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. 2018 Jun 10;293(30):11709–11726. doi: 10.1074/jbc.RA118.001897

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© 2018 Chu et al.

Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections.

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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.