Unraveling Potential Hosts of SARS-CoV-2 with ACE2 Orthologs

Experiment

Phylogenetic Analysis and Sequence Alignment

To determine the host range of SARS-CoV-2, ACE2 orthologs possessing critical amino acid residues relevant for viral entry were obtained from NCBI nucleotide database and analyzed in MEGA-X (v10.05) software.

Binding of ACE2 to SARS-CoV-2 S Protein

A549 cells were transduced with various ACE2 orthologs and incubated with the recombinant S1 domain of SARS-CoV-2 S protein to examine the interaction of SARS-CoV-2 protein with ACE2 orthologs. The cDNAs encoding ACE2 orthologs were codon-optimized and synthesized by GenScript.

Function of ACE2 Orthologs in SARS-CoV-2 Entry

To determine which ACE2 orthologs help SARS-CoV-2 enter host cells, genetic complementation experiments were conducted in A549 cells. A549 cells ectopically expressing individual ACE2 orthologs were infected with SARS-CoV-2. The investigators also ectopically expressed the serine protease TMPRSS2 in A549 cells to assess its role in ACE2 mediated viral entry. The human cDNA encoding TMPRSS2 was synthesized by GenScript.

Genetic Factors within ACE2 Preventing SARS-CoV-2 Entry

To identify ACE2 orthologs’ genetic determinants preventing SARS-CoV-2 infection in New World monkeys, ACE2 protein residues that contact the S protein were compared with that of susceptible species.

Results

From a total of 300 species analyzed, 80 ACE2 orthologs, all from mammalian species, had 5 amino acids critical for SARS-CoV-2 binding. A total of 48 representative ACE2 orthologs were ectopically expressed in A549 cells and evaluated for their ability to support SARS-CoV-2 entry. The data showed that most ACE2 orthologs from 48 species, including ferrets, cats, dogs, cattle, etc., could bind the S protein and promote viral entry except those derived from Koala and 3 species of New World monkeys. Ectopic expression of TMPRSS2 in combination with New World monkeys’ ACE2 orthologs could not render A549 cells permissive to SARS-CoV-2 entry. Yinghui et al. went further to explore the genetic determinants of ACE2 orthologs from New World monkeys restricting SARS-CoV-2 entry. The results indicate that residues at the ACE2–S interface differ between New World monkeys and susceptible species. Specifically, residues H41 and E42, in ACE2 orthologs from New World monkey species may fail to support hydrogen bonding interactions with SARS-CoV-2 S protein needed for viral entry.