Fig 1.
Transduction or infection of cells with CHIKV Env pseudotyped vector particles.
A: 293T, CHO-K1, and pgsA-745 cells were seeded in 24-well plates and transduced with gfp-encoding CHIKV Env-pseudotyped lentiviral vector particles. The cells were analyzed by flow cytometry for GFP expression 48 hrs post transduction. The proportion of GFP-positive cells is given relative to the proportion of positive cells following transduction with VSV-G-pseudotyped vectors (control). Data represent the average of three independent experiments. *** (P ≤ 0.001) indicates significant differences in transduction rates compared to CHO-K1 cells. B: CHO-K1 and pgsA-745 cells were seeded in 24-well plates and infected with CHIKV-mCherry at an MOI of 1. The viral replication was determined 6 and 24 hrs post-infection, respectively, by flow cytometry detecting mCherry. Data represent the average of three independent experiments. ** and *** indicate significant differences in infection rates to CHO-K1 cells after 6 and 24 hrs, respectively. * (P ≤ 0.05) and ** (P ≤ 0.01).
Fig 2.
Transduction of cells with CHIKV Env pseudotyped vectors in the presence of soluble GAGs.
293T (left), CHO-K1 (right), and pgsA-745 (bottom) cells were seeded in 384-well plates and transduced with CHIKV Env-pseudotyped vectors transferring a luciferase gene. Before addition to the cells, the vector particles were incubated with DX or one of the indicated GAGs for 30 minutes at 4°C (DX and GAGs in five 3-fold dilutions, ranging from 500.0 to 6.2 μg/μl). One day after transduction, the luciferase expression of the cells was detected by a luminometer [20]. The results are given as percentages of the untreated control values. The experiment was carried out twice in triplicate, and one representative triplicate result is shown.
Fig 3.
Infection and cell entry by CHIKV-mCherry-490 in the presence of soluble GAGs.
A: 293T cells were seeded in 24-well plates and 500 μg/ml of the indicated GAG was added. Afterwards, cells were infected with CHIKV-mCherry-490 using an MOI of 1. The viral replication was determined 6 hrs post-infection by flow cytometry detecting mCherry. B: 500 μg/ml of the indicated GAG and CHIKV-mCherry-490 (MOI 1) were incubated together at 4°C for 30 minutes. After addition to 293T cells, another incubation of 30 minutes at 4°C followed. Then, the unbound virus together with the respective GAG were washed away and fresh medium was added to the cells. The viral replication was determined 6 hrs post-infection by flow cytometry detecting mCherry. Data represent the average of three independent experiments. *** and **** indicate significant differences in infection rates to untreated control cells. ** (P ≤ 0.01) and *** (P ≤ 0.001).
Fig 4.
Purification of recombinant CHIKV E2 domains A, B, and C, and E2ex. The CHIKV E2 domains A, B, and C, and E2ex were cloned into the pET-15b vector, and the proteins were expressed in E. coli and purified under native (B, C) or denaturing conditions (A, E2) by HPLC (Ni-NTA beads). For A and E2, ion-exchange chromatography was additionally performed after the HPLC. Subsequently, they were dialyzed against PBS and concentrated. Shown is an SDS-PAGE with subsequent Coomassie staining of the purified proteins. BSA dilutions were used to estimate the protein concentration. Domain A has a molecular mass of 26.5 kDa, B of 8.5 kDa, C of 10.7 kDa, and E2ex of 40.4 kDa.
Fig 5.
Binding of recombinant CHIKV E2 domains A, B, and C, and E2ex to CHO-K1 and pgsA-745 cells. CHO-K1 (top) and pgsA-745 (bottom) cells were incubated with 10 μg of the indicated recombinant proteins. Binding was measured by flow cytometry using an anti-His-tag and an anti-mouse FITC conjugated antibody. The results are shown relative to the control (anti-His-tag and anti-mouse FITC antibodies alone). A value of one represents the mean FITC value of the control and is labeled by the dashed line. Values above this line indicate binding. Data represent the average of three independent experiments. * and ** indicate significant differences to the recombinant protein-free controls. n.s. means not significant. * (P ≤ 0.05) and ** (P ≤ 0.01). Protein sA served as a negative control [23].
Fig 6.
Binding of recombinant CHIKV E2 protein domains A, B, and C, and E2ex to 293T, CHO-K1, and pgsA-745 cells in the presence of soluble glycosaminoglycans (GAGs). 10 μg of the indicated recombinant proteins were incubated with the indicated soluble GAGs (500 μg/ml) for 30 minutes at 4°C. 293T (top), CHO-K1 (middle), and pgsA-745 (bottom) cells were then incubated with this mixture. Binding was measured by flow cytometry using an anti-His-tag and an anti-mouse FITC conjugated antibody. The results are shown as relative values to the control (incubation of cells with the respective recombinant protein alone). A value of one represents the mean FITC value of the control and is labeled by the dashed line. Values above this line indicate inhibition of binding. Data represent the average of three independent experiments. * and ** indicate significant differences to the GAG-free controls. n.s. means not significant. * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001) and **** (P ≤ 0.0001).
Fig 7.
Binding of recombinant Fc-fusion proteins containing CHIKV E2 domains A, B, and C to CHO-K1 and pgsA-745 cells. A: Fc-E2-fusion proteins and Fc protein were expressed from HEK293T cells, affinity purified by protein-A chromatography and separated by SDS-PAGE under reducing conditions. The Western blot was detected with an HRP-labeled anti-human IgG antibody. The calculated molecular weights are: Fc 29.2 kDa; Fc-CHIKV-E2-B 35,0 kDa; Fc-CHIKV-E2-C 37.1 kDa and Fc-CHIKV-E2-A 53 kDa. B: Separation of the Fc-fusion proteins under native conditions. The Western blot was detected with an HRP-labeled anti-human IgG antibody. C: CHO-K1 (black) and pgsA-745 (grey) cells were incubated with the indicated recombinant Fc-fusion proteins as single measurements. Binding was measured by flow cytometry using an anti-human IgG FITC conjugated antibody. The results are shown as fold induction compared to Fc binding. Two volumes were use either 400 μl or 100 μl (low). A value of one represents the mean FITC value of the control and is labeled by the dashed line. Values above this line indicate binding. Data represent an average experiment of three independent experiments performed.
Fig 8.
Binding of Fc-fusion proteins containing variants of the CHIKV E2 domain A to CHO-K1 and pgsA-745 cells.
A: Fc-E2 domain A-fusion proteins and Fc protein were expressed from HEK293T cells, affinity purified by protein-A chromatography and separated by SDS-PAGE. The Western blot was detected with an HRP-labeled anti-human IgG antibody. The calculated molecular weights are: Fc-CHIKV-E2-A, E79K and E166K 53 kDa; Fc-CHIKV-E2-A-ß 43,4 kDa. B: Separation of the Fc-fusion proteins under native conditions. The Western blot was detected with an HRP-labeled anti-human IgG antibody. C: CHO-K1 (black) and pgsA-745 (grey) cells were incubated with the indicated recombinant Fc-fusion proteins. Binding was measured by flow cytometry using an anti-human IgG FITC conjugated antibody. The results are shown as fold induction compared to Fc binding. A value of one represents the mean FITC value of the control and is labeled by the dashed line. Values above this line indicate binding. Data represent an average experiment of three independent experiments performed.