Figure 1.
Virus-producing constructs used to make pseudotyped lentiviruses.
(A) Schematic diagrams of constructs encoding the lentiviral backbone FUGW and envelope glycoproteins. Ubi: the human ubiquitin-C promoter; GFP: enhanced green fluorescence protein; WRE: the woodchuck hepatitis virus posttranscriptional regulatory element (WRE) to increase the level of transcription; ΔU3: deleted U3 region that results in the transcriptional activation of the integrated viral LTR promoter; pA: polyadenylation signal; E1, E2, 6k, E3: SFV-glycoprotein (E1 for fusion, E2 for receptor binding, 6k a linker, and E3 is a signal sequence). The VSV-G expressing plasmid contains the rabbit β-globin intron and poly(A) signal. (B) Viral supernatants harvested from virus-producing cells transiently transfected with GFP-vpr, SFV-G, or VSV-G, and other necessary packaging constructs, were coated to a poly-lysine containing coverslip by centrifugation. The resulting coverslips were then rinsed and immunostained with an anti-SFV-G antibody (red) to label the glycoproteins and imaged using a laser confocal microscope.
Figure 2.
Lentiviral transduction of DC-SIGN-expressing 293T cells.
(A) Expression of DC-SIGN in 293T (solid fill) and 293T.DCSIGN (open fill) was detected by flow cytometry. (B) Transduction titer of lentiviruses were quantified by serially diluting fresh viral supernatants of FUGW/SFVG and FUGW/VSVG and used to transduce 2×104 293T.DC-SIGN (open bars) or parental 293T cells (solid bars). Three days later, the transduction efficiency was measured by analyzing GFP expression using flow cytometry where the corresponding viral titer was calculated. Values are given as the mean of triplicates ± S.E.
Figure 3.
Effects of DC-SIGN or L-SIGN expression.
(A) Expression of L-SIGN and DC-SIGN in 3T3 (solid fill), 3T3.DCSIGN (open fill) and 3T3.LSIGN (gray fill), respectively, was detected using cross reactive DC-SIGN/L-SIGN antibody and quantified by flow cytometry. (B) Effects of DC-SIGN or L-SIGN expression on the infectivity of pseudotyped lentiviruses. SFV-G- and VSV-G-pseudotyped lentiviruses were normalized by p24 and spin-inoculated with LSIGN- or DCSIGN-expressing 3T3 cells; the parental 3T3 cells were included as controls. Three days later, the transduction efficiency was measured by analyzing GFP expression. Fold increase in percentage of GFP-positive cells is shown based on 3T3 cells where FUGW/SFVG transduced 6.6±0.7% and FUGW/VSVG transduced 72.2±1.0%. (C) Specificity of binding to DC-SIGN. [35S]-methionine-labeled FUGW/SFVG or FUGW/VSVG were incubated with 3T3 or DC-SIGN/L-SIGN-expressing cells at 4°C. Cells were washed and 35S radioactivity of the resuspended cells was quantitated with a liquid scintillation counter. Fold increase in [35S] bound viral particles is shown based on 3T3 cells with 3.59±0.27% and 25.23±1.25% of the total CPM of virus bound for FUGW/SFVG and FUGW/VSVG, respectively, where values are given as the mean of triplicates ± S.E.
Figure 4.
Specific inhibitors prevent DC-SIGN-mediated infection.
In dose-response experiments, 293T.DCSIGN cells were treated with SFV-G (solid circles) or VSV-G (open triangles) lentiviruses in the presence of increasing concentrations of mannan (A), or NH4Cl (B). (C) 293T.DCSIGN cells were incubated with antibodies at a concentration of 5 µg/ml, 15 nM Bafilomycin A1, 5 mM EDTA, or 10 µg/ml soluble DC-SIGN at 37°C for 30 min, and then inoculated with SFV-G (filled bars) or VSV-G (open bars) lentiviruses at an MOI∼0.8 for 8 hrs or lentiviruses incubated with 25 µg/mL ConA (1 h at 37°C). Subsequently, the supernatants were replaced and incubated with fresh medium for two days before being analyzed for GFP expression. The relative transduction was determined based on non-treated controls and values are given as the mean of triplicates ± S.E.
Figure 5.
Transduction of MoDCs by lentiviruses is inhibited with anti-DCSIGN antibody and mannan.
Human monocyte-derived DCs (MoDCs) were generated by culturing respective precursor cells in the presence of GM-CSF and IL-4. (A) The adherent cells (1×106) were cultured for 2 days and then DC-SIGN expression was detected by flow cytometry. (B) Human MoDCs (1×106) were incubated for 1 hour with mannan (200 µg/mL), anti-DC-SIGN(R) antibody (20 µg/mL) or without any reagents. The cells were then infected with FUGW/SFVG or FUGW/VSVG (MOI = 10) for 8 hours in the presence of blocking reagents. GFP expression was assayed by flow cytometry five days post-transduction where one representative figure is shown with values given as the mean of triplicates ± S.E.
Figure 6.
Transduction by SFV-G lentiviruses produced in DMJ treated cells.
3T3 (2×104), 3T3-DCSIGN (2×104) and MoDCs (1×106) were spin-infected with FUGW/SFVG produced in 293T cells without DMJ(−) or with DMJ(+) treatment. GFP expression was assayed by flow cytometry three days post-transduction where values are given as the mean of triplicates ± S.E.