PROJECT SUMMARY Viral spillover from animal reservoirs into humans can decimate public health systems and cripple the world economy, as evident with the current SARS-CoV-2 pandemic. Continued wildlife habitat destruction, human expansion, and routine global travel keep increasing the likelihood that another viral pandemic will occur again within the next few decades. Beta coronaviruses are an incredibly diverse family of viruses observed across Asia, Europe, and Africa, that have proven capable of zoonotic spillover into humans as they have caused multiple worldwide outbreaks over the last two decades. We still lack the fundamental understanding of the molecular and genetic factors that dictate the molecular compatibilities that determine which beta coronaviruses are most likely to jump into humans in the future. The ability of SARS-like beta coronaviruses to utilize ACE2 as a receptor for cell entry is a major factor determining the extent of coronavirus tropism across species or within the tissues of an organism. While SARS-CoV-2 has been heavily studied, almost nothing is known about most other members of this virus family. Traditional studies can only test a handful of conditions at a time, incompletely sampling the vast range of relevant experimental conditions, particularly for the hundreds of uncharacterized beta coronaviruses. Large- scale, minimally-biased, cell-based entry assays are needed to model how these factors converge to dictate the probability of infection. We will pair new methods in cell engineering and synthetic biology with DNA-sequencing enabled multiplex genetic assays to perform a series of large-scale infection assays revealing the factors determining susceptibility to beta coronavirus entry. These large-scale experiments will reveal how ACE2 sequence and cell surface density impact the efficiency of virus entry. By testing a library of receptor binding domain sequences identified from ecological surveillance of bat coronaviruses, we will identify which viruses possess sufficient affinity to human ACE2 to potentiate cross-species transmission, and create a catalog describing all of the different ways these viruses have evolved their sequences to engage ACE2. By modeling the relationship between spike and ACE2 protein sequence, expression level, and efficiency of cell entry, we will identify potential animal reservoirs for SARS-CoV-2 and other SARS-like bat coronaviruses, and predict which viruses have sufficient binding with human ACE2 to potentially spark the next pandemic.

PROJECT NARRATIVE We do not know enough about how efficiently the spike proteins from diverse SARS-like beta coronaviruses need to bind with the ACE2 host cell receptor proteins encoded by different animals to successfully jump across host species and spark pandemic zoonotic spillovers. Using cutting-edge synthetic biology paired with multiplex infection assays, we will perform a series of large-scale genetic studies to reveal how differences in ACE2 protein sequence and cell surface expression levels interact with coronavirus spike protein sequences from various bat coronaviruses to understand how often each virus infects those cells. This molecular and genetic information will reveal which animals can serve as hidden reservoirs for potentially zoonotic SARS-like coronaviruses deserving heightened surveillance.