The COVID-19 (Coronavirus disease-2019) pandemic, caused by the SARS-CoV-2 coronavirus, is a significant threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and MERS-CoV. Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analysis for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 Orf9b, an interaction we structurally characterized using cryo-EM. Combining genetically-validated host factors with both COVID-19 patient genetic data and medical billing records identified important molecular mechanisms and potential drug treatments that merit further molecular and clinical study.

The SARS-CoV-2 virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryogenic electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.

Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC 50) of 4.0 nM (180 ng ml −1). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy.