Fig 1: Analysis of KSHV interaction with caveolin-1 and CD109.(A and B) Three-dimensional confocal microscopy images of the colocalization of KSHV with CD109 (A) or caveolin-1 (Cav-1) (B). Senescent HuARLT cells infected with KSHV for 1 hour were stained with KSHV K8.1 antibody and target proteins, and Z-axis scanning was performed at 4 μm intervals, generating more than 10 scans. The bar-shaped images at the edges represent cross-sections, guided by white solid lines in the stacked image. (C) Quantitative analysis of the colocalization of KSHV with CD109 or caveolin-1 by Manders’ colocalization coefficient in KSHV-infected senescent HuARLT cells. n = 10. ***P < 0.001 (D) SPR analysis of KSHV binding to immobilized recombinant CD109. Data are representative of 3 independent experiments. (E) Neutralization of KSHV infectivity using a recombinant CD109 protein. Data are representative of 3 independent experiments. Data are presented as mean ± SD. n = 3. ***P < 0.001, Dunnett’s test for multiple comparisons.
Fig 2: KSHV binding to the cell surface of CAV1-KO and CD109-KO HuARLT cells.(A) Representative confocal images of KSHV virus particles in CAV1-KO and CD109-KO HuARLT cells. Scale bars: 10 μm. (B) Analysis of the number of KSHV particles that bind to the cell surface per cell in the images in A and Supplemental Figure 13. Data are representative of 10 independent experiments. Data are shown as mean ± SD. n = 10. ***P < 0.001, Dunnett’s test for multiple comparisons. (C) Quantification of the cell-surface–bound KSHV genome in the KSHV-infected cells by quantitative PCR. After 1 hour of KSHV infection, the cells were immediately scraped, and genomic DNA extracted after washing. The KSHV genome was quantified in the extracted DNA by quantitative PCR using primers for KSHV ORF26. Data are representative of 3 independent experiments. Data are shown as mean ± SD. n = 3. **P < 0.01; ***P < 0.001, Dunnett’s test for multiple comparisons.
Fig 3: Establishment and characterization of KO clones for the candidate proteins in HuARLT cells.(A) Schematic diagram of the gRNA sequence for caveolin-1 (CAV1), integrin α2 (ITGA2), F11R, and CD109. The gRNA-recognizing site is indicated as the CRISPR target sequence. (B) Western blot analysis of each KO protein. WT, WT HuARLT cell; KO, KO HuARLT cell. (C) Target sequence analysis in WT and KO HuARLT cells. The PCR products containing the gRNA targeting region from the genomic DNA of WT and KO cells were cloned into a T-vector. The sequences of 10 colonies were analyzed by Sanger sequencing. The mutated region is indicated by a box.
Fig 4: Analysis of KSHV infectivity in KO clones of CAV1, ITGA2, F11R, and CD109.An equivalent quantity of KSHV was used to infect the same number of WT and KO HuARLT cells, with or without induction of senescence. KSHV was prepared as GFP infectious units of 1 to infect approximately 90% of nonsenescent WT cells, followed by the infection of a 2-fold serially diluted virus into each group of conditioned cells. KSHV infectivity was measured using GFP expression. (A and B) Fluorescence microscopic visualization of KSHV-infected cells for each KO clone in nonsenescent (+dc, A) and senescent (–dc, B) HuARLT cells. CAV1, caveolin-1; ITGA2, integrin-α2. Scale bar: 100 μm. (C and D) Flow cytometric analysis of KSHV-infected cells for each KO clone in nonsenescent (+dc, C) and senescent (–dc, D) HuARLT cells. At the indicated GFP infectious units (GIU), the percentage of KSHV-infected cells was compared among each group. Data are representative of 3 independent experiments. Data are shown as mean ± SD. n = 3. ***P < 0.001, Dunnett’s test for multiple comparisons.
Fig 5: KSHV gH/gL interacts with CD109.Co-IP analysis of CD109 with KSHV glycoproteins gB (A), gH/gL (B), and K8.1 (C). HEK-293T cells were cotransfected with the indicated plasmids for 24 hours, after which cell lysates were subjected to IP using anti-FLAG magnetic agarose beads. The resulting complexes were then analyzed by immunoblotting (IB) using the indicated antibodies. Data are representative of 3 independent experiments.
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