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. 2023 Jun 15;11(3):e0348322.
doi: 10.1128/spectrum.03483-22. Epub 2023 May 18.

Spike-Independent Infection of Human Coronavirus 229E in Bat Cells

Marcus G Mah #  1 , Martin Linster #  1 , Dolyce H W Low  1 , Yan Zhuang  1 , Jayanthi Jayakumar  1 , Firdaus Samsudin  2 , Foong Ying Wong  1 , Peter J Bond  2   3 , Ian H Mendenhall  1 , Yvonne C F Su  1 , Gavin J D Smith  1   4   5   6
Affiliations

Spike-Independent Infection of Human Coronavirus 229E in Bat Cells

Marcus G Mah et al. Microbiol Spectr. .

Abstract

Bats are the reservoir for numerous human pathogens, including coronaviruses. Despite many coronaviruses having descended from bat ancestors, little is known about virus-host interactions and broader evolutionary history involving bats. Studies have largely focused on the zoonotic potential of coronaviruses with few infection experiments conducted in bat cells. To determine genetic changes derived from replication in bat cells and possibly identify potential novel evolutionary pathways for zoonotic virus emergence, we serially passaged six human 229E isolates in a newly established Rhinolophus lepidus (horseshoe bat) kidney cell line. Here, we observed extensive deletions within the spike and open reading frame 4 (ORF4) genes of five 229E viruses after passaging in bat cells. As a result, spike protein expression and infectivity of human cells was lost in 5 of 6 viruses, but the capability to infect bat cells was maintained. Only viruses that expressed the spike protein could be neutralized by 229E spike-specific antibodies in human cells, whereas there was no neutralizing effect on viruses that did not express the spike protein inoculated on bat cells. However, one isolate acquired an early stop codon, abrogating spike expression but maintaining infection in bat cells. After passaging this isolate in human cells, spike expression was restored due to acquisition of nucleotide insertions among virus subpopulations. Spike-independent infection of human coronavirus 229E may provide an alternative mechanism for viral maintenance in bats that does not rely on the compatibility of viral surface proteins and known cellular entry receptors. IMPORTANCE Many viruses, including coronaviruses, originated from bats. Yet, we know little about how these viruses switch between hosts and enter human populations. Coronaviruses have succeeded in establishing in humans at least five times, including endemic coronaviruses and the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In an approach to identify requirements for host switches, we established a bat cell line and adapted human coronavirus 229E viruses by serial passage. The resulting viruses lost their spike protein but maintained the ability to infect bat cells, but not human cells. Maintenance of 229E viruses in bat cells appears to be independent of a canonical spike receptor match, which in turn might facilitate cross-species transmission in bats.

Keywords: 229E; coronavirus; evolution; pandemic; receptor; receptor usage; spike; zoonotic.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Rhileki cells are susceptible and permissive to 229E infection. (A) Serial passage of six 229E isolates at an MOI of 0.01 in Rhileki cells. Viral genome copies of the inoculum (circles) and at 6 days postinoculation (square) are plotted. The inoculum for subsequent passages was standardized based on genome copy numbers. (B) Viral titers (TCID50/mL) in Caco2 cells of C2, C2R10, and C2R10C1 viruses after serial passage. (C) Viral titers (TCID50/mL) in Rhileki cells of C2R1 and C2R10 viruses after serial passage. Black dashed lines represent the limit of detection.
FIG 2
FIG 2
Schematic of nucleotide sequences within the spike and ORF4 gene regions. Color-filled rectangles represent parts of the genome in C2R1, C2R5, and C2R10 viruses that are present in the respective passage. Virus isolate names are in bold. Horizontal black lines indicate deleted parts of the genome with their respective size denoted by Δ; starts and ends of the deletions are indicated by the respective nucleotide position. Stop codons are denoted by an asterisk (*).
FIG 3
FIG 3
Spike protein expression and virus neutralization. (A) Western blot images of culture supernatants of Rhileki cells and Caco2 cells infected with C2R1, C2R5, C2R10, and C2R10C1 and stained with 229E anti-spike (S) and anti-nucleocapsid protein (N) antibodies. (B) Virus neutralization test, with virus titers (TCID50/mL) of C2 viruses in Caco2 cells (blue), C2 viruses in Rhileki cells (black), C2R10 viruses in Rhileki cells (brown), C2R10 viruses in Caco2 cells (red), and C2R10C1 virus in Caco2 cells for the isolate SG/2613/2011 only (green). The 229E anti-spike rabbit polyclonal sera at concentrations of 1.25 μg/mL to 80 μg/mL was incubated with the respective virus 1 h before inoculation. Blacked dashed lines indicate the limit of detection.
FIG 4
FIG 4
Genetic diversity of the SG/2613/2011 spike gene across passages. (A) Nucleotide sequences from position 21059 to 21100 and the corresponding amino acid translation within the S1 domain of the spike gene. Depending on the passage of the virus, the diversity is comprised of one to three uracil insertions at nucleotide position 21064 that alter the reading frame compared to the wild-type sequence (turquoise, line 1), GNFY-NE. With one (orange, line 2) or two (teal, line 3) uracil insertions, the reading frame shifts and leads to an early stop codon GNFL-Q* (E177*) and GNFFIAL* (L171*), respectively. At three (mustard, line 4) uracil insertions, the reading frame is restored with an additional phenylalanine GNFFY-NE. Double forward slashes (//) indicate 8 amino acid residues not shown for clarity of presentation. (B) Pie charts describe the percentage of viral genome reads with uracil insertions for C2 (100% no insertions, turquoise, n = 8,268), C2R5 (11.1% no insertions; 70.9% single insertion, orange; 18.0% double insertion, teal, n = 323), C2R10 (75.8% single insertion; 24.2% double insertion, n = 33), and C2R10C1 (11.2% no insertions; 88.8% triple insertion, mustard, n = 347) viruses. Figure was created with https://BioRender.com.
FIG 5
FIG 5
Effect of cathepsin inhibitors on C2R10 virus growth on Rhileki cells. Rhileki cells were pretreated with either 50 μM MDL28170 (turqoise) or 10 μM E64d (blue) before infection with C2R10 viruses at an MOI of 0.003. Viral genome copies were measured at 3 dpi. Cell viability towards a range of concentrations of the tested compounds were assessed. Cell viability: dimethyl sulfoxide (DMSO), 100%; 50 μM MDL28170, 89.3%; 10 μM E64d, 100%. The experiment was repeated three independent times and in triplicates. Error bars represent standard error of the mean; *, P = 0.0163; **, P < 0.005; ***, P < 0.0001.
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