Bilateral entry and release of Middle East respiratory syndrome coronavirus induces profound apoptosis of human bronchial epithelial cells

Abstract

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) infects human bronchial epithelial Calu-3 cells. Unlike severe acute respiratory syndrome (SARS)-CoV, which exclusively infects and releases through the apical route, this virus can do so through either side of polarized Calu-3 cells. Infection results in profound apoptosis within 24 h irrespective of its production of titers that are lower than those of SARS-CoV. Together, our results provide new insights into the dissemination and pathogenesis of MERS-CoV and may indicate that the virus differs markedly from SARS-CoV.

Fig 1

Human bronchial epithelial Calu-3 cells, colonic LoVo epithelial cells, and CD26/DPP4-expressing human Jurkat T cells are permissive for MERS-CoV/EMC-2012 infection. (A) Confluent Calu-3 cells, LoVo cells, alveolar A549 cells, ACE2-expressing A549 cells, embryonic kidney 293 cells, and ACE2-expressing 293 cells, grown in 12-well plates, were infected with MERS-CoV/EMC-2012 at an MOI of 0.1 for 24 h before harvesting of cell-free supernatants for assessing infectious viral titers by the standard Vero E6-based 50% tissue culture infective dose (TCID₅₀) assay. (B) The “one-step” growth curves of MERS-CoV/EMC-2012 and SARS-CoV (Urbani) were established by infecting confluent Calu-3 cells with an MOI of 3, as described above. Cell-free supernatants were harvested at the indicated time points for determining the yields of infectious progeny viruses. (C) Phenotypes of Jurkat T cells transfected with control-versus-CD26/DPP4-encoding plasmid. (D) CD26/DPP4 expression confers the susceptibity of Jurkat T cells to productive MERS-CoV/EMC-2012 infection. (E) Trypan blue exclusion assay was used to simultaneously monitor the viability and mortality of infected Jurkat T cells. **, P < 0.01; ***, P < 0.001 (Student's t test). Data are representative of two independently conducted experiments.

Fig 2

Prior IFN-β treatment effectively inhibits the replication of both SARS-CoV and MERS-CoV and attenuates MERS-CoV-induced CPE. Confluent monolayers of Calu-3 cells, grown in 12-well plates, were treated with recombinant (r) IFN-β for 16 h before being infected with either SARS-CoV or MERS-CoV/EMC-2012 (MOI = 0.1). The resulting cell-free supernatants were harvested at 24 h postinfection (pi) to determine the titers of infectious viruses as described for Fig. 1 (A), whereas the infected monolayer was fixed with paraformaldehyde (4%)–phosphate-buffered saline (PBS) for documentation of MERS-CoV-induced CPE (B). *, P < 0.05; ***, P < 0.001 (Student's t test).

Fig 3

MERS-CoV, but not SARS-CoV, rapidly induces apoptosis of infected Calu-3 cells. Calu-3 cells grown in chamber slides were subjected to either SARS-CoV or MERS-CoV/EMC-2012 (MOI = 0.1) or remained uninfected for 24 h before we analyzed characteristic markers of apoptosis by confocal microscopy. Specifically, the morphology of the nuclei was analyzed after staining with 4′,6-diamidino-2-phenylindole (DAPI), whereas the expression of cleaved caspase 3 (CCP3) was evaluated after staining with Alexa 488-conjugated anti-CCP3 antibodies. The resulting specimens were examined using confocal microscopy at a high magnification. (A and B) Nuclei of mock-infected cells (A) or SARS-CoV-infected cells (B) possess a homogeneously round and smooth morphology without detectable CCP3 expression. (C) In contrast, we readily detected in MERS-CoV/EMC-2012-infected cells prominent nuclear margination, chromatin condensation (arrows), and typical apoptotic bodies (arrowheads) accompanied by positive staining for CCP3 (bright green) in the cytoplasm. (D) Additionally, FITC-conjugated annexin V and propidium iodide (PI) staining was used to discriminate between early apoptotic and late apoptotic/necrotic cells in MERS-CoV/EMC-2012-infected (MOI = 0.1 and 3) and uninfected Calu-3 cultures at both 12 and 24 h pi. Magnification, ×100.

Fig 4

Unbiased apical and basoalteral expression of CD26/DPP4 enables bilateral MERS-CoV entry and release from polarized Calu-3 cells. (A) Confluent Calu-3 cells grown in the chamber slides were subjected to staining for CD26/DPP4 expression. The images of CD26/DPP4 surface expression (green) and nuclear staining (DAPI; blue) were captured by z-stack scanning from the bottom (A1), the middle (A2), and the top (A3) by using an inverted confocal microscope (Zeiss LSM 710). It is clear that CD26/DPP4 is uniformly expressed on the cell membrane of Calu-3 cells. (B and C) Highly polarized Calu-3 cells grown on filter inserts were inoculated with SARS-CoV (B) or MERS-CoV/EMC-2012 (C), at an MOI of 0.1, through either the apical or basolateral surface. At 24 h after infection, the culture supernatants collected from the apical and basolateral chambers were assessed for the contents of infectious viral particles by the TCID₅₀ assay. *, P < 0.05; ***, P < 0.001 (Student's t test). The results are derived from representative findings for two independently conducted experiments.