Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses

Abstract

Over the past 20 years, several coronaviruses have crossed the species barrier into humans, causing outbreaks of severe, and often fatal, respiratory illness. Since SARS-CoV was first identified in animal markets, global viromics projects have discovered thousands of coronavirus sequences in diverse animals and geographic regions. Unfortunately, there are few tools available to functionally test these viruses for their ability to infect humans, which has severely hampered efforts to predict the next zoonotic viral outbreak. Here, we developed an approach to rapidly screen lineage B betacoronaviruses, such as SARS-CoV and the recent SARS-CoV-2, for receptor usage and their ability to infect cell types from different species. We show that host protease processing during viral entry is a significant barrier for several lineage B viruses and that bypassing this barrier allows several lineage B viruses to enter human cells through an unknown receptor. We also demonstrate how different lineage B viruses can recombine to gain entry into human cells, and confirm that human ACE2 is the receptor for the recently emerging SARS-CoV-2.

Fig. 1. Betacoronavirus lineage B entry with human ACE2 is clade specific.

a, Betacoronaviruses, including SARS-CoV, interact with the host-cell receptor via the RBD in spike (Protein Data Bank ID: 5X5B; 2AJF). b, Engineered silent mutations in SARS spike facilitated replacement of the RBD sequence. SARS spike amino acid numbers are indicated in black for the silent cloning sites and orange for the RBD. c, Outline of the experimental workflow. d, Western blot of producer cell lysates and concentrated reporter particles. The labels along the top show the origin of the RBD in the SARS-CoV spike protein. e, Cladogram of the 29 spikes tested. Cells expressing either human ACE2 or empty vector were infected with pseudotyped VSV reporter particles, and luciferase was measured and normalized to no spike as a readout for cell entry. The data are representative of three technical replicates. Vertical bars indicate mean values of all three replicates and horizontal bars indicate s.d. Source data

Fig. 2. Trypsin enhances lineage B entry in various cell lines.

a–c, African green monkey (AGM; a), human (b) or bat cells (c) were infected with VSV particles pseudotyped with lineage B chimeric spikes. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without spike. Data in all panels are representative of three technical replicates. Vertical bars indicate mean values of all three replicates and horizontal bars indicate s.d. GI, gastrointestinal. Source data

Fig. 3. Lineage B entry into cells with known CoV receptors.

a, Schematic of known coronavirus spikes and their receptors. N-terminal RBD found in spike S1 of lineage A betacoronaviruses and gammacoronaviruses is indicated in orange. C-terminal RBD found in spike S1 of alphacoronaviruses and lineage B betacoronaviruses is indicated in blue. CECAM1, carcinoembryonic antigen-related cell adhesion molecule 1; HCoV, human coronavirus; IBV, infectious bronchitis virus; MHV, mouse hepatitis virus; TGEV, transmissible gastroenteritis coronavirus. b, Pseudotyped particles were either left untreated or treated with trypsin and subsequently used to infect BHK cells expressing the indicated coronavirus receptors. WT, wild type. c, Expression and pseudotype incorporation of SARS-S–SARS-CoV-2 RBD chimeras. SARS-S, SARS-CoV spike. The top labels indicate the origin of the RBD in the SARS spike protein. d, Pseudotypes were used to infect BHK cells expressing known receptors without protease treatment. The y axis labels indicate the origin of the RBD in the SARS spike protein. Data for all panels represent three technical replicates. Vertical bars indicate mean values of all three replicates and horizontal bars indicate s.d. Source data

Fig. 4. Lineage B clade-specific determinants for human ACE2 usage.

a, Schematic overview of clades 1, 2 and 3 of the betacoronavirus lineage B RBD. Shown in yellow are the 14 residues that contact ACE2. Loop deletions are shown for clades 2 and 3. b, Structure of human ACE2 and the SARS-S RBD (Protein Data Bank ID: 2AJF), with the loops highlighted in grey. c, VSV pseudotypes were used to infect BHKs transfected with either human ACE2 or empty vector. The data are representative of three technical replicates. Vertical bars indicate mean values of all three replicates and horizontal bars indicate s.d. d, Western blot of producer cell lysates and concentrated pseudotyped particles. The top labels show the source of the RBD in the spike protein. Source data

Fig. 5. Comparison of chimeric and full-length clade 2 and 3 spikes.

a, Design of full-length spike sequences from As6526 (clade 2) and BM48-31 (clade 3). b, Western blot of producer cell lysates and concentrated pseudotype particles. c, Pseudotypes were left untreated or treated with trypsin and subsequently used to infect Huh-7.5 cells. d, Pseudotypes with the indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect BHK cells expressing human ACE2. The data are representative of three technical replicates. Vertical bars indicate s.d. and horizontal bars indicate mean values of all three replicates. Source data

Extended Data Fig. 1. Lineage B RBD panel assembly and phylogenetic analysis.

a, Coronavirus sequences were downloaded from NCBI and further parsed to 29 unique RBD variants. b, Virus isolate name, accession number, clade, host species and location of identification listed for the 29 unique lineage B RBDs used in this study. c, Cladograms for the spike RBD and coronavirus RNA-dependent RNA polymerase (nsp12). d, Overview of experimental timeline.

Extended Data Fig. 2. Additional cell lines tested without protease.

a, Additional human and cell lines or b, bat-derived cell lines derived from other species were infected with VSV-reporter particles pseudotyped with chimeric spikes and luciferase was measured a readout for cell entry. Trypsin was not used in these infections. c, All cell lines tested in this study supported entry and reporter expression of VSV-g pseudotyped particles. For all panels shown are the data for 3 techinical replicates, horizontal bars indicate the s.d. and vertical bars indicate the mean value of all 3 replicates. Source data

Extended Data Fig. 3. 2019-nCoV uses human ACE2 to enter cells.

VSVΔG-luciferase/GFP particles were pseudotyped with the indicated spikes and used to infect BHKs transfected with known coronavirus receptors. Microscopy images were taken 20 hours post-infection. Scale bar indicates 1000 um.

Extended Data Fig. 4. Lineage B panel RBD sequence features.

a, Amino acid sequences corresponding to SARS-spike residues 317 through 500 were aligned with ClustalW. Contact points between SARS-spike and human ACE2 are indicated with an (*). Clade 2 sequences are shown as compared to clade 2 As6526, with identical residues indicated with a (.) and sites that vary between clade 2 viruses highlighted in purple. Loop deletions are highlighted in orange. b, Amino acid alignment of 2019-nCoV RBD and consensus RBD sequences for clade 1 and 2 and BM48-31 (clade 3). Loop deletions are highlighted in orange.

Extended Data Fig. 5. Model of lineage B entry.

a, SARS spike-clade 1 RBD enters cells expressing ACE2 and a host protease capable of cleaving SARS spike (left panel). While clade 2 RBDs can bind an unknown host receptor, the SARS spike backbone is incompatible with the receptor-associated protease, resulting in a lack of cleavage and entry (middle panel). The addition of exogenous protease may overcome the lack of endogenous protease cleavage of spike, resulting in receptor-dependent entry. Alternatively, the addition of exogenous protease may activate the receptor to facilitate entry. b, Replacing the RBD of As6526 spike with the clade 1 RBD allows for ACE2 interaction, but the As6526 spike backbone is incompatible with the ACE2-associated protease (left panel). Addition of exogenous protease overcomes protease incompatibility, allowing for ACE2-mediate entry (right panel).