Covalent Trimers of the Internal N-terminal Trimeric Coiled-coil of gp41 and Antibodies Directed against Them Are Potent Inhibitors of HIV Envelope-mediated Cell Fusion

Abstract

(transmembrane subunit of HIV envelope) and gp120 (surface envelope glycoprotein of HIV) (1). Both proteins therefore present highly attractive targets for the development of antiviral agents as well as broadly neutralizing antibodies. HIV Envmediated cell fusion involves a complex series of events. gp120 first binds to CD4 and a chemokine receptor; this triggers conformational changes in the gp120-gp41 complex that lead to the insertion of the gp41 fusion peptide into the target membrane and ultimately to cell fusion (2, 3).The structure of the ectodomain of gp41 (e-gp41) in its fusogenic/post-fusogenic state has been solved by both NMR (4) and crystallography (5-8) and consists of a trimer of hairpins comprising an internal parallel trimeric coiled-coil of N-terminal helices (residues 542-592 of HIV-1 Env (4)) surrounded by antiparallel C-terminal helices (residues 623-663 of HIV-1 Env (4)) (left-hand side of Fig. 1). The formation of the fusogenic/post-fusogenic state of e-gp41 provides the driving force for the apposition of the virus and cell membranes, thereby promoting membrane fusion (2). Prior to the formation of the fusogenic trimer of hairpins, e-gp41 exists in a "prehairpin" intermediate state (2, 9 -12), so-called because the C region of e-gp41 (corresponding to the C-helices in the fusogenic/postfusogenic state) is not yet associated with the internal trimeric coiled-coil of N-helices (Fig. 1, middle). In the prehairpin intermediate state both the internal trimeric coiled-coil (9, 10) and the C region of e-gp41 are accessible to inhibitors (13-15).There are three classes of fusion inhibitors that target the prehairpin intermediate state of e-gp41 (right-hand side of Fig. 1). Class 1 inhibitors (shown in blue at the top right of Fig. 1) are directed against the internal trimeric coiled-coil of N-helices. Examples of class 1 inhibitors include peptides derived from the C-helices such as C34 (residues 628 -661 of HIV-1 Env) 2 and T20 (also referred to as DP178, residues 638 -673 of HIV-1 Env which extends 10 residues beyond the C-terminal end of the C-helix), both of which have IC 50 values in the low nanomolar range (16 -18). T20 is currently in the final stages of * This work was supported by the Intramural AIDS Targeted Antiviral Program of the Office of the Director of the National Institutes of Health (to G. M. C. and C. A. B.). 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.‡ To whom correspondence may be addressed: Laboratory of Chemical Physics, Bldg. 5, Rm. B1-30I, NIDDK, National Institutes of Health, Bethesda, MD 20892-0510. Tel.: 301-496-0782; Fax: 301-496-0825; E-mail: mariusc@intra.niddk.nih.gov. § To whom correspondence may be addressed: Laboratory of Bioorganic Chemistry, Bldg. 8, Rm. 1A-02, NIDDK, National Institutes of Health, Bethesda, MD 20892-0820. Tel.: 301-594-5187; E-mail: cb194k@nih.gov. 1 The...

Conformational Changes in HIV-1 gp41 in the Course of HIV-1 Envelope Glycoprotein-Mediated Fusion and Inactivation

“…In addition to structural information, a wealth of HIV Env-mediated fusion data, which includes inhibition by peptides that mimic the sequences of the N-and C-helical regions (13)(14)(15)(16)(17)(18)(19)(20)(21) and fusion kinetics (22), has lent credence to this model. In the absence of complete structural information, some of the details of the HIV-1 Env-mediated fusion reaction have been inferred from immunochemical, biochemical, and mutagenic analysis.…”

Quaternary protein mimetics of gp41 elicit neutralizing antibodies against HIV fusion‐active intermediate state

“…17,20,21 In a viral infection assay, 3a mimetics representing both C-and N-terminal heptad-repeat regions were substantially more potent inhibitors than the corresponding monomers. Furthermore, truncated protein mimetics 3a-N11 and 3a-C12 were shown to be ineffective viral inhibitors (Figure 4).…”

“…In addition, N-HR is highly conserved among viral isolates whereas C-HR is only moderately conserved; 15 however, both have been targeted for antiviral peptide development. 8,[16][17][18][19][20][21][22] . Peptides such as N36, C34, and T20, which overlaps part of C34, are thought to act as dominant inhibitors by binding to the prehairpin state and forming peptide-gp41 complexes that interfere with transition to the six-helix-bundle postfusion state.…”

“…10,20,[23][24][25][26] First, synthetic peptide antigens representing monomeric gp41 peptides are conformationally unstable. Since stability and bioactive conformation are intimately associated with trimeric quaternary structure, they are difficult to duplicate using monomeric peptides.…”

“…Consequently, attempts to exploit these peptides as immunogens, alone or with additional variable regions, have been unsuccessful. [23][24][25][26] Golding et al 10 and Louis et al 20 showed that antibodies gp41 Protein Mimetics Elicit Neutralizing Antibodies 321 generated against complexed peptides or engineered trimeric peptides based on the N-HR or C-HR domains possess viral neutralizing activity. These results suggest that targeting the bioactive conformations of the N-HR or C-HR domains is a viable vaccine design strategy.…”