Wednesday, January 15, 2025
1/15/2025
PROJECT SUMMARY The continuous adaptation of SARS-CoV-2 generates variants of concern (VOC). New VOCs arise independently and outcompete previous ones, consistent with ever increasing viral fitness. Most VOC adaptations reside in the spikes (S), the complex multidomain glycoprotein trimers that bind viruses to cells and mediate virus-cell membrane fusion. Well-characterized adaptations in the S receptor binding domains (RBDs) alter RBD structural dynamics, receptor affinities, and antibody interactions. However, adaptations in other S domains remain largely under-evaluated. This includes the S2 domains that execute virus-cell membrane fusion. This proposal aims to elucidate consequences of VOC adaptive changes in the S2 domains. The current VOC, omicron (o), has accumulated over six S2 mutations. Our central objective is to evaluate the ways these and other S2 adaptations alter S protein dynamics to facilitate human cell entry. We will utilize a collection of viral membrane fusion and cell entry assay systems to determine whether S2 changes reset the dynamics of transient S protein intermediate states. These transitory states include RBD elevations that control receptor binding and S2 refoldings from prefusion to fusion intermediate and through to postfusion configurations. Our assay systems will employ fusion-competent virus-like particles (VLPs) for sensitive evaluation of entry steps, soluble receptors as probes for RBD exposures, HR2 peptides for detection of specific S transitional intermediates, replication-competent VSVSARS-CoV-2-S for selecting HR2 peptide-resistant variants, and SARS-CoV-2 replicons and recombinant SARS-CoV-2 viruses for convincingly assigning specific S2 amino acid substitutions to consequential redirection of virus-cell entry pathways. Our preliminary results suggest that omicron S2 adaptations operate allosterically to alter S-receptor interactions. The first aim will determine how S2 fusion domains control RBDs and their interactions with receptors. We will identify specific VOC S2 mutations that control receptor reactivity and S stability. We will determine how these mutations change viral entry requirements. Our initial results also suggest that omicron S2 mutations change the pace of the conformational transitions facilitating membrane fusion. The second aim will determine how S2 mutations control dynamics of fusion domains. We will determine whether the rates of S2 structural transitions vary between VOCs. We will also select and characterize variants resistant to inhibitors of the S2 structural dynamics and will find out whether these resistant variants have unique requirements for the receptors and proteases that determine cell susceptibility to infection. The results of this study will clarify currently obscure selective forces driving the human adaptation of past (α, β, γ, δ), current (ο), and future VOCs. We expect the results will illuminate properties of the membrane fusion – inducing S2 domains in ways that reveal new targets for inhibition of CoV entry.
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