ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions

Abstract

SARS-CoV-2 uses the human ACE2 receptor (hACE2), with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility in vitro and in vivo. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K mutations, to match hACE2, rescued SARS-CoV-2 replication. hACE2-lentivirus transduction of C57BL/6J, IFNAR-/- and IL-28RA-/- mice lungs indicated type I and III IFN receptor knockout had minimal effect on virus replication. However, RNA-Seq illustrated that they were required for inflammatory responses in C57BL/6J mice, which were similar to those seen in COVID-19 patients. Finally, we illustrate the utility of hACE2 transduction for vaccine evaluation in C57BL/6J mice. The hACE2-lentivirus system thus has broad application in SARS-CoV-2 research, in particular allowing access to GM mice, while also offering the inherent advantages of lentiviral transduction such as stable genomic integration.

Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2 spike mediated infection

“…In contrast, K31 is positively selected in bats [33], with multiple Rhinolophus species such as R. ferrumequinum, R. landeri, and R. pusillus encoding D31 (S6 Fig) . Notably, Glu, Asn, and Thr residues are also observed at position 31 in other bat species, including high intraspecies variation within R. sinicus (S6 Fig) . Despite the importance of K31 for SARS-CoV-2 infection of cells expressing human ACE2, its effect in the context of the bat ACE2 alleles has not been characterized, and it remains to be seen whether these residues are affecting SARS-CoV-2 infection within bat cells. Notably, the conversion of murine ACE2 to N31K and H353K renders Mus musculus, house mouse cells permissible for SARS-CoV-2 entry, supporting the critical role of variants at this position across species [34].…”

Tracking inflammation resolution signatures in lungs after SARS-CoV-2 omicron BA.1 infection of K18-hACE2 mice

“…Minor intrusion of red blood cells and serum into bronchi, and some alveolar edema were also ). RTqPCR was undertaken for 3 genes and two time points to validate the RNA-Seq data, with highly significant concordance emerging for the two methods (S4a Fig) . We have previously reported high levels of concordance between these two methods in other studies [60,66].…”

“…Mice were weighed and monitored as described [60]. Mice were euthanized using CO 2 , lungs were removed, with the left lung fixed in formalin for histology, the right lung inferior lobe placed in RNAlater for RNA-Seq and the remaining lobes used for tissue titers determination by CCID 50 assays using Vero E6 cells as described [57,60].…”

“…BA.1 viral stocks were propagated in Vero E6 cells as described [60]. UV inactivation of the BA.1 virus was undertaken using the UVC 500 Ultraviolet Crosslinker (Hoefer) (dose 7650 J/m 2 UVC) as described [60]. Virus stocks were titered using cell culture infectious dose 50% (CCID 50 ) assays [61].…”

“…Tissue titers were determined as described [60]. Briefly, 5 fold serial dilutions of clarified tissue homogenates were applied in duplicates to Vero E6 cells in 96 well plates.…”