hydrophobic, interhelical interactions, and several critical contacts are made between residues that form a deep cavity in the N-terminal trimer and the C-helix residues that pack into this cavity. In addition, the trimer-of-hairpins structure has an extensive network of hydrogen bonds within a conserved glutamine-rich layer of poorly understood function. Formation of the trimer-of-hairpins structure is thought to directly force the viral and target membranes together, resulting in membrane fusion and viral entry. We test this hypothesis by constructing four series of gp41 mutants with disrupted interactions between the N- and C-helices. Notably, in the three series containing mutations within the cavity, gp41 activity correlates well with the stability of the N-C interhelical interaction. In contrast, a fourth series of mutants involving the glutamine layer residue Gln-653 show fusion defects even though the stability of the hairpin is close to wild-type. These results provide evidence that gp41 hairpin stability is critical for mediating fusion and suggest a novel role for the glutamine layer in gp41 function

Biophysical and mutational analysis of gp41-mediated membrane fusion and its inhibition

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005.Includes bibliographical references.The experiments described in this thesis were designed to elucidate the manner in which the HIV-1 envelope protein (Env) initiates infection of host cells, and to develop inhibitors of viral entry. Env comprises two non-covalently attached subunits, gpl20 and gp41, that associate as a trimer on the virion surface. Once gp 120 contacts the target cell, gp41 undergoes extensive conformational changes to mediate fusion of viral and cellular membranes. First, a short hydrophobic stretch of residues at the gp4 1 N-terminus insert into the target membrane, anchoring the protein in both viral and cellular membranes. This 'prehairpin' intermediate structure exposes an N-terminal a-helical coiled coil that is the target of promising antiviral peptides and small molecules. A previously unstudied region of N-terminal trimer was stabilized by fusion to a trimeric scaffold peptide and biophysically characterized (Chapter 2). This hybrid peptide itself potently inhibited HIV fusion, and the basis for this inhibition was assessed by studying mutant molecules. Efforts to use this peptide as an immunogen to elicit anti-gp41 antibodies are also outlined. Similar design strategies may be useful in developing N-terminal peptide inhibitors and HIV vaccine candidates, and in screening for antiviral molecules that bind to this region. As fusion progresses, the prehairpin intermediate resolves into a hairpin structure. This critical transition involves interaction of the N-terminal coiled coil with the gp41 C-terminal region. Evidence suggests that this N-C association provides the energy necessary to promote juxtaposition and merging of viral and cellular membranes.(cont.) This hypothesis was tested using a biophysical and cell biological approach (Chapter 3), in which residues essential for this transition were mutated and analyzed. These studies confirm the hypothesis and highlight the importance of specific hydrophobic and polar contacts between the N- and C- terminal gp41 regions. This work contributes to a detailed understanding of the gp41 fusion machinery; furthermore, it shows that such knowledge can be used to design effective viral entry inhibitors. Finally, Chapter 4 places this work in the context of a broad overview of current drug and vaccine developments, and addresses some of the significant challenges that confront HIV researchers.by Tara R. Suntoke.Ph.D

Effects of Genital Ulcer Disease and Herpes Simplex Virus Type 2 on the Efficacy of Male Circumcision for HIV Prevention: Analyses from the Rakai Trials

Ron Gray and colleagues analyze data from two circumcision trials in Uganda to assess how HSV-2 status and genital ulcer disease affect the procedure's ability to reduce HIV infection