Isolation of ACE2-dependent and -independent sarbecoviruses from Chinese horseshoe bats

Abstract

While the spike proteins from SARS-CoV and SARS-CoV-2 bind to host ACE2 to infect cells, the majority of bat sarbecoviruses cannot use ACE2 from any species. Despite their discovery almost 20 years ago, ACE2-independent sarbecoviruses have never been isolated from field samples, leading to the assumption these viruses pose little risk to humans. We have previously shown how spike proteins from a small group of ACE2-independent bat sarbecoviruses may possess the ability to infect human cells in the presence of exogenous trypsin. Here, we adapted our earlier findings into a virus isolation protocol, and recovered two new ACE2-dependent viruses, RsYN2012 and RsYN2016, as well as an ACE2-independent virus, RsHuB2019. Although our stocks of RsHuB2019 rapidly acquired a tissue-culture adaption that rendered the spike protein resistant to trypsin, trypsin was still required for viral entry, suggesting limitations on the exogenous entry factors that support bat sarbecoviruses. Electron microscopy revealed ACE2-independent sarbecoviruses have a prominent spike corona and share similar morphology to other coronaviruses. Our findings demonstrate a broader zoonotic threat posed by sarbecoviruses and shed light onto the intricacies of coronavirus isolation and propagationin vitro.SIGNIFICANCESeveral coronaviruses have transmitted from animals to people and 20 years of virus discovery studies have uncovered thousands of new coronavirus sequences in nature. Most of the animal-derived sarbecoviruses have never been isolated in culture due to cell incompatibilities and a poor understanding of thein vitrorequirements for their propagation. Here, we built on our growing body of work characterizing viral entry mechanisms of bat sarbecoviruses in human cells and have developed a virus isolation protocol that allows for exploration of these understudied viruses. Our protocol is robust and practical, leading to successful isolation of more sarbecoviruses than previous approaches and from field samples that had been collected over a 10-year longitudinal study.

ACE2-independent sarbecovirus cell entry is supported by TMPRSS2-related enzymes and reduces sensitivity to antibody-mediated neutralization

“…Further, we demonstrate that trypsin treatment can confer infectivity of certain animal sarbecoviruses for human cells. Entry of these viruses is ACE2-independent and trypsin-dependent and the latter phenotype is determined by the RBD, confirming previous studies conducted with smaller numbers of spike proteins 21,31–33 .…”

“…However, it is not fully clear whether certain animal species can be identified as potential reservoirs or intermediate hosts for animal sarbecoviruses based on exceptionally broad receptor activity of their ACE2 orthologues. Recent studies provided evidence that the exposure of certain sarbecovirus S proteins to trypsin can facilitate ACE2-independent viral entry into human cells, a process that is determined by the receptor binding domain (RBD), and that equipping these S proteins with a multibasic cleavage site, a major virulence determinant of SARS-CoV-2 16,30 , is insufficient for trypsin-independent entry 21,31–33 . However, these analyses were confined to small numbers of S proteins and it has not been resolved whether cellular proteases other than trypsin can cleave and activate trypsin-dependent sarbecovirus S proteins for host cell entry.…”

Viral entry is a weak barrier to zoonosis

“…Our results challenge the view that incompatibilities between viral entry factors across host proteases, which are required for spike activation and play a key role in unlocking the human infectivity of some coronaviruses 14,51,52 .…”

“…Known coronavirus receptors such as ACE2 and DPP4 were expressed in few cell lines, in contrast to ubiquitous viral receptors such as DAG1, LDLR, NPC1, and EFNA1, among others. Moreover, coronavirus entry involves additional RBP-specific factors such as host proteases, which are required for spike activation and play a key role in unlocking the human infectivity of some coronaviruses 14,51,52 .…”

Engineering customized viral receptors for various coronaviruses

“…Subsequently, we investigated whether CVRs can facilitate efficient propagation of authentic coronavirus requiring strict culture conditions. RsHuB2019A, a relative of HKU3, is an ACE2-independent bat sarbecoviruses recently isolated from field samples 3 . Isolation and propagation of this virus was carried out in Huh-7 under a serum-free culture condition with exogenous trypsin, with viral infection being difficult to detect while maintaining normal cell morphology (Fig.…”