N-Linked Glycosylation Facilitates Sialic Acid-Independent Attachment and Entry of Influenza A Viruses into Cells Expressing DC-SIGN or L-SIGN ▿

  1. Patrick C. Reading1,2,*
  1. 1Department of Microbiology and Immunology, The University of Melbourne, 3010 Victoria, Australia
  2. 2WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria 3051, Australia
  3. 3Westmead Millennium Institute, Sydney, 2145 New South Wales, Australia
  1. FIG. 1.
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    FIG. 1.

    Lec2 cells are deficient in cell surface SA, do not bind influenza virus, and are largely resistant to influenza virus infection. (A) FACS histograms showing SA-(α-2,3)-Gal expression on parental cells (CHO Pro-5) compared to Lec2 CHO cells. Cells were incubated in either media alone (mock, black histograms), or in media supplemented with 200 mU of bacterial sialidase/ml (gray histograms) and, after washing, the binding of b-MAA was determined. Dashed lines represent Pro5 or Lec2 cells stained with streptavidin-APC conjugate alone. (B) The binding of influenza virus BJx109 to CHO Pro5 cells (white histogram) and Lec2 CHO cells (gray histogram) was determined by flow cytometry, as described in Materials and Methods. The dashed line represents CHO Pro5 cells stained with streptavidin-APC conjugate alone. (C) Influenza virus does not infect Lec2 CHO cells efficiently. Monolayers of CHO Pro5 cells (CHO cells) or Lec2 CHO cells (Lec2 cells) were incubated with 107 or 106 PFU (indicated as high or low dose, respectively) of BJx109 for 1 h at 37°C. Monolayers were washed, incubated 6 to 8 h and then fixed and stained by immunofluorescence for expression of newly synthesized viral NP. The data represent the mean percent infection (± the SEM from a minimum of four independent fields per chamber and are representative of at least three independent experiments.

  2. FIG. 2.
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    FIG. 2.

    Total and cell surface expression of DC-SIGN and L-SIGN by Lec2 CHO cell transfectants. (A) FACS histograms showing expression of human C-type lectins on the surface of transfected Lec2 cells (gray histograms). White histograms represent control-transfected Lec2 cells expressing intracellular OVA and stained for DC-SIGN/L-SIGN, as indicated. (B) Western blot analysis showing expression and migration pattern of DC-SIGN and L-SIGN in lysates from transfected Lec2 cells. Cell lysates from Lec2-OVA (Lec2-control), Lec2-DC-SIGN, or Lec2-L-SIGN cells were resolved by SDS-PAGE under nonreducing conditions and transferred to a PVDF membrane. To detect human C-type lectins, the blot was incubated with MAb DC28, which is cross-reactive with both DC-SIGN and L-SIGN, and developed as described in Materials and Methods. Arrows indicate bands corresponding to the approximate molecular masses of DC-SIGN and L-SIGN (ca. 49 to 55 kDa). The data are representative of at least three independent experiments.

  3. FIG. 3.
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    FIG. 3.

    DC-SIGN and L-SIGN expressed on the surface of Lec2 cells retain Ca2+-dependent lectin activity. Lec2 cells expressing intracellular OVA (thin black line) or cell surface DC-SIGN or L-SIGN (thick black line in upper and lower panels, respectively) were incubated with b-mannan at 4°C for 30 min in the presence of 5 mM CaCl2. After washing, cell surface b-mannan was detected by using streptavidin-APC. Lec2-DC-SIGN and Lec2-L-SIGN cells were also incubated with b-mannan in the presence of 10 mM EDTA, and the binding of b-mannan was determined (dashed line). The data are representative of at least two independent experiments.

  4. FIG. 4.
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    FIG. 4.

    DC-SIGN and L-SIGN expressed by Lec2 CHO cells mediates Ca2+-dependent binding to influenza virus. (A) Binding of influenza virus BJx109 to Lec2-control (thin black lines), Lec2-DC-SIGN (thick black line, upper panel), or Lec2-L-SIGN cells (thick black line, lower panel) was determined at 4°C in the presence of 5 mM CaCl2. Binding of BJx109 to Lec2-DC-SIGN or Lec2-L-SIGN cells was also determined at 4°C in the presence of 10 mM EDTA (dashed lines). The data are representative of at least two independent experiments. (B) VOPBA demonstrating Ca2+-dependent binding of influenza virus BJx109 to proteins corresponding to DC-SIGN and L-SIGN. Whole-cell lysates from Lec2-control, Lec2-DC-SIGN, or Lec2-L-SIGN cells were electrophoresed by SDS-PAGE under nonreducing conditions, transferred to PVDF membrane, and probed with influenza virus BJx109 in the presence of either 5 mM CaCl2 (Ca2+) or 10 mM EDTA. Arrows indicate bands corresponding to the approximate molecular masses of DC-SIGN and L-SIGN (ca. 49 to 55 kDa). The data are representative of two independent experiments.

  5. FIG. 5.
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    FIG. 5.

    DC-SIGN and L-SIGN expression in Lec2 cells enhances influenza virus infection. (A and B) Monolayers of Lec2-DC-SIGN, Lec2-L-SIGN, or Lec2-control cells were incubated with 107 PFU of influenza virus for 60 min at 37°C. Monolayers were washed, incubated 6 to 8 h and then fixed and stained by immunofluorescence for expression of newly synthesized viral NP. (A) Data represent the mean percent infection (± the SEM) from no less than four independent fields per chamber and are pooled from two separate experiments. (*, Significantly enhanced compared to Lec2-control cells [P < 0.05, one-way ANOVA].) (B) Lec2-DC-SIGN and Lec2-L-SIGN cells stained for viral NP (FITC) and double-stranded nucleic acids (propidium iodide [PI]) at 2 and 8 h postinfection (×100 magnification). (C) vRNA and mRNA levels of influenza A virus M gene in infected cells were determined by qRT-PCR. The data represent the mean fold increase (± the SEM) in vRNA and mRNA copy number between 2 and 8 h for each cell line. Assays were performed in triplicate and are representative of two independent experiments. (D) Enhanced infection of Lec2-DC-SIGN and Lec2-L-SIGN does not result in amplification and release of infectious virus. Cell monolayers were infected in chamber slides as described above. Cell supernatants were collected at 2 and 24 h postinfection, and titers of the infectious virus were determined by standard plaque assay. The data show the mean fold increase in virus titer (PFU/ml) between 2 and 24 h (± the SEM). Assays were performed in triplicate.

  6. FIG. 6.
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    FIG. 6.

    Infection of Lec2-DC-SIGN and Lec2-L-SIGN cells by influenza virus infection is blocked by mannan but not by treatment of cells with bacterial sialidase. Monolayers of (i) parental CHO Pro 5 cells, (ii) Lec2-control, (iii) Lec2-DC-SIGN, or (iv) Lec2-L-SIGN cells in eight-well chamber slides were treated with either 10 mg of mannan/ml at 37°C for 60 min or 200 mU of bacterial sialidase/ml or were incubated with medium alone (no treatment) before infection with 107 PFU of BJx109. The percentage of infected cells was determined by using immunofluorescence at 6 to 8 h postinfection. The data represent the mean percent infection (± the standard deviation) from no less than four independent fields per chamber and are pooled from three independent experiments. (*, Significantly reduced compared to no treatment controls [P < 0.05, one-way ANOVA].)

  7. FIG. 7.
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    FIG. 7.

    Influenza viruses differ in their ability to infect Lec2-DC-SIGN and Lec2-L-SIGN cells. (A) Virus strains BJx109 and PR8 were compared for their ability to bind ConA by ELISA. Biotin-labeled ConA was added to wells coated with increasing concentrations of BJx109 or PR8 in buffer alone or buffer supplemented with 5 mg of mannan/ml (+ mannan). Wells coated with BJx109 or PR8 were also treated with 0.011 M sodium meta-periodate (+ periodate) for 20 min prior to the addition of ConA. Equivalent coating levels of BJx109 and PR8 were confirmed using MAb 165, which binds to the host-derived carbohydrate antigen characteristic of egg-grown influenza viruses (data not shown). MAbs directed to HA of BJx109 or PR8 confirmed that levels of virus were similar following treatment with periodate (data not shown). (B) Monolayers of parental CHO Pro5 (i), Lec2-control (ii), Lec2-DC-SIGN (iii), or Lec2-L-SIGN (iv) cells were infected with 5 × 106 PFU of RG-PR8, RG-PR8-Beij/89 HA/NA, RG-PR8-Beij/89 HA, or RG-PR8-Beij/89 NA. (C) Monolayers of parental CHO Pro5 (i), Lec2-control (ii), Lec2-DC-SIGN (iii), or Lec2-L-SIGN (iv) cells were infected with 3 × 106 PFU of either New Cal/99 or Sol Is/06. For panels B and C, the percentage of infected cells was determined by using immunofluorescence at 6 to 8 h postinfection as described above. The data represent the mean percent infection (± the standard deviation) from no fewer than four independent fields per chamber and are pooled from two independent experiments. (#, <1% of cells infected; *, significantly reduced compared to both RG-PR8-Beij/89 HA/NA and RG-PR8-Beij/89 HA [P < 0.05, one-way ANOVA].)

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  1. Accepted manuscript posted online 29 December 2010, doi: 10.1128/JVI.01705-10 J. Virol. vol. 85 no. 6 2990-3000
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