New Biophysical Approaches Reveal the Dynamics and Mechanics of Type I Viral Fusion Machinery and Their Interplay with Membranes

Abstract

Protein-mediated membrane fusion is a highly regulated biological process essential for cellular and organismal functions and infection by enveloped viruses. During viral entry the membrane fusion reaction is catalyzed by specialized protein machinery on the viral surface. These viral fusion proteins undergo a series of dramatic structural changes during membrane fusion where they engage, remodel, and ultimately fuse with the host membrane. The structural and dynamic nature of these conformational changes and their impact on the membranes have long-eluded characterization. Recent advances in structural and biophysical methodologies have enabled researchers to directly observe viral fusion proteins as they carry out their functions during membrane fusion. Here we review the structure and function of type I viral fusion proteins and mechanisms of protein-mediated membrane fusion. We highlight how recent technological advances and new biophysical approaches are providing unprecedented new insight into the membrane fusion reaction.

Keywords: biophysics; dynamics; hemagglutinin; influenza; mechanisms of membrane fusion; membrane fusion; structural mechanics; type I fusion protein; viral fusion.

Figure 1

Architecture of a type I fusion protein. (A) The structures of the influenza hemagglutinin (HA) fusion protein in the pre-fusion (PDB 3HMG) and post-fusion (PDB 1QU1) states highlight the dramatic pH-dependent reorganization that drives the membrane fusion reaction. The pre-fusion state is metastable with respect to the post-fusion state. In the pre-fusion state, the HA1 receptor binding domain (RBD) (shown in light grey volume and blue ribbon) forms a “clamp” interaction with the HA2 fusion subunit, thereby stabilizing the high-energy, “spring-loaded” HA2 fusion domain (highlighted by the HA1-HA2 interface shown in grey). The HA2 N-terminal fusion peptide (FP shown in dark magenta) forms a “hook” within the fusion domain, lashing adjacent protomers together. Once destabilized by low pH, these interactions are lost and the HA reorganizes to the post-fusion state where the N-terminal FP and transmembrane domain (TMD) are colocalized in the newly fused membrane. The post-fusion state is characterized by the trimer of hairpins formed by the two heptad repeat regions (HR1 and HR2). (B) Comparison of the pre-fusion (top) and post-fusion (bottom) structures of diverse type I fusion proteins reveals the conservation of core architectural features including the “clamp” interaction between the RBD and fusion domain and reorganization of the two heptad repeats into a trimer of hairpins. Shown are the pre- and post-fusion structures of the HIV-1 Env (PDB 5FUU and 1I5X), Coronavirus (CoV) S (PDB 5W9J and 6B3O), and Lassa virus glycoprotein complex (GPC) (PDB 5VK2 and 5OMI).

Figure 2

Structural mechanics of influenza-HA-fusion activation. (A) Single molecule FRET (sm-FRET) monitoring of HA during fusion activation shows the transition from the pre-fusion state (1) (high FRET) through an obligate and dynamic intermediate (2) (intermediate FRET and reversible low FRET) to the irreversible post-fusion state (3) (low FRET) (figure modified with permission from Das et al., 2018). (B) Cartoon model describing HA-fusion activation shows the formation of a dynamic intermediate ensemble, as supported by sm-FRET and hydrogen/deuterium-exchange mass spectrometry (HDX-MS). Transitions between states are labeled according to those observed by sm-FRET (panel A) and the dynamic intermediate state is depicted according to the HDX-MS study (panel C). (C) Pulse-labeling HDX-MS reveals the formation of a dynamic intermediate state in fusion-active HA on infectious influenza virions. In the neutral pH pre-fusion state (left—grey envelope) the HA2 B-loop peptide becomes labeled with a moderate level of deuterium as it is a structured loop in the pre-fusion state. After incubation at pH 5.10 for 1 min the B-loop displays three unique HDX states corresponding to the pre-fusion state (grey—moderate level of deuterium exchange), post-fusion helical bundle (blue—very low level of deuterium exchange), and dynamic intermediate (red—high level of deuterium exchange). After continued incubation at low pH the HA2 B-loop transitions monotonically to the post-fusion state (blue).

Figure 3

Dynamic conformational changes in Ebola virus GP during viral entry. (A) sm-FRET monitoring of Ebola GP without the mucin-like domain (GPΔmuc) revealed the GP was highly dynamic under equilibrium conditions and reversibly transitioned between three states: the high-FRET state (H) corresponding to the pre-fusion conformation, intermediate-FRET state (I), and low-FRET state (L). Population FRET histograms show the equilibrium distribution of all observed FRET states. Transition density plots (TDP) (far right) reveal the direct transitions between each FRET state for all observed trajectories. (B) Removal of the glycan cap resulted in lower occupancy of the high-FRET state and increased occupancy of the intermediate-FRET state. The glycan cap (GP_CL) also displayed reduced transitions between all FRET states indicating lowered conformational and structural dynamics. (C) Niemann-Pick C1 (NPC1) receptor binding to GP_CL further biased the equilibrium distribution towards the intermediate-FRET state and reduced transitions between states. Receptor binding did not result in irreversible transitions but rather quenched the conformational dynamics of GP. Figure modified with permission from [105].

Figure 4

Visualizing influenza virus membrane fusion. Intermediates throughout the membrane fusion were visualized by cryo-electron tomography (cryo-ET) and the sequence of membrane remodeling was elucidated. (A) Cryo-ET images highlight key intermediates during membrane fusion including HA bridging (1), membrane pinching (2), and formation of a tightly docked interface (3) (left to right) (scale bar = 50 nm). (B) Cartoon model describing the membrane ultrastructure and sequence of intermediates during influenza viral membrane fusion.