Functional assessment of cell entry and receptor usage for lineage B β-coronaviruses, including 2019-nCoV

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 novel 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 2019-nCoV, 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 2019-nCoV.

Figure 1:. β-Coronavirus lineage B entry with human ACE2 is clade-specific

a, β-Coronaviruses, including SARS-CoV, interact with the host cell receptor via the Receptor Binding Domain (RBD) in spike (PDB: 5×5B, 2AJF). b, SARS-CoV spike was engineered with silent mutations to facilitate cloning novel RBD sequences in place of the SARS spike RBD. SARS spike amino acid numbers are indicated for silent cloning sites and the RBD in grey and orange, respectively. c, outline of experimental workflow. d, Westernblot of producer cell lysates and concentrated reporter particles. e, BHK cells were transfected with either human ACE2 or empty vector, subsequently infected with VSV-reporter particles pseudotyped with chimeric spikes, luciferase was measured and normalized to no pseudotype as a readout for cell entry. Shown are the data for 3 replicates.

Figure 2:. Trypsin enhances lineage B entry in various cell lines

a, Primate cells, b, human cells or c, bat cells were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without pseudotype. Shown are the data for 3 replicates.

Figure 3:. Lineage B entry into cells with known CoV receptors

a, Schematic of known coronavirus RBDs and their receptors. b, Pseudotyped particles were either left untreated and treated with trypsin and subsequently used to infect BHK cells transfected with the coronavirus receptor indicated. Shown are data from 3 replicates. c, Expression and pseudotype incorporation of SARS-S-2019-nCoV RBD chimeras. d, Pseudotypes were used to infect cells expressing hACE2, hDPP4, hAPN, or empty vector, without protease treatment.

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

a, Schematic overview of clade 1, 2 and 3. Highlighted in yellow are the 14 residues that contact human ACE2. Deletions in loops 1 and 2 are indicated for clades 2 and 3. b, Structure of human ACE2 and the SARS-S RBD (PDB: 2AJF), with loops highlighted in gray. c, VSV pseudotypes were generated with the indicated RBD and used to infect BHKs transfected with either human ACE2 or empty vector. Shown are data for 3 replicates. d, Westernblot of producer cell lysates and concentrated pseudotyped particles.

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

a, Full length spike sequences from As6526 (clade 2) and BM48–31 (clade 3) were codon optimized, FLAG tagged and synthesized. Silent mutations flanking the RBD facilitated replacing the native RBD with the clade 1 consensus RBD. b, Westernblot of producer cell lysates and concentrated pseudotypes particles. c, Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect Huh-7.5 cells. Shown are data for 3 replicates. d, Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect BHK cells transfected with human ACE2. Shown are data for 3 replicates.