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. 2004 Nov 2;101(44):15748-53.
doi: 10.1073/pnas.0403812101. Epub 2004 Oct 20.

CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus

Scott A Jeffers  1 , Sonia M Tusell, Laura Gillim-Ross, Erin M Hemmila, Jenna E Achenbach, Gregory J Babcock, William D Thomas Jr, Larissa B Thackray, Mark D Young, Robert J Mason, Donna M Ambrosino, David E Wentworth, James C Demartini, Kathryn V Holmes
Affiliations

CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus

Scott A Jeffers et al. Proc Natl Acad Sci U S A. .

Abstract

Angiotensin-converting enzyme 2 (ACE2) is a receptor for SARS-CoV, the novel coronavirus that causes severe acute respiratory syndrome [Li, W. Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran, M., Sullivan, J. L., Luzuriaga, K., Greenough, T. C., et al. (2003) Nature 426, 450-454]. We have identified a different human cellular glycoprotein that can serve as an alternative receptor for SARS-CoV. A human lung cDNA library in vesicular stomatitis virus G pseudotyped retrovirus was transduced into Chinese hamster ovary cells, and the cells were sorted for binding of soluble SARS-CoV spike (S) glycoproteins, S(590) and S(1180). Clones of transduced cells that bound SARS-CoV S glycoprotein were inoculated with SARS-CoV, and increases in subgenomic viral RNA from 1-16 h or more were detected by multiplex RT-PCR in four cloned cell lines. Sequencing of the human lung cDNA inserts showed that each of the cloned cell lines contained cDNA that encoded human CD209L, a C-type lectin (also called L-SIGN). When the cDNA encoding CD209L from clone 2.27 was cloned and transfected into Chinese hamster ovary cells, the cells expressed human CD209L glycoprotein and became susceptible to infection with SARS-CoV. Immunohistochemistry showed that CD209L is expressed in human lung in type II alveolar cells and endothelial cells, both potential targets for SARS-CoV. Several other enveloped viruses including Ebola and Sindbis also use CD209L as a portal of entry, and HIV and hepatitis C virus can bind to CD209L on cell membranes but do not use it to mediate virus entry. Our data suggest that the large S glycoprotein of SARS-CoV may use both ACE2 and CD209L in virus infection and pathogenesis.

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Figures

Fig. 1.
Fig. 1.
Expression of SARS-CoV genomic and messenger RNA in CHO cells transduced with a human lung cDNA library, and binding of SARS-CoV spike glycoprotein to cell membranes. (A) Multiplex RT-PCR at 1 and 16 h PI with SARS-CoV. Multiplex RT-PCR was performed as in ref. . Control CHO cells or CHO cells transduced with retrovirus pseudotypes containing a human lung cDNA library were inoculated with SARS-CoV or mock inoculated. The viral genomic and subgenomic RNAs and cellular mRNA encoding GAPDH were reverse transcribed and amplified. Negative images of the amplicons are shown. The bottom gel shows Vero E6 monkey kidney cells, a positive control for SARS-CoV infection, in which subgenomic viral RNA increased at 16 h PI. The minute amount of subgenomic viral RNA in the 1-h sample was probably due to a low level of subgenomic viral RNA contamination of the input virus. In the fourth lane of the upper gel, CHO library cells that were sorted twice for S590 binding also showed an increase in subgenomic viral RNA at 16 h PI. These cells were further sorted for S590 binding, and single-cell clones were produced. Four of these cell lines showed an increase in subgenomic viral RNA at 16 h PI: clones 2.15, 2.22, 2.27, and 2.37. The proviral integrants were amplified by RT-PCR of RNA from the cloned cell lines. Sequencing results in Table 1 showed that all four of the SARS-CoV-susceptible cell lines contained CD209L. Clone 2.27 cells that had the largest increase in subgenomic viral RNA were further studied. (B) Flow cytometric analysis of soluble SARS-CoV S1180 protein binding to CHO (Upper) and clone 2.27 (Lower) cells.
Fig. 2.
Fig. 2.
Expression of CD209L protein by CHO cells transduced with a human lung cDNA library. (A) Flow cytometric analysis of clone 2.27 cells with antibodies specific for CD209L and DC-SIGN. Antibodies specific for DC-SIGN, CD209L, and an antibody that recognizes both DC-SIGN and CD209L were used to determine which glycoprotein was expressed on clone 2.27 cells. Only the antibodies that recognized CD209L bound to the clone 2.27 cells. (B) Immunoblots for ACE2 or CD209L of cell lines used in this study. Cell lysates from CHO cells, clone 2.27 cells, CHO cells stably expressing CD209L, CHO cells transiently transfected (Tf) with CD209L, and Vero E6 cells were run on a 10% SDS/PAGE gel and blotted onto poly(vinylidene difluoride). The blots were probed with polyclonal antibodies specific for the N or C termini of CD209L or for ACE2. The antibodies were detected by horseradish peroxidase-conjugated donkey anti-goat IgG antibodies and chemiluminescence.
Fig. 3.
Fig. 3.
Multiplex RT-PCR analysis of cell lines at various time points after SARS-CoV inoculation or mock inoculation. (A) Multiplex RT-PCR analysis was performed as in Fig. 1 on samples collected at 1, 24, 48, and 72 h PI from clone 2.27, Vero E6, and CHO cells (A) and from CHO cells transiently transfected (Tf) with a plasmid containing CD209L cDNA (B). Negative images are shown.
Fig. 4.
Fig. 4.
Immunofluorescent labeling of SARS-CoV N protein in CHO and clone 2.27 cells inoculated with SARS-CoV. Scattered infected cells showed cytoplasmic expression of the virus nucleocapsid protein.
Fig. 5.
Fig. 5.
Expression of CD209L in human lung. (A) Localization of CD209L to the capillaries (arrows) in the alveolar wall by immunohistochemistry. (B) DAPI (4′,6-diamidino-2-phenylindole) staining to demonstrate cell nuclei along the alveolar wall. (C) Expression of CD209L in hyperplastic type II alveolar (arrow) and endothelial (red arrow) cells by peroxidase immunohistochemistry in a lung of a cigarette smoker. (D) There was no labeling of these cells with control goat IgG.
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