HIV-1 Membrane Fusion Mechanism:  Structural Studies of the Interactions between Biologically-Active Peptides from gp41

Abstract

Two synthetic peptides corresponding to sequences in HIV-1LAI gp41, (aa558-595) and T20 (aa 643-678), are strong inhibitors of HIV-1 viral fusion, having EC50 values of 1 microgram/mL and 1 ng/mL, respectively. Previous work suggested that T21 forms a coiled-coil structure in PBS solution, while T20 is primarily nonhelical, and that the inhibitory action of these peptides occurs after the interaction between the viral gp120 protein and the cellular CD4 receptor [Wild, C.T., Shugars, D. C., Greenwell, T. K., McDanal, C. B., Matthews, T. J. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9770 and references therein]. The current study uses sedimentation equilibrium (SE), circular dichroism (CD), and viral-fusion assays to quantitatively investigate peptide structure and peptide-peptide interactions. SE analyses of T21 (1-100 microM) indicate that the peptide self associates via a monomer/dimer/tetramer equilibrium; in addition, T20 is monomeric in the range of 1-10 microM and exhibits a complicated monomer/tetramer equilibrium between 20 and 100 microM. Singular value decomposition analyses of the CD spectra of T21 and T20 indicate that the helical content of these peptides in PBS solution is 90% and 20%, respectively. A structural interaction between the two peptides is detected by CD at several concentration ratios of T20:T21. These experiments emphasize that T20 interacts specifically with the tetrameric form of T21. Truncated forms of T20 also exhibit structural interactions with T21 at varying concentration ratios. The ability of T20 and the truncated peptides to interact structurally with tetrameric T21 correlates with antiviral activity. Implications of these findings are discussed in terms of proposed mechanisms of membrane fusion inhibition and the structural changes which occur in gp41 during membrane fusion.

Role of the Envelope Genetic Context in the Development of Enfuvirtide Resistance in Human Immunodeficiency Virus Type 1-Infected Patients

“…ENF is a 36-amino-acid peptide derived from the sequence of HR2 (24,44). ENF binding to HR1 is thought to compete with the folding of the HR2 domain onto HR1, thus preventing Env-mediated membrane fusion (1,5,17).…”

“…Similar to results with other antiviral agents, however, ENF-resistant HIV-1 variants may emerge under the selective pressure of ENF (26,32,39,42) whenever the treatment fails to completely suppress viral replication in vivo. Understanding the determinants of, and the constraints to, the development of resistance to ENF is essential for the optimization of the clinical use of this new class of inhibitors.ENF is a 36-amino-acid peptide derived from the sequence of HR2 (24,44). ENF binding to HR1 is thought to compete with the folding of the HR2 domain onto HR1, thus preventing Env-mediated membrane fusion (1,5,17).…”

C-terminal Octylation Rescues an Inactive T20 Mutant

“…Thus, we investigated whether octylation altered the secondary structure of the wild type and mutant peptides by means of CD. HIV-1 gp41-derived T20 has been reported to lack a well defined structure in aqueous environment (21). However, a 13-residue C-terminal fragment of HIV-1 gp41-derived T20 was recently shown to form a stable monomeric 3(10) helix by NMR (29).…”

“…However, the understanding of its detailed mechanism of inhibition has been hampered by the following. (i) T20 lacks the residues that bind to the coiled coil C-terminal cavity; (ii) the known crystal and solution structures of fragments of HIV or SIV gp41 ectodomains do not fully include the regions corresponding either to T20 or to its putative binding site at the N terminus of the coiled coil; and (iii) in addition to a primary target site within the N-terminal heptad repeat (21), T20 has been postulated to bind to a second site in gp41, presumably a non-specified region at the C terminus of the ectodomain close to the viral membrane (22,23). Indeed, that T20 acts in close proximity to the membrane is supported by the studies of von Laer and co-workers (24).…”

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

“…A major difference, arguably, lies in their level of structural organization. Peptides possess and mimic stable secondary structures 17,48 whereas quaternary protein mimetics possess and mimic stable tertiary or quaternary structures. The gp41 prefusion state is a quaternary structure formed by oligomerization of three copies of gp41.…”