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. 2013 May;87(10):5502-11.
doi: 10.1128/JVI.00128-13. Epub 2013 Mar 6.

The spike protein of the emerging betacoronavirus EMC uses a novel coronavirus receptor for entry, can be activated by TMPRSS2, and is _targeted by neutralizing antibodies

Stefanie Gierer  1 , Stephanie Bertram, Franziska Kaup, Florian Wrensch, Adeline Heurich, Annika Krämer-Kühl, Kathrin Welsch, Michael Winkler, Benjamin Meyer, Christian Drosten, Ulf Dittmer, Thomas von Hahn, Graham Simmons, Heike Hofmann, Stefan Pöhlmann
Affiliations

The spike protein of the emerging betacoronavirus EMC uses a novel coronavirus receptor for entry, can be activated by TMPRSS2, and is _targeted by neutralizing antibodies

Stefanie Gierer et al. J Virol. 2013 May.

Abstract

The novel human coronavirus EMC (hCoV-EMC), which recently emerged in Saudi Arabia, is highly pathogenic and could pose a significant threat to public health. The elucidation of hCoV-EMC interactions with host cells is critical to our understanding of the pathogenesis of this virus and to the identification of _targets for antiviral intervention. Here we investigated the viral and cellular determinants governing hCoV-EMC entry into host cells. We found that the spike protein of hCoV-EMC (EMC-S) is incorporated into lentiviral particles and mediates transduction of human cell lines derived from different organs, including the lungs, kidneys, and colon, as well as primary human macrophages. Expression of the known coronavirus receptors ACE2, CD13, and CEACAM1 did not facilitate EMC-S-driven transduction, suggesting that hCoV-EMC uses a novel receptor for entry. Directed protease expression and inhibition analyses revealed that TMPRSS2 and endosomal cathepsins activate EMC-S for virus-cell fusion and constitute potential _targets for antiviral intervention. Finally, EMC-S-driven transduction was abrogated by serum from an hCoV-EMC-infected patient, indicating that EMC-S-specific neutralizing antibodies can be generated in patients. Collectively, our results indicate that hCoV-EMC uses a novel receptor for protease-activated entry into human cells and might be capable of extrapulmonary spread. In addition, they define TMPRSS2 and cathepsins B and L as potential _targets for intervention and suggest that neutralizing antibodies contribute to the control of hCoV-EMC infection.

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Figures

Fig 1
Fig 1
EMC-S is expressed in transfected cells and incorporated into lentiviral particles. (A) Plasmids encoding EMC-S with a C-terminal tag were transfected into 293T, HeLa, and BHK-21 cells, as indicated. The transfected cells were lysed, and the lysates were analyzed by Western blotting using tag-specific monoclonal antibodies. Cells transfected with empty plasmid (pcDNA) served as a negative control. (B) SARS-S or EMC-S with a C-terminal V5 tag was coexpressed with HIV-1 Gag protein in 293T cells. Cells expressing Gag-Pol alone were used as a control (pcDNA). At 48 h posttransfection, virus-like particles were harvested, concentrated via ultrafiltration and high-speed centrifugation through a sucrose cushion, and analyzed by Western blotting using a V5-specific antibody and a p55-Gag-reactive antibody as a loading control. The results are representative of at least three independent experiments.
Fig 2
Fig 2
Lentiviral pseudotypes bearing EMC-S transduce a broad spectrum of human cells. Lentiviral pseudotypes carrying the glycoprotein of VSV or EMC as well as pseudotypes bearing no glycoprotein (pCAGGS) were normalized for equal p24 capsid protein content and used for transduction of the indicated human cell lines (A) or unstimulated and PMA-stimulated THP-1 cells and primary human monocyte-derived macrophages (B). Cells were incubated with pseudotypes for 8 h, followed by replacement of the infection medium with fresh culture medium. After 72 h, cell lysates were prepared and analyzed for luciferase activity. The results shown are representative of three (A) or two (B) experiments performed in triplicate, using two independent pseudotype preparations. Error bars indicate standard deviations (SD).
Fig 3
Fig 3
EMC-S does not employ previously described coronavirus receptors for entry into _target cells. 293T cells were transiently transfected with empty plasmid (pCAGGS) or plasmids expressing the indicated coronavirus receptors. The cells were then transduced with infectivity-normalized pseudotypes bearing VSV-G, SARS-S, 229E-S, MHV-S, or EMC-S. Luciferase activities in cell lysates were determined at 72 h postinfection. Similar results were obtained in an independent experiment. Transduction of cells transfected with receptor-encoding plasmids is shown as fold enhancement relative to transduction of cells transfected with empty plasmid, which was set as 1.
Fig 4
Fig 4
EMC-S-driven cellular entry depends on the activity of cathepsin B and/or L. MRC5 _target cells were preincubated with the indicated concentrations of ammonium chloride, MDL28170, dimethyl sulfoxide (DMSO), or phosphate-buffered saline (PBS) for 60 min, followed by inoculation with infectivity-normalized pseudotypes bearing the glycoprotein of VSV, MLV, hCoV-EMC, or EBOV. Luciferase activity in cell lysates was measured at 72 h postinfection. The results of a representative experiment performed in triplicate are shown. Transduction efficiency is shown relative to transduction of PBS-treated cells, which was set as 100%. Error bars indicate SD. Similar results were obtained in another experiment using different pseudotype preparations.
Fig 5
Fig 5
TMPRSS2 activates EMC-S for cathepsin B/L-independent entry into _target cells. (A) 293T cells were transfected with empty plasmid (−control) or cotransfected with plasmids encoding EMC-S and the indicated proteases. Forty-eight hours after transfection, cells were either left untreated or treated with trypsin, and lysates were analyzed by Western blotting using a V5 antibody or a β-actin antibody as a loading control. The results are representative of three independent experiments with different plasmid preparations. (B) Incorporation of cleaved EMC-S forms into lentiviral particles was assessed by coexpression of EMC-S (with a C-terminal V5 tag) with the indicated proteases and HIV-1 Gag. Virus-like particles were concentrated from the culture supernatants and analyzed by Western blotting using a V5 antibody, with a p55-Gag antibody used as a loading control. The experiment shown is representative of three independent experiments. (C) 293T cells were transiently transfected with empty plasmid (pcDNA) or a plasmid encoding TMPRSS2 and used as _target cells for transduction by lentiviral vectors bearing EMC-S or VSV-G (as a control). Prior to infection, _target cells were incubated with the indicated protease inhibitors. Luciferase activities in cell lysates were measured at 72 h postinfection. The average of three experiments (two for VSV-G-mediated transduction of TMPRSS2-expressing cells) performed in triplicate is shown. Transduction of untreated cells was set as 100%. Error bars indicate standard errors of the means (SEM). (D) Caco-2 cells, which express endogenous TMPRSS2, were incubated with DMSO or the indicated protease inhibitors, followed by transduction with lentiviral pseudotypes bearing EMC-S or VSV-G (as a control). At 72 h postinfection, luciferase activities in cell lysates were measured. The results of a representative experiment performed in triplicate are shown; error bars indicate SD. Comparable results were observed in two independent experiments.
Fig 6
Fig 6
Host cell entry driven by EMC-S is neutralized by serum from an hCoV-EMC-infected patient. Pseudotypes bearing the glycoproteins of VSV, SARS-CoV, and hCoV-EMC, normalized for equal infectivity, were preincubated with the indicated serum dilutions in triplicate for 30 min at room temperature. Thereafter, the mixtures were added to 293T-ACE2 cells for 8 h, followed by replacement of the infection medium by fresh culture medium. Luciferase activities were determined after 72 h postinfection. The results shown are representative of three independent experiments. Error bars indicate SD.
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