Structural Analysis and Assembly of the HIV-1 Gp41 Amino-Terminal Fusion Peptide and the Pretransmembrane Amphipathic-At-Interface Sequence

Abstract

The amino-terminal region within the HIV-1 gp41 aromatic-rich pretransmembrane domain is an amphipathic-at-interface sequence (AIS). AIS is highly conserved between different viral strains and isolates and recognized by the broadly neutralizing 2F5 antibody. The atomic structure of the native Fab2F5-bound AIS appears to involve a nonhelical extended region and a beta-turn structure. We previously described how an immunogenic complex forms, based on the stereospecific interactions between AIS and the gp41 amino-terminal fusion peptide (FP). Here, we have analyzed the structure generated by these interactions using synthetic hybrids containing AIS and FP sequences connected through flexible tethers. The monoclonal 2F5 antibody recognized FP-AIS hybrid sequences with an apparently higher affinity than the linear AIS. Indeed, these hybrids exhibited a weaker capacity to destabilize membranes than FP alone. A combined structural analysis, including circular dichroism, infrared spectroscopy, and two-dimensional infrared correlation spectroscopy, revealed the existence of specific conformations in FP-AIS hybrids, predominantly involving beta-turns. Thermal denaturation studies indicated that FP stabilizes the nonhelical folded AIS structure. We propose that the assembly of the FP-AIS complex may act as a kinetic trap in halting the capacity of FP to promote fusion.

Induction of Antibodies Specific for Gp41 of HIV-1 by Gene Gun DNA Vaccination

“…Other authors had shown an interaction between MPER and FPPR during infection [18][19][20][21][22]. These findings suggested that the interaction between the MPER and the FPPR during infection may generate a conformation able to induce 2F5/4E10-like neutralising antibodies.…”

“…There may be several reasons for the failure of many of the attempts to induce 2F5/4E10-like neutralising antibodies including our new approach: First, although it has been shown, that peptides derived from the FPPR increase the binding of mAb 2F5 to its epitope in the MPER/ E2 domain [14] and there is an interaction between MPER and FFPPR [18][19][20][21][22], it is still unknown whether this interaction is also required for the induction of 2F5/4E10-like broadly neutralising antibodies. Second, it remains unclear, whether the DNA immunisation generated the correct membrane associated context.…”

“…Interaction of both antibodies with lipids, their long hydrophobic CDR3 loop and cross-reactivity with cardiolipin have been discussed controversially as possible reasons for the failure of all previous immunisation attempts [15][16][17]. Since there is evidence for intramolecular interactions between the MPER and the FPPR during infection and neutralisation [18][19][20][21][22] and a peptide corresponding to the sequence of the FPPR has been shown to increase binding of mAb 2F5 to its epitope [14], it was suggested that the FPPR may be required to generate a conformation which will be able to induce broadly neutralising antibodies of the 2F5 and 4E10.…”

Identification of the LWYIK Motif Located in the Human Immunodeficiency Virus Type 1 Transmembrane gp41 Protein as a Distinct Determinant for Viral Infection

“…On the other hand, this pre-TM region may act in conjunction with other sequences in the gp41 ectodomain to maintain the native and fusion states of the Env. Lorizate et al showed that the fusion peptide and pre-TM sequence can coassemble into a defined complex that acts as a prefusion clasp to restrict fusion peptide-mediated fusion (39,40). The Trp-rich membrane-proximal region and the N-terminal fusion peptide-proximal polar segment were recently shown to act synergistically to form a fusion-competent prefusion gp120-gp41 complex and stabilize the membrane-interactive end of the trimeric hairpin fusion core (2).…”

The Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 4E10 Monoclonal Antibody Is Better Adapted to Membrane-Bound Epitope Recognition and Blocking than 2F5

“…Hydropathy plots calculated according to the Wimley-White (WW) scale (81) actually revealed a fully hydrophobic-at-interface sequence within gp41 MPER, previously termed the pretransmembrane domain (PreTM) (39,50,59,70,71). Thus, the gp41 region spanning the 2F5 and 4E10 epitopes has a specific affinity for the membrane interface, the lipid bilayer region between the high-polarity water phase and the low-polarity hydrocarbon core (79).Subsequent development of WW scale applications, including the simultaneous computation of interfacial affinity and hydrophobic moments (38,58), revealed that the MPER was segmented into two subdomains, a phenomenon that was pro-* Corresponding author. Mailing address:…”