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. 2021 Jul 16;17(7):e1009715.
doi: 10.1371/journal.ppat.1009715. eCollection 2021 Jul.

Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2 spike mediated infection

Nidhi Shukla  1 , Sarah M Roelle  1 , Vinicius G Suzart  1 , Anna M Bruchez  1 , Kenneth A Matreyek  1
Affiliations

Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2 spike mediated infection

Nidhi Shukla et al. PLoS Pathog. .

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Improved cell engineering platform expressing human ACE2.
A) Schematic of the newly described LLP-Int-iCasp9-Blast landing pad (top), and the ACE2 expression construct used in the majority of this study (bottom). Key: Tet, Tet-inducible promoter; BFP, Blue fluorescent protein; Int, Bxb1 integrase; IRES, Internal Ribosome Entry Site; iCasp9, inducible caspase 9; Bsd, Blasticidin-S deaminase; CMV, Cytomegalovirus promoter; rtTA, reverse tet transactivator; H2A, Histone 2A; Pac, Puromycin N-acetyltransferase; attP and attB are Bxb1 recombination sites. Inverted red triangles denote viral 2A-like sequences allowing co-translational separation of the polyprotein. Thick vertical lines are transcriptional terminator sequences. B) Percentages of recombined cells in various HEK 293T Bxb1 landing pad cells, in the absence (left) or presence (right) of negative selection agent AP1903. Red and blue indicate the absence and presence of exogenous Bxb1 integrase expression plasmid, respectively. n = 4; error bars denote 95% confidence intervals. C) Representative smoothed histograms of the flow cytometry mean fluorescence intensities of cells where GFP was expressed by plasmid transfection (top), lentiviral transduction (middle), or recombination into the landing pad (bottom). Light and dark green correspond to days 3 and 5 after plasmid transfection, respectively. For the lentivirus transduced and Bxb1 recombined cells, the cells had been generated a week before the first time-point. D) Representative mCherry fluorescence distribution of ACE2-recombined cells, as captured by flow cytometry, in cells left uninduced (orange) or induced to express ACE2 from the Tet-inducible promoter using 2 μM doxycycline (black). E) Geometric means of green fluorescence of SARS-CoV-2 RBD-sfGFP -stained ACE2 expressing cells. n = 2 and 8 for uninduced and induced cells, respectively. Error bars denote 95% confidence intervals. F) Representative scatterplot of mCherry fluorescence and RBD-sfGFP staining of ACE2 recombinant cells. The green line denotes the linear correlation between red and green MFI for the double-positive population. Pearson’s r2 = 0.44. G) Percent of mCherry positive cells that were also positive for SARS-CoV-2 RBD-sfGFP staining, for 5 repeat staining experiments. H) Fold pseudovirus infection of ACE2 overexpressing cells, normalized to infection of parental HEK 293T cells. n = 3 for SARS-CoV and SARS-CoV-2 spikes, error bars denote 95% confidence intervals.
Fig 2
Fig 2. ACE2 variant expressed at high abundance levels.
A) The location of ACE2 (orange) K31 and K353 residues (grey) in the interface with SARS-CoV-2 RBD (blue); with the three-dimensional structure provided by PDB 6M17. B) Schematic showing the coding region of the ACE2 expression construct, as well as the dEcto negative control construct lacking the entire extracellular domain. C) ACE2 variant constructs encoding the consensus Kozak, stained with SARS-CoV-2 RBD-sfGFP. n = 4 or more, error bars denote 95% confidence intervals. D) Pseudovirus infection rates of ACE2 variants encoding the consensus Kozak, normalized to cells encoding WT ACE2. n = 6, error bars denote 95% confidence intervals. E) Scatterplots depicting pseudovirus infection or SARS-CoV-2 RBD-sfGFP staining of cells encoding consensus Kozak ACE2 preceding WT, K31D, or K353D ACE2, compared to published binding studies with SARS-CoV or SARS-CoV-2 RBDs.
Fig 3
Fig 3. Tuning of ACE2 cellular abundance using suboptimal Kozak sequences.
A) (Top) Schematic of consensus and suboptimal Kozak sequences preceding the ACE2 coding region. (Bottom) Representative flow cytometry smoothed histograms of negative control cells, or cells expressing WT ACE2 encoded behind a consensus or suboptimal Kozak sequence, stained with SARS-CoV-2 RBD-sfGFP. B) Representative Western blot showing the differences in total ACE2 protein abundance in engineered cells. C) Cell surface staining using SARS-CoV-2 RBD-sfGFP for cells with contrasting Kozak sequences (left), or encoding ACE2 variants (right). n = 4 or more, error bars denote 95% confidence intervals. D) Pseudovirus infection of cells with contrasting Kozak sequences (left), or encoding ACE2 variants in the context of a suboptimal Kozak (right). n = 6 or more, error bars denote 95% confidence intervals. Asterisks denote samples that exhibited a p-value less than 0.01 in a one-sample T-test from the infection value of 1.
Fig 4
Fig 4. ACE2 variant effects on binding and infection.
A) Structure of the SARS-CoV-2 RBD in complex with ACE2, from PDB 6M17. Positions of variants tested in the manuscript are shown as spheres colored according to their order from the N- to C-terminus. (Bottom) A zoomed in view of the RBD:ACE2 interface, where the majority of the tested variants are found. B) Infection of a panel of ACE2 variants of residues near the spike interaction surface, separated into variants currently unobserved (top) and observed (bottom) in publicly available human genomics datasets. n = 5 or more replicates, error bars denote 95% confidence intervals. C) Scatter plot showing relative pseudovirus infection rates (y-axis) and the mean value of the published SARS-CoV-2 RBD-sfGFP staining (x-axis) for WT ACE2 and its variants. D) Scatter plot showing normalized infection of SARS-CoV-2 (x-axis) or SARS-CoV (y-axis) spike pseudoviruses. E) Scatter plot comparing the pseudovirus infection values of SARS-CoV or SARS-CoV-2 pseudoviruses with their average allele frequencies observed in the GnomAD and BRAVO exome databases. The grey line in panels C and D, denote a hypothetical perfect correspondence between assays with a slope of 1.
Fig 5
Fig 5. Comparison of ACE2 variant susceptibilities to SARS-CoV-2 replication with SARS-CoV-2 spike pseudovirus infection.
A) Tissue culture infectious dose 50 assays, where dilutions of SARS-CoV-2 were added to 96-wells containing HEK 293T cells expressing the denoted WT or variant ACE2 proteins. TCID50 values were normalized to values corresponding to infection of WT ACE2 cells assessed in the same experiment. Samples colored in blue are those where no cytotoxicity was observed in any dilution, and these samples were given a nominal count of 1 to denote the limit of detection of the assay, and were then normalized accordingly. The horizontal dashes indicate geometric means across experiments. Error bars denote 95% confidence intervals. N = 3 or more replicates. B) Scatter plot comparing SARS-CoV-2 pseudovirus infection and geometric means of normalized TCID50 values shown in panel A. Blue lines indicate representative threshold values that separate permissive from non-permissive ACE2 variants. C) Scatter plot comparing published SARS-CoV-2 RBD-sfGFP binding values to geometric means of normalized TCID50 values.
Fig 6
Fig 6. ACE2 variant dependencies for SARS-CoV-2 spike variants.
A) Relative titers of D614G and N501Y SARS-CoV-2 spike pseudoviruses, relative to WT. n = 7. B) Relative infectivity of WT, D614G, N501Y SARS-CoV-2 spike pseudoviruses, normalized to infectivity in WT ACE2 cells. VSV-G pseudoviruses were included as a negative control. n = 7. C) Scatter plot showing normalized infectivities of D614G (x-axis) and WT (y-axis) SARS-CoV-2 spike pseudoviruses. D) Scatter plot showing normalized infectivity of N501Y (x-axis) and WT (y-axis) SARS-CoV-2 spike pseudoviruses. The grey line in panels C and D, denote a hypothetical perfect correspondence between assays with a slope of 1. E) Structure of the RBD:ACE2 interface (pdb: 6m17), with the RBD shown as a cyan ribbon, and ACE shown as an orange cartoon with a semi-transparent surface representation. Residues of ACE variants highlighted in Figs 4E and 6D are shown as sphere representations. N501 on the RBD is shown as blue spheres.
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