FIG 5.

GBP1 disrupts the formation of actin filaments, thus inhibiting the nuclear delivery of KSHV virions. (A) The five stably transfected wild-type and mutant GBP1-overexpressing cell lines, grown in six-well plates, were incubated with KSHV.BAC16 for 6 h, washed twice with PBS, and then incubated with trypsin-EDTA for 5 min at 37°C. The cells were spun down and then washed once with PBS. Total DNA was isolated and subjected to qPCR. (B) Collected cells were resuspended in 500 μl hypotonic buffer and incubated on ice for 15 min, and 25 μl 10% NP-40 was added, followed by vortexing for 10s. The cell nuclei were spun down, and the pellet was collected. Histone H3 was used as the marker for the nucleus, while α-tubulin and GAPDH were used as the markers for the cytoplasm. (C) The five cell lines were infected with KSHV as described above for panel A. The nuclear fraction was isolated, and the number of nucleus-associated KSHV genome copies was estimated as described in Materials and Methods. (D) SLK-Con and SLK-GBP1-wt cells were fixed and stained with rat anti-GBP1 antibody, followed by incubation with goat anti-mouse IgG(H+L) Alexa Fluor 555 (red). The actin filaments were then stained with Cytopainter phalloidin-iFluor 488 (green) for 90 min. Slides were photographed by using a digital camera and software. (E) SLK-Con and SLK-GBP1-wt cells were incubated with KSHV for 6 h. Cells were fixed and stained with mouse anti-ORF65 antibody and then further stained with the secondary antibody goat anti-mouse IgG(H&L) Alexa Fluor 555 (red), followed by incubation with Cytopainter phalloidin-iFluor 488 (green) for 90 min.