A spike-binding protein as a versatile tool to detect and inhibit transmissible gastroenteritis virus

The Spike protein of coronaviruses (CoVs) plays an essential role in viral entry and is the main target of antibodies produced following vaccination or natural infection. Most of the knowledge on the function of Spike proteins is incoming from the beta-CoV genus causing severe diseases in humans. Indeed, numerous tools such as pseudoviruses have been developed to measure beta-CoV Spikes activity simply and rapidly, and several ligands of different natures binding to these Spike proteins (such as antibodies or nanobodies) have been characterized both structurally and functionally. However, far fewer tools are available to study Spike proteins from other CoV genera such as alpha-CoVs, which include important animal viruses, sometimes emerging and potentially zoonotic. In this study, we developed and validated a pseudovirus system to study the viral entry of a porcine alpha-CoV, transmissible gastroenteritis virus (TGEV). Using phage display screening, we then identified, from a library of biosynthetic ligands known as "alpha-Reps," small proteins capable of binding to an S1-TGEV-Fc construct. One of these alpha-Reps, TGE-D9, inhibited pseudo-TGEV entry in a dose-dependent manner and was able to bind the Spike protein displayed at the surface of viral particles, providing a potential alternative to antibodies for the detection of virions. These data pave the way for functional and structural studies to understand the binding and mode of action of these synthetic ligands on the TGEV and other alpha-CoV Spike proteins, complementary with other ligands of different natures, to deepen our understanding of alpha-CoV Spike properties.