DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus

Abstract

The lectins DC-SIGN and DC-SIGNR can augment viral infection; however, the range of pathogens interacting with these attachment factors is incompletely defined. Here we show that DC-SIGN and DC-SIGNR enhance infection mediated by the glycoprotein (GP) of Marburg virus (MARV) and the S protein of severe acute respiratory syndrome coronavirus and might promote viral dissemination. SIGNR1, a murine DC-SIGN homologue, also enhanced infection driven by MARV and Ebola virus GP and could be targeted to assess the role of attachment factors in filovirus infection in vivo.

FIG. 1.

Modulation of viral infection by lectin expression. (A) Characterization of cell lines expressing lectins upon induction. The indicated cell lines were generated by stable transfection of T-REx cells (Invitrogen). Lectin expression was induced by doxycycline (0.1 μg/ml) and quantified by fluorescence-activated cell sorting with a monoclonal antibody directed against a C-terminal AU-1 antigenic tag. Control cells are shown in light grey, uninduced cells are shown in dark grey, and doxycycline-induced cells are shown in black. (B) Infection of lectin-expressing cell lines. Lectin expression was induced with doxycycline, and the indicated cell lines were infected with lentiviral pseudotypes bearing the indicated glycoproteins. The lentiviral genome encodes the luciferase gene which is expressed under control of the viral promoter upon integration of the viral genome into the cellular chromosome. Luciferase activity in cell lysates was quantified 3 days after infection. Infection of lectin-expressing cells is presented relative to infection of T-REx control cells. Upon infection of control cells with pseudotypes bearing SARS-CoV S, MARV GP, VSV-G, or no GP, 548, 2,125, 2,197 and 35 cps were measured. A representative experiment is shown; similar results were obtained in two independent experiments. Error bars indicate standard deviations (SD). EBOZ, EBOV Zaire subspecies. (C) Inhibition of infection of DC-SIGN-expressing T-REx cells. DC-SIGN-expressing and control T-REx cells were induced with doxycycline, preincubated for 30 min with the indicated antibodies or mannan at a final concentration of 20 μg/ml, and infected with SARS-CoV S-bearing pseudotypes. Luciferase activity was assessed 3 days after infection. The results are shown relative to inhibition with control murine IgG; similar results were obtained in an independent experiment.

FIG. 2.

SARS-CoV S1 interactions with transiently expressed ACE2, DC-SIGN, or DC-SIGNR. 293T cells were transiently calcium phosphate transfected with the indicated receptors and incubated with purified S1-Ig fusion protein and anti-DC-SIGN MAb (DC-11) or anti-ACE2 antiserum at 0.1 μg/ml. Following washing, ACE2 expression, lectin expression, and S1-Ig binding were analyzed by flow cytometry.

FIG. 3.

Interaction of SARS-CoV S-bearing pseudotypes with DC-SIGN and DC-SIGNR expressed on nonpermissive cells. (A) Infection of nonpermissive QT6 cells expressing DC-SIGN, DC-SIGNR, or ACE2. Quail-derived QT6 cells were transiently transfected with the indicated expression vectors and infected with pseudotypes bearing SARS-CoV S, VSV-G, or EBOV subspecies Zaire (EBOZ) GP. Luciferase activity in cell lysates was determined 3 days after infection. Results are presented relative to entry into control transfected cells. A representative experiment is shown; similar results were obtained in an independent experiment. Error bars indicate SD. (B) DC-SIGN-mediated transmission of SARS-CoV S-bearing pseudotypes. B-THP cells expressing DC-SIGN or control B-THP cells were incubated with SARS-CoV S-bearing pseudotypes in the presence or absence of mannan, washed, and cocultivated with Huh-7 target cells. Alternatively, B-THP and Huh-7 cells were directly infected. A representative experiment is shown; comparable results were obtained in an independent experiment. Error bars indicate SD.

FIG. 4.

SARS-CoV interactions with DCs. (A) SARS-CoV infection of DCs. Immature monocyte-derived DCs and Vero cells were seeded onto chamber slides, cultivated overnight, and challenged with SARS-CoV (Frankfurt strain). Infected cells were detected by immunostaining with a nucleocapsid-specific antibody 2 days after infection. (B) DC-mediated transfer of SARS-CoV S-harboring pseudotypes. Immature monocyte-derived DCs were pulsed with SARS-CoV S-bearing pseudotypes in the presence or absence of mannan, washed, and cocultivated with permissive Huh-7 cells. Alternatively, DCs and Huh-7 cells were directly infected. A representative experiment is shown, and similar results were obtained in an independent experiment. Error bars indicate SD.