Bottom-up Assembled Synthetic SARS-CoV-2 Miniviruses Reveal Lipid Membrane Affinity of Omicron Variant Spike Glycoprotein.

The ongoing COVID-19 pandemic has been brought on by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike glycoprotein (S), which decorates the viral envelope forming a corona, is responsible for the binding to the angiotensin-converting enzyme 2 (ACE2) receptor and initiating the infection. In comparison to previous variants, Omicron S presents additional binding sites as well as a more positive surface charge. These changes hint at additional molecular targets for interactions between virus and cell, such as the cell membrane or proteoglycans on the cell surface. Herein, bottom-up assembled synthetic SARS-CoV-2 miniviruses (MiniVs), with a lipid composition similar to that of infectious particles, are implemented to study and compare the binding properties of Omicron and Alpha variants. Toward this end, a systematic functional screening is performed to study the binding ability of Omicron and Alpha S proteins to ACE2-functionalized and nonfunctionalized planar supported lipid bilayers. Moreover, giant unilamellar vesicles are used as a cell membrane model to perform competitive interaction assays of the two variants. Finally, two cell lines with and without presentation of the ACE2 receptor are used to confirm the binding properties of the Omicron and Alpha MiniVs to the cellular membrane. Altogether, the results reveal a significantly higher affinity of Omicron S toward both the lipid membrane and ACE2 receptor. The research presented here highlights the advantages of creating and using bottom-up assembled SARS-CoV-2 viruses to understand the impact of changes in the affinity of S for ACE2 in infection studies.

Protein-Based Patterning to Spatially Functionalize Biomimetic Membranes.

The bottom-up reconstitution of proteins for their modular engineering into synthetic cellular systems can reveal hidden protein functions in vitro. This is particularly evident for the bacterial Min proteins, a paradigm for self-organizing reaction-diffusion systems that displays an unexpected functionality of potential interest for bioengineering: the directional active transport of any diffusible cargo molecule on membranes. Here, the MinDE protein system is reported as a versatile surface patterning tool for the rational design of synthetically assembled 3D systems. Employing two-photon lithography, microswimmer-like structures coated with tailored lipid bilayers are fabricated and demonstrate that Min proteins can uniformly pattern bioactive molecules on their surface. Moreover, it is shown that the MinDE system can form stationary patterns inside lipid vesicles, which allow the targeting and distinctive clustering of higher-order protein structures on their inner leaflet. Given their facile use and robust function, Min proteins thus constitute a valuable molecular toolkit for spatially patterned functionalization of artificial biosystems like cell mimics and microcarriers.

Extracellular Cues Govern Shape and Cytoskeletal Organization in Giant Unilamellar Lipid Vesicles.

Spontaneous and induced front-rear polarization and a subsequent asymmetric actin cytoskeleton is a crucial event leading to cell migration, a key process involved in a variety of physiological and pathological conditions such as tissue development, wound healing, and cancer. Migration of adherent cells relies on the balance between adhesion to the underlying matrix and cytoskeleton-driven front protrusion and rear retraction. A current challenge is to uncouple the effect of adhesion and shape from the contribution of the cytoskeleton in regulating the onset of front-rear polarization. Here, we present a minimal model system that introduces an asymmetric actin cytoskeleton in synthetic cells, which are resembled by giant unilamellar lipid vesicles (GUVs) adhering onto symmetric and asymmetric micropatterned surfaces. Surface micropatterning of streptavidin-coated regions with varying adhesion shape and area was achieved by maskless UV photopatterning. To further study the effects of GUV shape on the cytoskeletal organization, actin filaments were polymerized together with bundling proteins inside the GUVs. The micropatterns induce synthetic cell deformation upon adhesion to the surface, with the cell shape adapting to the pattern shape and size. As expected, asymmetric patterns induce an asymmetric deformation in adherent synthetic cells. Actin filaments orient along the long axis of the deformed GUV, when having a length similar to the size of the major axis, whereas short filaments exhibit random orientation. With this bottom-up approach we have laid the first steps to identify the relationship between cell front-rear polarization and cytoskeleton organization in the future. Such a minimal system will allow us to further study the major components needed to create a polarized cytoskeleton at the onset of migration.

Synthetic virions reveal fatty acid-coupled adaptive immunogenicity of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 infection is a major global public health concern with incompletely understood pathogenesis. The SARS-CoV-2 spike (S) glycoprotein comprises a highly conserved free fatty acid binding pocket (FABP) with unknown function and evolutionary selection advantage1,2. Deciphering FABP impact on COVID-19 progression is challenged by the heterogenous nature and large molecular variability of live virus. Here we create synthetic minimal virions (MiniVs) of wild-type and mutant SARS-CoV-2 with precise molecular composition and programmable complexity by bottom-up assembly. MiniV-based systematic assessment of S free fatty acid (FFA) binding reveals that FABP functions as an allosteric regulatory site enabling adaptation of SARS-CoV-2 immunogenicity to inflammation states via binding of pro-inflammatory FFAs. This is achieved by regulation of the S open-to-close equilibrium and the exposure of both, the receptor binding domain (RBD) and the SARS-CoV-2 RGD motif that is responsible for integrin co-receptor engagement. We find that the FDA-approved drugs vitamin K and dexamethasone modulate S-based cell binding in an FABP-like manner. In inflammatory FFA environments, neutralizing immunoglobulins from human convalescent COVID-19 donors lose neutralization activity. Empowered by our MiniV technology, we suggest a conserved mechanism by which SARS-CoV-2 dynamically couples its immunogenicity to the host immune response.

A time-resolved live cell imaging assay to identify small molecule inhibitors of FGF2 signaling.

Fibroblast growth factor 2 (FGF2) is a cell survival factor with crucial functions in tumor-induced angiogenesis. Here, we describe a novel time-resolved FGF2 signaling assay based upon live cell imaging of neuroblastoma cells. To validate this system, we tested 8960 small molecules for inhibition of FGF2 signaling with kinetic resolution. Hit compounds were validated in dose-response experiments for FGF2 signaling, FGF receptor antagonism, downstream ERK phosphorylation and FGF2-dependent chemoresistance in a cellular leukemia model system. The new screening system for FGF2 signaling inhibitors has unique features, deselecting compounds with pleiotropic effects on cell proliferation and, along with the experimental pipeline reported, great potential for the discovery of new classes of FGF2 signaling inhibitors that block FGF2 dependent tumor cell survival.