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. 2018 Jul 27;293(30):11709-11726.
doi: 10.1074/jbc.RA118.001897. Epub 2018 Jun 10.

Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

Hin Chu  1   2 , Che-Man Chan  1   2 , Xi Zhang  2 , Yixin Wang  2 , Shuofeng Yuan  2 , Jie Zhou  1   2 , Rex Kwok-Him Au-Yeung  3 , Kong-Hung Sze  1   2 , Dong Yang  2 , Huiping Shuai  2 , Yuxin Hou  2 , Cun Li  2 , Xiaoyu Zhao  2 , Vincent Kwok-Man Poon  2 , Sze Pui Leung  2 , Man-Lung Yeung  1   2   4   5 , Jinghua Yan  6 , Guangwen Lu  7 , Dong-Yan Jin  8 , George Fu Gao  6   9 , Jasper Fuk-Woo Chan  10   2   4   5 , Kwok-Yung Yuen  11   2   4   5   12
Affiliations

Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

Hin Chu et al. J Biol Chem. .

Abstract

Coronavirus tropism is predominantly determined by the interaction between coronavirus spikes and the host receptors. In this regard, coronaviruses have evolved a complicated receptor-recognition system through their spike proteins. Spikes from highly related coronaviruses can recognize distinct receptors, whereas spikes of distant coronaviruses can employ the same cell-surface molecule for entry. Moreover, coronavirus spikes can recognize a broad range of cell-surface molecules in addition to the receptors and thereby can augment coronavirus attachment or entry. The receptor of Middle East respiratory syndrome coronavirus (MERS-CoV) is dipeptidyl peptidase 4 (DPP4). In this study, we identified membrane-associated 78-kDa glucose-regulated protein (GRP78) as an additional binding target of the MERS-CoV spike. Further analyses indicated that GRP78 could not independently render nonpermissive cells susceptible to MERS-CoV infection but could facilitate MERS-CoV entry into permissive cells by augmenting virus attachment. More importantly, by exploring potential interactions between GRP78 and spikes of other coronaviruses, we discovered that the highly conserved human GRP78 could interact with the spike protein of bat coronavirus HKU9 (bCoV-HKU9) and facilitate its attachment to the host cell surface. Taken together, our study has identified GRP78 as a host factor that can interact with the spike proteins of two Betacoronaviruses, the lineage C MERS-CoV and the lineage D bCoV-HKU9. The capacity of GRP78 to facilitate surface attachment of both a human coronavirus and a phylogenetically related bat coronavirus exemplifies the need for continuous surveillance of the evolution of animal coronaviruses to monitor their potential for human adaptations.

Keywords: GRP78; MERS-CoV; attachment factors; bat CoV-HKU9; coronavirus; coronavirus spike; infection; infectious disease; viral attachment; viral evolution; viral infection; virology; virus.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Identification of GRP78 as a target membrane protein of the MERS-CoV spike. A, silver staining of membrane proteins of BEAS2B cells transfected with pcDNA–MERS-CoV–S1–V5. Membrane extracts were immunoprecipitated (IP) with V5 antibody and Sepharose A/G beads, followed by washing and eluting with glycine (lane 1). Sepharose beads were boiled in sample buffer after glycine elution (lane 2). Membrane extracts were immunoprecipitated with mouse isotype control and Sepharose A/G beads (lane 3). B, expression of MERS-CoV–S1-V5 was detected by Western blotting (WB) with an anti-ERS-CoV spike antibody. C, silver staining of membrane proteins of BEAS2B cells. The membrane extracts were immunoprecipitated with purified recombinant MERS-CoV–S1–FLAG protein using anti-FLAG M2 antibody and Sepharose A/G beads, followed by washing and eluting with 3× FLAG peptides (lane 1). Sepharose beads were boiled in sample buffer after 3× FLAG peptide elution (lane 2). Membrane extracts were immunoprecipitated with mouse isotype control and Sepharose A/G beads (lane 3). D, 5 μg of sedimented membrane extracts were run on SDS-PAGE and subjected to Western blots using antibodies against the plasma membrane marker (EGFR and pan-cadherin), endoplasmic reticulum marker (calreticulin), Golgi marker (giantin), and nucleus marker (lamin A). E, gel fragment indicated by the red arrowhead in A and C was excised for LC-MS/MS analysis. MS/MS data were searched against all mammalian protein databases in NCBI and Swiss-Prot. The protein was identified as GRP78 with significant hits over different domains of the sequence.
Figure 2.
Figure 2.
GRP78 interacts with the MERS-CoV spike. A, BHK21 cells were transfected with pcDNA–GRP78–V5 (lanes 1 and 2) or empty vector (lane 3). The cell lysate was immunoprecipitated (IP) with either purified recombinant MERS-CoV–S1–FLAG protein (lanes 1 and 3) or E. coli bacterial alkaline phosphatase (BAP)-FLAG protein (lane 2) pre-adsorbed onto anti-FLAG M2-agarose beads. The precipitated protein complex was detected using the anti-FLAG antibody or the anti-V5 antibody. B, reciprocal co-IP was performed using GRP78 as the bait protein. Purified MERS-CoV–S1–FLAG (lanes 1 and 3) or BAP–FLAG proteins (lane 2) were immunoprecipitated with overexpressed GRP78–V5 or pcDNA–V5 proteins pre-adsorbed on anti-V5 Sepharose beads. The precipitated protein complex was detected using the anti-FLAG antibody or the anti-GRP78 antibody. C, membrane fraction of Huh7 cells was extracted and immunoprecipitated with either MERS-CoV–S1–FLAG(lanes 1 and 3) or BAP–FLAG (lane 2). D, reciprocal co-IP was performed using GRP78 as the bait. Mouse IgG was used in place of the membrane extract as a negative control. E, endogenous co-IP was performed in MERS-CoV- or mock-infected Huh7 and BEAS2B cells. Immunoprecipitation was performed using the anti-GRP78 antibody, the anti-MERS-CoV spike antibody, or the mouse isotype control. The precipitated protein complexes were detected with the anti-MERS-CoV spike antibody or the anti-GRP78 antibody. WB, Western blotting.
Figure 3.
Figure 3.
GRP78 is abundantly expressed on the cell surface of mammalian cells. Surface GRP78 expression was detected on mammalian cell lines with flow cytometry with no cell permeabilization. The immunostaining was performed for human lung cell lines (A), human extrapulmonary cell lines, human primary macrophages, and human primary T cells (B), as well as nonhuman cell lines (C). D, percentage of GRP78-positive cells quantified with DPP4 included for comparisons. E, MFI of GRP78 on the cell surface was quantified with isotype and DPP4 staining included as controls. F, sequence homology between human GRP78 and GRP78 in other mammals. Gates in A–C represented the percentage of GRP78-positive cells. Data in D and E represented mean and standard deviation from three independent experiments.
Figure 4.
Figure 4.
Co-expression of GRP78 and DPP4 in human tissues. Immunostaining of GRP78 and DPP4 was performed on paraffin slides of normal human tissues. GRP78 was labeled with a polyclonal rabbit anti-GRP78 antibody, and DPP4 was labeled with a polyclonal goat anti-DPP4 antibody. Cell nuclei were labeled with DAPI. The co-expression of GRP78 and DPP4 was detected in the bronchus (A), bronchiole (B), and alveolus (C). The co-localization of GRP78 and DPP4 was examined at a higher magnification in D. Images were acquired with a Carl Zeiss LSM 710 system. Bars, 50 μm for A–C. Bars, 5 μm for D.
Figure 5.
Figure 5.
GRP78 is involved in MERS-CoV entry. Pseudovirus antibody blocking assays were performed in Huh7 (A) and BEAS2B (B) cells. A titration of GRP78 or isotype control antibodies from 0 to 2.5 μg/ml was added and pre-incubated with Huh7 and BEAS2B cells for 1 h at 37 °C. MERS–S-pseudovirus or VSV-G–pseudovirus was subsequently added at a ratio of 100 LP per cell for 1 h. Luciferase activity was determined at 72 h post-inoculation and was normalized to that of the mock-treated cells. C, antibody blocking assay was performed in Huh7 cells using infectious MERS-CoV. Huh7 cells were pre-incubated with antibodies at the indicated concentration for 1 h at 37 °C. The cells were then challenged with MERS-CoV at 1 m.o.i. 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 with qPCR, and the result was normalized to that of the mock-treated cells. D, Huh7 or BEAS2B cells were treated with 75 nm GRP78, DPP4, or scrambled siRNA for 2 consecutive days. The knockdown efficiency was evaluated with Western blottings. E, siRNA-treated Huh7 or BEAS2B cells were infected with MERS-CoV at 1 m.o.i. for 1 h at 37 °C. After 1 h, the cells were harvested, and virus entry was evaluated with qPCR analysis. The result was normalized to that of the scrambled siRNA-treated cells. siRNA-treated BEAS2B cells were infected with MERS-CoV at 0.1 m.o.i. for 1 h at 37 °C. The cell lysates (F) and supernatants (G) were harvested at 24 and 48 h post-infection. MERS-CoV replication was evaluated with qPCR analysis. H, siRNA-treated MDM or HFL was infected with MERS-CoV at 1 m.o.i. for 2 h at 37 °C. After 2 h, the cells were harvested, and virus entry was evaluated with qPCR analysis (I). The result was normalized to that of the scrambled siRNA-treated cells. siRNA-treated MDM or HFL was infected with MERS-CoV at 0.1 m.o.i. for 1 h at 37 °C. The cell lysates (J) and supernatants (K) were harvested at 24 h post-infection. MERS-CoV replication was evaluated with qPCR analysis. In all panels, data represented mean and S.D. from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisks when p < 0.05. ns means not significant.
Figure 6.
Figure 6.
GRP78 is an attachment factor of MERS-CoV. A, to assess the role of GRP78 on MERS-CoV attachment, GRP78-overexpressing AD293 and BHK21 cells were challenged with MERS-CoV at 15 m.o.i. for 2 h at 4 °C. After 2 h, the cells were washed, detached with 10 mm EDTA on ice, and fixed in 4% paraformaldehyde before immunolabeling for flow cytometry. B, percentage of MERS-CoV N–positive AD293 and BHK21 cells was quantified for MERS-CoV attachment. C, to assess the role of GRP78 on MERS-CoV entry, GRP78-overexpressing AD293 and BHK21 cells were challenged with MERS-CoV at 5 m.o.i. for 2 h at 37 °C. After 2 h, the inoculum was replaced with culture media, and the cells were incubated for another 4 h before harvesting for flow cytometry. D, percentage of MERS-CoV N–positive AD293 and BHK21 cells was quantified for MERS-CoV entry. B and D, percentage of MERS-CoV N–positive cells among GRP78-transfected (GRP78+) cells was calculated as (%GRP78+N+ cells/(%GRP78+N+ cells + %GRP78+N− cells)) × 100%. The percentage of MERS-CoV N–positive cells among GRP78-nontransfected (GRP78−) cells was calculated as (%GRP78−N+ cells/(%GRP78−N+ cells + %GRP78−N− cells)) × 100%. Data represented mean and S.D. derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisks when p < 0.05. ns means not significant.
Figure 7.
Figure 7.
GRP78 is up-regulated on the surface of MERS-CoV–infected cells. A, Huh7 cells were infected with MERS-CoV at 0.01 and 0.1 m.o.i. and were harvested for flow cytometry analysis at 24 h post-infection. B, percentage of MERS-CoV N–positive cells was quantified. C, in parallel, cell surface and total DPP4 and GRP78 among mock- or MERS-CoV–infected samples were analyzed by flow cytometry. D, percentage of DPP4-positive cells and GRP78-positive cells in mock- or MERS-CoV–infected samples were quantified. Total DPP4 and GRP78 staining was performed by first permeabilizing the cells with 0.1% Triton X-100, whereas the surface DPP4 and GRP78 staining was performed in the absence of cell permeabilization. The gate in A represented the percentage of MERS-CoV N–positive cells. The gates in C represented the percentage of DPP4- (upper panels) and GRP78 (lower panels)-positive cells. Data represented mean and S.D. derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisks when p < 0.05. ns means not significant.
Figure 8.
Figure 8.
GRP78 interacts with the bCoV-HKU9 spike and serves as an attachment factor for bCoV-HKU9. A, BHK21 cells were transfected with pcDNA–GRP78–V5 (lanes 1 and 2) or empty vector (lane 3). Co-IP between GRP78 and bCoV-HKU9 spike was performed using GRP78 as the bait protein. Purified bCoV–HKU9–S1–FLAG (lanes 1 and 3) or BAP–FLAG proteins (lane 2) were immunoprecipitated (IP) with overexpressed GRP78–V5 or pcDNA–V5 proteins pre-adsorbed on anti-V5–Sepharose beads. The precipitated protein complex was detected using the anti-V5 antibody or the anti-FLAG antibody. B, co-IP between GRP78 and SARS-CoV spike was performed using GRP78 as the bait protein. Purified SARS-CoV–S1–FLAG (lanes 1 and 3) or BAP–FLAG proteins (lane 2) were immunoprecipitated with overexpressed GRP78–V5 or pcDNA–V5 proteins pre-adsorbed on anti-V5–Sepharose beads. The precipitated protein complex was detected using the anti-V5 antibody or the anti-FLAG antibody. C, HKU9–S-pseudovirus entry assays were performed in a number of mammalian cell lines. Mock-inoculated and MERS–S-pseudovirus–inoculated cells were included as negative and positive controls, respectively. HKU9–S-pseudovirus and MERS–S-pseudovirus were added at a ratio of 100 LP per cell for 1 h. Luciferase activity was determined at 72 h post-inoculation. D, HKU9–S-pseudovirus attachment efficiency was evaluated in Caco2 and RLK cells. HKU9–S-pseudovirus was inoculated on Caco2 and RLK cells at 100 LP per cell for 2 h at 4 °C. After 2 h, the cells were washed, fixed, and immunolabeled for flow cytometry. HKU9–S-pseudovirus binding was identified with an in-house mouse bCoV-HKU9 spike immune serum. E, HKU9–S-pseudovirus entry in L929 and BHK21 cells was assessed with or without GRP78 overexpression. HKU9–S-pseudovirus was inoculated at 100 LP per cell for 1 h at 37 °C. Luciferase activity was determined at 72 h post-inoculation. F and G, antibody-blocking assay for HKU9–S-pseudovirus binding was performed in RLK cells. RLK cells were pre-incubated with the rabbit anti-GRP78 antibody and the rabbit control IgG from 0 to 5 μg/ml. After the pre-incubation, HKU9–S-pseudovirus was inoculated to the cells at 100 LP per cell for 2 h at 4 °C. The cells were then washed, fixed, and immunolabeled for flow cytometry. HKU9–S-pseudovirus binding was identified with an in-house mouse bCoV-HKU9 spike immune serum. The percentage of bCoV-HKU9 spike-positive cells was quantified in H, and the MFI of the bCoV-HKU9 spike on the cell surface was quantified in I. Gates in D, F, and G represented the percentage of HKU9 spike-positive cells. Data represented mean and S.D. derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisks when p < 0.05. WB, Western blot. ns means not significant.
Figure 9.
Figure 9.
Sialic acids and GRP78 act independently to facilitate the surface attachment of MERS-CoV. A, Huh7 cells were treated with neuraminidase from C. perfringens, with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with MERS–S-pseudovirus and assessed at 72 h post-infection for pseudovirus entry. B, RLK cells were treated with neuraminidase from Clostridium perfringens, with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with HKU9–S-pseudovirus and assessed at 72 h post infection for pseudovirus entry. Pseudovirus entry was quantified using a microplate reader as relative light units (RLU). Data represented mean and standard deviation derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisk marks when p < 0.05.
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