Fig 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.

Fig 3: 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.

Fig 4: 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.

Fig 5: 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.