FIG. 3.

DC-SIGN-dependent uptake of the SARS-CoV S-pseudotyped lentiviral vector, and cell-mediated transfer and infection of target cells. (A) Binding of purified SARS-CoV S glycoprotein to cell lines. A total of 10⁶ African green monkey kidney cells (Vero), human T-cell leukemia cells (A3R5 and MT2), or THP-1 myelomonocytic leukemia cells expressing wild-type or mutant forms of DC-SIGN (THP-DC-SIGN or THP-DC-SIGNΔ35, respectively) were incubated with purified S(1190)-Myc-His glycoprotein for 20 min on ice. Binding of S protein to the cells was detected by using an FITC-labeled anti-His (COOH-terminal) antibody (blue) (dilution, 1:100; Invitrogen). A FITC-labeled IgG isotype was used as a control (red). Data were analyzed by flow cytometry. (B) Direct viral entry (left) and cell-mediated virus transfer (right) of the SARS-CoVS-pseudotyped lentiviral vector from THP-1, THP-DC-SIGN, and THP-DC-SIGNΔ35 cells. (Left) Susceptibilities of Vero, A3R5, MT2, THP-1, THP-DC-SIGN, and THP-DC-SIGNΔ35 cells to SARS-CoV S-pseudotyped lentiviral vector infection were measured after transduction by use of the luciferase reporter. (Right) Cell-mediated pseudoviral transfer by THP-1, THP-DC-SIGN, or THP-DC-SIGNΔ35 cells (3 × 10⁴) was also assessed by incubating the cells with the SARS-CoV S-pseudotyped lentiviral vector for 2 h at 37°C, followed by three washes before addition of the respective cells to the indicated target Vero cells at a 1:1 ratio. Cells were collected 72 h later for luciferase assay. (C) Inhibition of direct infection and cell-mediated transfer of SARS-CoV S pseudolentivirus by a mouse anti-SARS-CoV S protein antiserum. (Left) The SARS-CoV S-pseudotyped lentiviral vector was exposed to a mouse control or anti-S specific antiserum at the indicated dilutions for 60 min at 37°C before being added to Vero cells. (Right) For cell-mediated transfer, THP-DC-SIGN cells were incubated with pseudoviruses as described in the legend to panel B, followed by incubation with Vero cells in the presence of a control or anti-SARS-CoV S specific mouse antiserum for 48 h. After 48 h, cells were collected for luciferase assays.