Evolution of ACE2-independent SARS-CoV-2 infection and mouse adaption after passage in cells expressing human and mouse ACE2

Abstract

Human <i>ACE2</i> Human angiotensin converting enzyme 2 (hACE2) is the key cell attachment and entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with the original SARS-CoV-2 isolates unable to use mouse <i>ACE2</i> (mACE2). Herein we describe the emergence of a SARS-CoV-2 strain capable of <i>ACE2</i>-independent infection and the evolution of mouse-adapted (MA) SARS-CoV-2 by <i>in vitro</i> serial passaging of virus in co-cultures of cell lines expressing hACE2 and mACE2. MA viruses evolved with up to five amino acid changes in the spike protein, all of which have been seen in human isolates. MA viruses replicated to high titers in C57BL/6J mouse lungs and nasal turbinates and caused characteristic lung histopathology. One MA virus also evolved to replicate efficiently in several <i>ACE2</i>-negative cell lines across several species, including clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) <i>ACE2</i> knockout cells. An E484D substitution is likely involved in <i>ACE2</i>-independent entry and has appeared in only ≈0.003 per cent of human isolates globally, suggesting that it provided no significant selection advantage in humans. <i>ACE2</i>-independent entry reveals a SARS-CoV-2 infection mechanism that has potential implications for disease pathogenesis, evolution, tropism, and perhaps also intervention development.