Using Molecular Evolution to Explore Alternative Receptors for SARS-CoV-2

Zoonotic viruses, which can spread from animals to humans, are responsible for recent human pandemics. Thus, it is critical to understand the molecular factors that allow viruses to infect host cells. Since 2020, SARS-CoV-2 likely spilled over from bats into the human population causing approximately 7 million deaths globally. While SARS-CoV-2 is known to canonically use angiotensin converting enzyme 2 to bind and enter cells, recent molecular studies have discovered 10 alternative human receptors. It is unclear whether these proteins represent biologically significant avenues for SARS-CoV-2 entry in humans or other animals. We hypothesize that host receptor sequence changes resulting from persistent evolutionary arms races with viruses can inform our understanding of SARS-CoV-2 receptor usage. We are assembling a library of all publicly available reference ortholog sequences of alternative receptors. We will use established molecular sequence evolution analysis tools (Hyphy and PAML) to determine if SARS-CoV-2-related viruses have applied a positive selective pressure on these receptors in bats or other vertebrates. To test predictions generated by these analyses, we will clone alternative receptor sequences from cellular RNA into expression plasmids and perform infection assays using VSV reporter viruses decorated with SARS-CoV-2-related sarbecovirus spikes.