Middle East Respiratory Syndrome Coronavirus Efficiently Infects Human Primary T Lymphocytes and Activates the Extrinsic and Intrinsic Apoptosis Pathways

Abstract

Middle East respiratory syndrome (MERS) is associated with a mortality rate of >35%. We previously showed that MERS coronavirus (MERS-CoV) could infect human macrophages and dendritic cells and induce cytokine dysregulation. Here, we further investigated the interplay between human primary T cells and MERS-CoV in disease pathogenesis. Importantly, our results suggested that MERS-CoV efficiently infected T cells from the peripheral blood and from human lymphoid organs, including the spleen and the tonsil. We further demonstrated that MERS-CoV infection induced apoptosis in T cells, which involved the activation of both the extrinsic and intrinsic apoptosis pathways. Remarkably, immunostaining of spleen sections from MERS-CoV-infected common marmosets demonstrated the presence of viral nucleoprotein in their CD3(+) T cells. Overall, our results suggested that the unusual capacity of MERS-CoV to infect T cells and induce apoptosis might partly contribute to the high pathogenicity of the virus.

Keywords: MERS-CoV; T lymphocytes; apoptosis; caspase; marmosets; spleen; tonsil.

Figure 1.

Middle East respiratory syndrome coronavirus (MERS-CoV) differentially infects subsets of human peripheral blood mononuclear cells (PBMCs). A, Human PBMCs were infected with MERS-CoV at 2 50% tissue culture infective doses per cell. At 24 hours after infection, infected cells were fixed with 4% paraformaldehyde (PA) and immunolabeled for detection of cell surface markers and MERS-CoV nucleoprotein (NP). The shaded curve and the solid line represent MERS-CoV NP expression from mock-infected and MERS-CoV–infected cells, respectively. The summary panel at the right represents the average percentage of infected cells from 3 different donors. B, Uninfected human PBMCs were fixed with 4% PA and immunolabeled for detection of surface DPP4 expression. The shaded curve and the solid line represented isotype and DPP4-specific staining, respectively. The summary panel at the right represents the average mean fluorescent intensity (MFI) from 3 different donors. In all panels, bars and error bars represented means and standard deviations, respectively. Statistical analyses were performed using the Student t test. *P < .001. Abbreviation: NK, natural killer.

Figure 2.

Middle East respiratory syndrome coronavirus (MERS-CoV) efficiently infects CD4⁺ and CD8⁺ T cells and downregulates surface dipeptidyl peptidase 4 (DPP4) in the infected cells. A, T cells were infected with MERS-CoV and severe acute respiratory syndrome CoV (SARS-CoV) at 2 50% tissue culture infective doses (TCID₅₀) per cell. Cells were fixed at 24 hours after infection and immunolabeled for detection of expression of MERS-CoV NP or SARS-CoV NP. The shaded curve and the solid line represent virus NP expression from mock-infected and MERS/SARS-CoV–infected cells, respectively. B, T cells were infected with MERS-CoV at 2 TCID₅₀ per cell. Cells were fixed at 24 hours after infection and immunolabeled for detection of CD4 or CD8 and MERS-CoV NP expression. The shaded curve and the solid line represent NP expression from mock-infected and MERS-CoV–infected cells, respectively. C, Uninfected T cells were fixed and immunolabeled for detection of CD4 or CD8 and surface DPP4 expression. The shaded curve and the solid line represent isotype and DPP4-specific staining, respectively. D, T cells were infected with MERS-CoV at 2 TCID₅₀ per cell. Cells were fixed at 24 hours after infection and immunolabeled for detection of CD4 or CD8, DPP4, and MERS-CoV NP expression. Surface or total DPP4 was detected by labeling the cells with the DPP4 antibody before or after cell permeabilization, respectively. The shaded curve represents isotype staining of DPP4. The solid line and dotted line represent DPP4 staining from mock-infected and MERS-CoV–infected cells, respectively. The DPP4 mean fluorescence intensity (MFI) in infected cells was calculated on the basis of CD4⁺/MERS-CoV NP–expressing or CD8⁺/MERS-CoV NP–expressing double-positive T-cell data. The DPP4 MFI in mock-infected cells was calculated on the basis of CD4⁺ or CD8⁺ T-cell data. The summary panels at the right represent the average percentage of infected cells (A and B), MFI (C), or percentage of mock MFI (D) from 3 different donors. In all panels, bars and error bars represent means and standard deviations. Statistical analyses were performed using the Student t test. *P < .05.

Figure 3.

Middle East respiratory syndrome coronavirus (MERS-CoV) induces apoptosis and substantial upregulation of active caspase 3 in infected T cells. A, T cells were infected with MERS-CoV or severe acute respiratory syndrome CoV (SARS-CoV) at 2 50% tissue culture infective doses (TCID₅₀) per cell. Cells were fixed at the indicated time points and immunolabeled for detection of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)–positive cells. B, The average percentage of TUNEL-positive cells from 3 different donors. C, T cells were infected with MERS-CoV at 2 TCID₅₀ per cell. Cells were fixed at the indicated time points and immunolabeled for detection of MERS-CoV nucleoprotein (NP) and active caspase 3. D, The average percentage of active caspase 3–positive cells among MERS-CoV NP⁺ cells from 3 different donors. In all panels, bars and error bars represent means and standard deviations. Statistical analyses were performed using the Student t test. *P < .01.

Figure 4.

The extrinsic and intrinsic caspase-dependent apoptosis pathways are activated in Middle East respiratory syndrome coronavirus (MERS-CoV)–infected T cells. T cells were infected with MERS-CoV at 2 50% tissue culture infective doses per cell. At the indicated time points, infected cells were labeled with MERS-CoV nucleoprotein (NP), as well as FITC-IETD-FMK (for caspase 8; A and B) and FITC-LEHD-FMK (for caspase 9; C and D) for 1 hour at 37°C. The average percentage of active caspase 8–positive (B) or active caspase 9–positive (D) cells among MERS-CoV NP–expressing cells from 3 different donors was illustrated. In all panels, bars and error bars represent means and standard deviations. Statistical analyses were performed using the Student t test. *P < .05.

Figure 5.

Middle East respiratory syndrome coronavirus (MERS-CoV)–induced caspase 3 activation in T cells occurs rapidly upon infection and is not inhibited by UV inactivation of the virus. A, T cells and Vero E6 cells were infected with MERS-CoV at 2 50% tissue culture infective doses (TCID₅₀) per cell. Cells were fixed at the indicated time points and immunolabeled for detection of MERS-CoV nucleoprotein (NP) and active caspase 3. B, T cells and Vero E6 cells were infected with MERS-CoV at 2 TCID₅₀ per cell. In parallel, equal volumes of UV-inactivated MERS-CoV were applied to Vero E6 cells and T cells. At 24 hours after infection, cells were harvested, fixed, and immunolabeled for detection of MERS-CoV NP and active caspase 3. The illustrated result was a representative of 2 independent experiments that showed similar results.

Figure 6.

T cells in human lymphatic organs, including the spleen and the tonsil, are highly susceptible to Middle East respiratory syndrome coronavirus (MERS-CoV) and MERS-CoV–induced apoptosis. Disassociated human spleen and tonsil cells were infected with MERS-CoV at 2 50% tissue culture infective doses per cell. Cells were fixed at 24 hours after infection and assessed for MERS-CoV nucleoprotein (NP; A), terminal deoxynucleotidyl transferase dUTP nick end labeling activity (B), and active caspase 3 (C) among CD3⁺ cells. The illustrated result was a representative of 2 independent experiments that showed similar results.

Figure 7.

Middle East respiratory syndrome coronavirus (MERS-CoV) infects CD3⁺ T cells in the spleen of infected common marmosets. Immunostaining was performed on paraformaldehyde-fixed and paraffin-embedded sections of spleen obtained on day 1 after virus challenge of animals. Colocalization between MERS-CoV nucleoprotein (NP; D and G) and CD3 (E and H) were detected in the spleen of the infected animals (arrows; D–F and G–I). CM1 and CM2 represent 2 individual common marmosets. MERS-CoV NP was not detected in the preimmune serum control (A–C). Bars represent 25 µm.