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. 2016 Sep 29;90(20):9114-27.
doi: 10.1128/JVI.01133-16. Print 2016 Oct 15.

Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5 Is an Important Surface Attachment Factor That Facilitates Entry of Middle East Respiratory Syndrome Coronavirus

Che-Man Chan  1 , Hin Chu  1 , Yixin Wang  2 , Bosco Ho-Yin Wong  2 , Xiaoyu Zhao  2 , Jie Zhou  1 , Dong Yang  2 , Sze Pui Leung  2 , Jasper Fuk-Woo Chan  3 , Man-Lung Yeung  1 , Jinghua Yan  4 , Guangwen Lu  4 , George Fu Gao  4 , Kwok-Yung Yuen  5
Affiliations

Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5 Is an Important Surface Attachment Factor That Facilitates Entry of Middle East Respiratory Syndrome Coronavirus

Che-Man Chan et al. J Virol. .

Abstract

The spike proteins of coronaviruses are capable of binding to a wide range of cellular targets, which contributes to the broad species tropism of coronaviruses. Previous reports have demonstrated that Middle East respiratory syndrome coronavirus (MERS-CoV) predominantly utilizes dipeptidyl peptidase 4 (DPP4) for cell entry. However, additional cellular binding targets of the MERS-CoV spike protein that may augment MERS-CoV infection have not been further explored. In the current study, using the virus overlay protein binding assay (VOPBA), we identified carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) as a novel cell surface binding target of MERS-CoV. CEACAM5 coimmunoprecipitated with the spike protein of MERS-CoV in both overexpressed and endogenous settings. Disrupting the interaction between CEACAM5 and MERS-CoV spike with anti-CEACAM5 antibody, recombinant CEACAM5 protein, or small interfering RNA (siRNA) knockdown of CEACAM5 significantly inhibited the entry of MERS-CoV. Recombinant expression of CEACAM5 did not render nonpermissive baby hamster kidney (BHK21) cells susceptible to MERS-CoV infection. Instead, CEACAM5 overexpression significantly enhanced the attachment of MERS-CoV to the BHK21 cells. More importantly, the entry of MERS-CoV was increased when CEACAM5 was overexpressed in permissive cells, which suggested that CEACAM5 could facilitate MERS-CoV entry in conjunction with DPP4 despite not being able to support MERS-CoV entry independently. Taken together, the results of our study identified CEACAM5 as a novel cell surface binding target of MERS-CoV that facilitates MERS-CoV infection by augmenting the attachment of the virus to the host cell surface.

Importance: Infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with the highest mortality rate among all known human-pathogenic coronaviruses. Currently, there are no approved vaccines or therapeutics against MERS-CoV infection. The identification of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) as a novel cell surface binding target of MERS-CoV advanced our knowledge on the cell binding biology of MERS-CoV. Importantly, CEACAM5 could potentiate the entry of MERS-CoV by functioning as an attachment factor. In this regard, CEACAM5 could serve as a novel target, in addition to dipeptidyl peptidase-4 (DPP4), in the development of antiviral strategies for MERS-CoV.

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Figures

FIG 1
FIG 1
CEACAM5 is a cell surface binding protein of MERS-CoV. (A) A549 and NIH 3T3 biotinylated cell membrane proteins were extracted, separated by 4 to 12% gradient gel, and transferred to PVDF membranes. WB, Western blotting. (B) Membrane proteins were probed with MERS-S-pseudotyped virus followed by detection of virus binding by incubation with immune serum directed against the MERS-CoV spike protein (left lane). Nonsusceptible cell membrane extracts from NIH 3T3 was included as a negative control (right lane). (C) The gel fractions corresponding to the positive signal at approximately 90 kDa were cut, electroeluted, and confirmed by VOPBA prior to submission for MS protein identification. (B and C) The position of CEACAM5 is indicated with asterisks. (D) Identified amino acid sequences and their corresponding positions.
FIG 2
FIG 2
Surface expression of CEACAM5 on mammalian cells. (A) The indicated human cell lines were fixed in 4% paraformaldehyde and immunolabeled for surface DPP4 and CEACAM5 expression. The shaded curves and the open curves represent isotype- and antigen-specific staining, respectively. Horizontal lines represent the gating for DPP4+ or CEACAM5+ cells. The numbers represent the percentage of positive cells. (B and C) The same fixation and immunostaining procedures were performed for human primary T cells (B) and a number of nonhuman mammalian cells (C). (D) Average percentages of DPP4/CEACAM5-positive cells. (E) Average DPP4/CEACAM5 MFIs were quantified. Rabbit isotype IgG was used in place of antigen-specific antibodies for the controls. (D and E) Shown are means and standard deviations of the results of three independent experiments.
FIG 3
FIG 3
Expression of CEACAM5 in human lung tissues. Immunostaining of CEACAM5 and DPP4 was performed on paraffin slides of normal human lung tissues. Representative images of CEACAM5 and DPP4 expression in various regions of the human lung are shown, including the bronchi (A), small airways (B), and alveoli (C). The arrowheads indicate alveolar macrophages with CEACAM5 and DPP4 coexpression. Bars, 50 μm.
FIG 4
FIG 4
CEACAM 5 interacts with MERS-CoV spike. (A) Surface and intracellular expression of CEACAM5-V5 were verified by flow cytometry. (B) For co-IP, BHK21 cells were transfected with CEACAM5-V5 (lanes 1 and 2 from left) or empty vector (lane 3). The cell lysate was immunoprecipitated with MERS-CoV S1-FLAG (lanes 1 and 3) or E. coli BAP-FLAG protein (lane 2) preadsorbed onto anti-FLAG M2 agarose beads prior to SDS-PAGE. The protein complex was detected by using the anti-FLAG antibody or the anti-V5 antibody. (C) Reciprocal co-IP was performed using CEACAM5-V5 as the bait. Purified MERS-CoV S1-FLAG (lanes 1 and 3) or BAP-FLAG (lane 2) proteins were immunoprecipitated with overexpressed CEACAM5-V5 or pcDNA-V5 protein preadsorbed onto anti-V5 Sepharose beads. Western blots were detected with the anti-FLAG or the anti-CEACAM5 antibody. (D) Endogenous co-IP was performed with MERS-CoV-infected or mock-infected Huh7 cell lysates using the rabbit anti-CEACAM5 antibody, the rabbit anti-MERS-CoV spike antibody, or the rabbit isotype IgG. Western blots were detected with the rabbit anti-MERS-CoV spike antibody or the rabbit anti-CEACAM5 antibody.
FIG 5
FIG 5
CEACAM5-specific antibody blocks MERS-CoV entry and replication. (A) The antibody blocking assay was performed in Calu3 cells using MERS-S-pseudotyped virus. Antibodies were diluted to 2 μg/ml and incubated with Calu3 cells for 1 h at 37°C. The pseudotyped viruses were then added at a ratio of 100 LP per cell for 1 h. Luciferase activity was determined at 48 h postinfection and was normalized to that of the mock-treated cells. (B) The antibody blocking assay was performed in Calu3 cells using MERS-CoV. Calu3 cells were preincubated with antibodies at the indicated concentrations for 1 h at 37°C. The cells were then inoculated with MERS-CoV at an MOI of 1 for 1 h at 37°C in the presence of the antibodies. After 1 h, the cells were washed and harvested. MERS-CoV entry was assessed by qPCR, and the result was normalized to that of the mock-treated cells. (C) Calu3 cells were treated with CEACAM5 antibody for a total of 2 h during preincubation and virus inoculation (−1 to 1) or after virus inoculation (1 to 3). MERS-CoV entry was assessed by qPCR. (D) The antibody blocking assay was performed in Caco2 cells. (E and F) The impact of CEACAM5 inhibition on MERS-CoV replication was investigated in Huh7 cells. Huh7 cells were preincubated with antibodies at 2 μg/ml for 1 h at 37°C. The cells were then inoculated with MERS-CoV at an MOI of 0.0005 for 1 h at 37°C in the presence of the antibodies. At the end of the inoculation period, the inoculum was replaced with culture medium containing the indicated antibodies. Cell lysates (E) and supernatants (F) were harvested at 1, 24, and 48 h postinfection. The virus genome copy number was determined with qPCR. ND, virus was not detected. The data are represented as means and standard deviations of the results of three independent experiments. The results for the anti-DPP4-, anti-CEACAM5-, and control IgG-treated samples were compared with those for the mock-treated samples. Statistical analyses were carried out using Student's t test. Statistical significance is indicated by the asterisks (P < 0.05).
FIG 6
FIG 6
CEACAM5 recombinant protein and siRNA knockdown of CEACAM5 inhibit MERS-CoV entry. (A and B) MERS-CoV at an MOI of 0.1 was preincubated with human recombinant proteins at the indicated concentrations for 1 h at 37°C. After the preincubation, the protein-virus mixture was added to Calu3 cells (A) or Huh7 cells (B) for 2 h at 37°C. The cell lysates were subsequently harvested for qPCR analysis. Human recombinant DPP4 and human recombinant IgG were included as positive and negative controls, respectively. Results from the human recombinant DPP4-, human recombinant CEACAM5-, and control human recombinant IgG-treated samples were compared with those of the mock-treated samples. (C) Huh7 cells were treated with CEACAM5 protein for a total of 3 h during preincubation and virus inoculation (−1 to 2) or after virus inoculation (2 to 5). MERS-CoV entry was assessed by qPCR. (D) Huh7 cells were treated with 100 nM gene-specific or scrambled siRNA for two consecutive days. (E and F) Summary of the reduction of surface CEACAM5 and DPP4 expression. (G) siRNA-treated Huh7 cells were subjected to MERS-CoV infection at an MOI of 1 for 2 h at 37°C. The cell lysates were subsequently harvested for qPCR analysis, and the result was normalized to those of the mock-treated cells. The results from the DPP4 siRNA-, CEACAM5 siRNA-, and scrambled-siRNA-treated samples were compared with those of the mock-treated samples. The data are represented as means and standard deviations of the results of three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance is indicated by the asterisks (P < 0.05).
FIG 7
FIG 7
CEACAM5 is an attachment factor for MERS-CoV. (A and B) To assess whether CEACAM5 is important for MERS-CoV entry, human CEACAM5 was overexpressed in BHK21 cells. Human CEACAM5-expressing BHK21 cells were then challenged with MERS-CoV at an MOI of 5 for 2 h at 37°C. The inoculum was replaced with culture medium, and the cells were incubated for another 4 h before being harvested for flow cytometry. (C and D) To assess whether CEACAM5 is important for MERS-CoV attachment, human CEACAM5-expressing BHK21 cells were challenged with MERS-CoV at an MOI of 30 for 2 h at 4°C before being harvested for flow cytometry. Human DPP4-expressing BHK21 cells were included as controls for both entry and attachment assays. (B and D) The data are represented as the percentages of MERS-CoV NP-positive BHK21 cells after infection with or without DPP4/CEACAM5 overepression. The means and standard deviations were derived from the results of three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance is indicated by the asterisks (P < 0.05).
FIG 8
FIG 8
CEACAM5 overexpression does not confer infectivity to nonpermissive cells on MERS-CoV but enhances MERS-CoV entry into permissive cells. (A and C) To verify the role of CEACAM5 in MERS-CoV entry, human CEACAM5 was overexpressed in BHK21 cells or AD293 cells. The cells were then challenged with MERS-CoV at an MOI of 1 for 2 h at 37°C. After 2 h, the cells were washed extensively and harvested for qPCR analysis. (B and D) To verify the role of CEACAM5 in MERS-CoV attachment, human CEACAM5-expressing BHK21 cells or human CEACAM5-expressing AD293 cells were challenged with MERS-CoV at an MOI of 1 for 2 h at 4°C. The cell lysates were then harvested for qPCR analysis. DPP4- and empty-vector (pcDNA3.1)-expressing BHK21 cells were included as controls. The data are represented as means and standard deviations of the results of three independent experiments. The results from the human DPP4-, human CEACAM5-, and empty-vector-overexpressing samples were compared with those of the mock-treated samples. Statistical analyses were carried out using Student's t test. Statistical significance is indicated by the asterisks (P < 0.05).
FIG 9
FIG 9
CEACAM5 overexpression does not facilitate SARS-CoV entry in VeroE6 cells. (A) VeroE6 cells were transfected with a CEACAM5-expressing plasmid or an empty vector. (B) The VeroE6 cells were challenged with SARS-S pseudovirus or VSV-G pseudovirus at a ratio of 100 LP per cell for 1 h. Luciferase activity from CEACAM5-transfected VeroE6 cells was determined at 48 h after pseudovirus challenge and was normalized to that of the empty-vector-transfected VeroE6 cells. (B) Means and standard deviations were derived from the results of three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance is indicated by the asterisks (P < 0.05).
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