Infection by enveloped viruses commences with a membrane fusion event between the viral envelope and target cell membrane. For many viruses, fusion is mediated by viral fusion proteins. Viral fusion proteins of enveloped viruses are found in three classes. Within class I, influenza HA and HIV-1 Env are well-studied examples. One distinguishing feature is that they contain an “external” helical fusion peptide (FP) that is exposed following a proteolytic cleavage event. In contrast, class II and III fusion proteins contain distinct internal fusion loops. This project focuses on the coronavirus (CoV) S protein, which is formally a class I fusion protein, but is distinct in that it has a novel “internal” FP that is exposed by a proximal cleavage event to initiate its interaction with the host membrane. This novel FP possesses a great deal of inherent flexibility in its ability to be cleaved by several proteases that are present in distinct microenvironments and cell types. This flexibility is exploited by coronaviruses to enable diverse entry pathways and it is also a primary determinant of viral tropism.

There is little mechanistic understanding of how this FP functions. In a broad sense, we know that upon cleavage at the S2’ position, the S protein takes an extended conformation with an exposed FP. This FP inserts into the target membrane, and then the S protein undergoes another conformational change that pulls the membranes together. The strength of the FP-mediated anchoring in the target membrane limits the pulling force that can be applied before the FP anchors are detached from the target membrane. Therefore, understanding this step of the fusion process, namely the interaction of the FP with the target membrane and what biochemical and biophysical aspects influence it, is key to understanding viral fusion and how to stop it. Towards this goal, our labs have established the critical role for calcium ions in inducing structure in the FP through interactions with well-conserved charged amino acids within its sequence. We have also shown that the presence of calcium ions correlates with FP membrane penetration, increased lipid ordering in the host membrane, and overall infection. We have also recently shown that other less-conserved amino acids can significantly influence the adhesion strength between the FP and host membrane. Therefore, we hypothesize molecular level interactions between the FP and the host membrane will also be influenced by cholesterol, lipid chemistry, and the FP amino acid sequence, specifically a hydrophobic stretch of amino acids and proximal residues that are predicted to influence the hydrophobic interactions with the membrane.

We aim to understand the relationship between FP sequence and target membrane composition on membrane fusion by using complementary techniques that enable examination of this process across scales, from the fusion peptide to the fusion protein to the whole virus. First, we will quantify, at the single molecule level using atomic force microscopy (AFM), the intermolecular interactions between FPs of varying sequence and membranes of varying composition. Next, using optical tweezers and total internal reflection fluorescence microscopy, we will measure fusion kinetics, rate constants for intermediates, and fusion efficiencies using tailored membrane model systems and whole virion/host cell systems that allow individual and population-level observations. Finally, we will integrate the findings of the first two aims with cell infectivity and cell-cell fusion assays.

This research will enhance fundamental understanding of viral membrane fusion by leveraging expertise in biophysical tools and virology. AFM allows molecular-scale assessment of the interaction forces between the FP and membrane. Optical Tweezer Force Spectroscopy allows molecular-scale assessment of individual forces between fusion proteins and a host membrane. Single-virion tracking measures membrane fusion, captures intermediate states, and probes conditions governing their formation. Integrating these results allows us to correlate molecular scale interactions with the fusion process in new ways that are applicable in many contexts.

These studies will reveal the roles of FP sequence and target membrane composition on viral fusion, informing understanding of viral infection. FPs across the CoV family are well conserved, so new knowledge will be broadly useful. Many viruses contain class-I fusion proteins; this research will also inform their fusion mechanisms. Patterns of amino acid sequences that correlate to enhanced fusion function and pathogenesis may assist in recognizing sequences that are predictive of future pandemic strains. Moreover, knowing what critical host membrane features make it susceptible to infection can assist in predicting cell tropism and factors beyond the receptor that favour viral entry. This data will aid antiviral development, vaccine technology, and diagnostics. Our educational plan creates an online outreach module on virus entry that will be used across our countries to inspire girls to pursue STEM.