Abstract
SARS-CoV and SARS-CoV-2 encode spike proteins that bind human ACE2 on the cell surface to enter target cells during infection. A small fraction of humans encode variants of ACE2, thus altering the biochemical properties at the protein interaction interface. These and other ACE2 coding mutants can reveal how the spike proteins of each virus may differentially engage the ACE2 protein surface during infection. We created an engineered HEK 293T cell line for facile stable transgenic modification, and expressed the major human ACE2 allele or 28 of its missense mutants, 24 of which are possible through single nucleotide changes from the human reference sequence. Infection with SARS-CoV or SARS-CoV-2 spike pseudotyped lentiviruses revealed that high ACE2 cell-surface expression could mask the effects of impaired binding during infection. Drastically reducing ACE2 cell surface expression revealed a range of infection efficiencies across the panel of mutants. Our infection results revealed a non-linear relationship between soluble SARS-CoV-2 RBD binding to ACE2 and pseudovirus infection, supporting a major role for binding avidity during entry. While ACE2 mutants D355N, R357A, and R357T abrogated entry by both SARS-CoV and SARS-CoV-2 spike proteins, the Y41A mutant inhibited SARS-CoV entry much more than SARS-CoV-2, suggesting differential utilization of the ACE2 side-chains within the largely overlapping interaction surfaces utilized by the two CoV spike proteins. These effects correlated well with cytopathic effects observed during SARS-CoV-2 replication in ACE2-mutant cells. The panel of ACE2 mutants also revealed altered ACE2 surface dependencies by the N501Y spike variant, including a near-complete utilization of the K353D ACE2 variant, despite decreased infection mediated by the parental SARS-CoV-2 spike. Our results clarify the relationship between ACE2 abundance, binding, and infection, for various SARS-like coronavirus spike proteins and their mutants, and inform our understanding for how changes to ACE2 sequence may correspond with different susceptibilities to infection.
Highlights
Zoonotic spillover of viruses from animal reservoirs can decimate public health systems and the global economy, as evident with the current SARS-CoV-2 pandemic
SARS-like coronaviruses, such as SARS-CoV-2, use their spike proteins to bind a common surface on the human ACE2 protein to gain entry and subsequently infect cells
We used site-specific genomic integration and expression of WT ACE2 or its missense variants, many of them previously observed in human exomes, to determine how ACE2 sequence and abundance correspond to infectability by SARS-CoV or SARS-CoV-2
Summary
Zoonotic spillover of viruses from animal reservoirs can decimate public health systems and the global economy, as evident with the current SARS-CoV-2 pandemic. Similar to MERS-CoV but dissimilar to SARS-CoV, the SARS-CoV-2 spike possesses a furin cleavage site that separates the S1 and S2 units during virus release from producer cells [4]. While this cleavage is not essential and can be performed by host proteases in the target cell, furin cleavage increased pathogenicity in animal models [5,6]. Some cells express cell-surface proteases such as TMPRSS2, capable of cleaving the spike protein and enabling virion entry at the plasma membrane[8,9,10,11]. The ACE2 binding step is believed to occur in either case, regardless of the second protease-cleavage step that decides the location of viral fusion and entry
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