Fusion of host and viral membranes is a critical step during infection by membrane-bound viruses. The HIV-1 glycoproteins gp120 (surface subunit) and gp41 (fusion subunit) represent the prototypic system for studying this process; in the prevailing model, the gp41 ectodomain forms a trimeric six-helix bundle that constitutes a critical intermediate and provides the energetic driving for overcoming barriers associated with membrane fusion. However, most structural studies of gp41 variants have been performed either on ectodomain constructs lacking one or more of membrane-associated segments (the fusion peptide, FP, the membrane-proximal external region, MPER, and transmembrane domain, TM), or on variants consisting of these isolated segments alone without the ectodomain. Several recent reports have suggested that the HIV-1 ectodomain, as well as larger construct containing the membrane-bound segments, dissociate from a trimer to a monomer in detergent micelles. Here we compare the properties of a series of gp41 variants to delineate the roles of the ectodomain, FP, and MPER and TM, all in membrane-mimicking environments. We find that these proteins are prone to formation of a monomer in detergent micelles. In one case, we observed exclusive monomer formation at pH 4 but conditional trimerization at pH 7 even at low micromolar (~5 μM) protein concentrations. Liposome release assays demonstrate that these gp41-related proteins have the capacity to induce content leakage, but that this activity is also strongly modulated by pH with much higher activity at pH 4. Circular dichroism, NMR, and binding assays with antibodies specific to the MPER provide insight into the structural and functional roles of the FP, MPER, and TM and their effect on structure within the larger context of the fusion subunit.

Fusion of host and viral membranes is a crucial step during infection by enveloped viruses. In the structurally-defined “class I″ viral glycoproteins, the formation of a highly stable α-helical bundle by the ectodomain of the fusion subunit (e.g., GP2 for Marburg virus, MARV) is postulated to provide the energetic driving force to overcome barriers associated with membrane fusion. Upon cell binding, the fusion subunit is proposed to form an extended intermediate that bridges both the viral and host membranes, and collapse of this extended intermediate brings the two membranes into proximity. While there is much high-resolution structural data available for prefusion and post-fusion structures of viral glycoproteins, little information is available about intermediate conformations especially in the context of the fusion loop/peptide (FL or FP) and membrane-proximal external region (MPER)/transmembrane (TM) segments. We present structural and functional studies on segments of MARV GP2 that encompass the FL and MPER/TM in detergent micelles and lipid bicelles. A protein that contains most elements of GP2 (“MGP2-full”) is α-helical in membrane-mimicking environments and has pH-dependent membrane lytic activity. MGP2-full is monomeric under such conditions, contrasting with the trimeric species that has been described previously for MARV GP2 ectodomain in aqueous buffer. Variants of MARV GP2 containing the N- and C-terminal halves (“MGP2-FNL” and “MGP2-CMT”, respectively) have similar properties. This work provides novel insight into conformational and membrane-perturbing properties of the MARV fusion subunit and how they may relate to viral membrane fusion.