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. 2021 Jan 4;12(1):29. doi: 10.1038/s41419-020-03303-1

Fig. 3. XPO1 knockdown blocks the KSHV lytic replication program but has no effect on KSHV infectivity, and virus entry and trafficking during primary infection.

Fig. 3

A HUVEC were infected with lentiviruses expressing shRNAs targeting XPO1 (shXPO1-1 and shXPO1-2) or control shRNA (shCtrl). Cells were selected with puromycin (1 µg/ml) for 48 h and analyzed by Western-blotting to monitor the knockdown efficiency. β-actin was used as the loading control. B, C HUVEC with XPO1 knockdown were infected with KSHV for 96 h. Supernatants were collected and used to titrate infectious virions by infecting fresh HUVEC. At 48 hpi, representative images were taken (B) and used for quantifying the numbers of GFP-positive cells (C). D HUVEC with XPO1 knockdown were infected with KSHV for 48 h. Cells were fixed and stained for LANA (red). The expression level and pattern of LANA are shown. Nuclei were stained with DAPI. Images were taken with a confocal microscopy (magnification ×600). E Quantification of LANA-positive cells based on images taken as described in (D). F HUVEC with knockdown of XPO1 were infected with KSHV for 6 h, fixed, and stained for nuclei with DAPI (blue) and KSHV particles with an antibody to ORF65 (red). Images were taken with a confocal microscopy (magnification ×1000). G The total number of KSHV particles successfully docked at the perinuclear region of each nucleus was quantified and analyzed based on images taken as described in (F). H HUVEC with XPO1 knockdown infected with KSHV for 6 h were collected and analyzed for viral genome copy numbers by qPCR using LANA primers. I–L HUVEC with XPO1 knockdown were infected with KSHV and then collected for RNA extraction at 48 hpi. The expression levels of KSHV transcripts were then examined by RT-qPCR. M HUVEC with XPO1 knockdown were infected with KSHV and collected at 48 hpi. The expression of viral proteins was then analyzed by Western blotting. β-actin was used as a loading control. *, **, and *** indicate P values of < 0.05, < 0.01, and < 0.001, respectively; NS not significant.