Fig 1: The role of the CD147 protein complex in different CHIKV strains and related alphaviruses. (A–K) stable KO cell lines defective of CD147 or SLC1A5 or both CD147 and SLC1A5 were used (A) infection with Asian strain CHIKV for 52 h (B) infection with IOL for 48 h (C) infection with WA CHIKV for 28 h. (D) infection with CHIKV carrying a reporter nanoluciferase gene for 28 h (E) infection with SINV for 24 h (F) infection with WEEV for 24 h (G) infection with EEEV for 28 h (H) infection with VEEV for 28 h (I) infection with ONNV for 48 h (J) infection with MAYV for 48 h (K) infection with RRV for 48 h. Detection of infected KO cells with E staining combined with flow cytometry. All graphs depict percentage E positive cells. (A–K) All graphs were normalized to the percentage E-positive WT HEK293T cells in highest virus concentration. Mean ± SD is shown (n = 6; 2 independent experiments with 3 technical replicates); *p < 0.05, **p < 0.01, ***p < 0.001 compared to WT HEK293T cells (L) Phylogenetic tree of Old and New world alphaviruses based on E2.
Fig 2: Effect of CD147 expression on CHIKV infection efficiency. (A) Stable knockout cell lines of CD147, SLC1A5 and CD147-SLC1A5 combined were infected with a reporter Nanoluc CHIKV for 24 h. Data is normalized to 293T WT. Mean ± SD is shown (n = 6; 2 independent experiments with 3 technical replicates). (B) Reintroduction of CD147 or SLC1A5 in the stable KO cell lines used in (A). Expression constructs had synonymous mutations at the target gRNA site. Both a transfection control (trans) and a no transfection control (empty) were used. Normalization with the no transfection control (empty) within each KO cell line. Mean ± SD is shown (n = 3; 3 independent transfections) (C) Binding assay with Nanoluc CHIKV on HEK293T and HEK293T ?CD147 cl1 cells. Cell associated viral RNA was measured after 45 min virus binding at 37°C using RT-qPCR. Mean ± SD is shown (n = 3; 1 experiment with 3 technical replicates) *p < 0.05, ***p < 0.001.
Fig 3: Protein structure comparison of CD147 and MXRA8. (A) Alignment of crystal structures of MXRA8 (PDB: 6JO7, green) and CD147 (PDB: 3B5H, blue) using the FATCAT algorithm (Ye and Godzik, 2004). (B) Alignment graph with four aligned fragment pairs (AFP) (red, purple, yellow, and green) and 3 twist (arrows). On the axes the length of the amino acid sequence of MXRA8 and CD147 is depicted. (C) Detailed alignment of the four AFPs shown in red, purple, yellow and green as in (B). The amino acid sequence of MXRA8 and CD147 is shown.
Fig 4: CHIKV envelope association with the CD147 complex (A) Scheme of the used AP-MS approach. (B) CHIKV envelope Interaction partners of interest are listed. Examination of their plasma membrane presence (PM), presence of a transmembrane domain (TM), and MiST score (MiST) are indicated. Components of the CD147 protein complex retrieved in the CHIKV envelope affinity purification are indicated. (C–F) Western Blot of affinity purification of Strep-tagged Envelope protein. Detection of E2 and the interactors CD147, CD98, and SLC1A5. Affinity purification of untransfected cells (empty) were included as controls. On each blot input lysates of untransfected cells (empty) or E2 expressing cells (E2) and affinity purifications (AP) of the empty or E2 cells were loaded. For the CD147, CD98 and SLC1A5 blots 0.4 µl of input lysates and 10 µl of APs was loaded. For the E2 blot 5 µl of input lysates and 10 µl of APs was loaded.
Supplier Page from DNASU for BSG (Homo sapiens) in pLX304 (Gateway V5-tagged lentiviral expression vector)