Fig 1.
Filamin A phosphorylation is induced by ORF45-mediated RSK activation.
A. Ectopic ORF45 WT and F66A, RN or RC mutated constructs were introduced into SLK cells by infection with recombinant lentiviruses. The cells were collected at 72 h post-infection, and whole cell lysates were analyzed by Western Blots as indicated. B. HEK293 cells were cotransfected with HA-RSK2 WT-, CA- or KD-expressing plasmids with empty vector or ORF45-expressing plasmids for 24 h, and then, the cells were serum-starved overnight, collected and subjected to western blot analysis as indicated. C. SLK cells and iSLK.219 cells were induced with 1 µg/ml Dox and 1 mM NaB. The cells were collected at different time points, and whole cell extracts were detected as indicated. D. Control cells, iSLK-Bac16, -STOP45, ORF45F66A, ORF45RN or ORF45RC cells were induced with Dox and NaB as described above, the cells were collected at 72 h after induction, and whole cell extracts were analyzed. E. HEK293 cells were infected with Bac16 or STOP45 virions (MOI = 10). At the different time points post-infection, the cells were collected and whole cell extracts were prepared and subjected to Western Blotting analysis as indicated. F. The iSLK.Bac16 or iSLK.STOP45 cells were induced with Dox and NaB for 48 h, and then fixed and stained with anti-pFilamin A antibody with Alexa-555 secondary antibody, and finally visualized with confocal fluorescence microscopy. The percentages of pFilamin A-positive cells in cells undergoing lytic reactivation were calculated in three independent experiments and shown. **, p < 0.01; t test. G. The iSLK.Bac16 cells were induced with Dox and NaB for 48 h, and then fixed and stained with anti-ORF45 antibody with Alexa-647 secondary antibody and anti-pFLNA antibody with Alexa-555 secondary antibody, and finally visualized with confocal fluorescence microscopy. The subcellular co-localization of ORF45 and pFilamin A was analyzed and shown.
Fig 2.
ORF45 reduces cell adhesion through RSK activation.
A. HEK293 cells were infected with IRES-zsGreen empty vector, ORF45 WT or F66A-expressing lentiviruses (MOI = 2). Forty-eight hours later, the cells were dissociated and seeded on a fibronectin-coated 96-well plate. After 1 h incubation, the unattached cells were removed by low shear washing, the attached cells were observed and recorded under fluorescence microscopy, and a representative image was shown. B. HEK293 cells transfected with ORF45 and RSK2 WT- or mutant-expressing plasmids were dissociated and seeded on a fibronectin-coated 96-well plate, and the relative focal adhesion was calculated and shown as mean ± standard deviation (SD). *, p < 0.05; **, p < 0.01; t test. C. Cell detachment is induced during lytic replication. iSLK.219 cells were seeded on SLK cell monolayers at a 1:50 ratio and then induced with Dox and NaB for 48 h. The living cells were fixed and directly visualized with confocal fluorescence microscopy. D. iSLK-Bac16 cells and iSLK-STOP45 cells were induced with Dox and NaB, or iSLK.STOP45 cells were infected with ORF45 WT or F66A-expressing lentiviruses (MOI = 5) and then cells were induced with Dox and NaB at 12 h post-infection. The cells were visualized with inverted microscopy at 72 h post-induction. The quantification of relative cell adhesion was calculated and shown. **, p < 0.01; t test.
Fig 3.
ORF45 promotes cell migration during early primary infection.
A-C. HUVECs were transduced with IRES-zsGreen empty vector, ORF45 WT- or F66A-expressing lentiviruses (MOI = 2). Forty-eight hours later, the cells were observed and recorded under fluorescence microscopy (A). A total of 5 × 104 cells/well were subjected to a transwell migration assay with DMEM containing 20% FBS. Cells were left for migration for 24 h and then fixed, stained with crystal violet and recorded by microscopy (B). Migrated cell numbers in each field were counted, and the means were analyzed and are shown (C). D-E. HUVECs were infected with empty or ORF45- or F66A-expressing lentiviruses (MOI = 5). Twelve hours post infection, the cells were subjected to wound healing assays. Representative images are shown (D), and the cells that migrated into the closure per field were counted. The means were analyzed and are shown (E). F. HUVECs were infected with purified Bac16, STOP45 or ORF45F66A recombinant viruses (MOI = 10). Sixty hours after infection, the cells were subjected to a wound healing scratch assay, and representative images are shown. The means of migrated cell numbers were analyzed and are shown (G).
Fig 4.
Filamin A phosphorylation is essential for ORF45-induced detachment and migration but not for virion production.
A-D. HEK293 cells were transfected with Filamin A WT or S2152A mutated constructs and then infected with empty vector or ORF45-expressing lentiviruses at 8 h after transfection. Twenty-four hours later, the cell morphology was observed by inverted microscopy, and representative images are shown (A). Thirty-six hours after infection, the cells were subjected to analysis for relative adhesion (B), gene expression by western blots (C), and cell migration in the wound healing scratch assay (D). E. Bac16-harboring 239T cells in 6-well plates were transfected with 1.6 μg of Filamin A WT or S2152A construct for 24 h, and then, the cells were induced with 20 ng/ml TPA and 0.3 mm NaB for lytic replication. The supernatants were collected at different time points, and then, the yield of progeny viruses was detected with quantitative PCR analysis.
Fig 5.
Filamin A KO and S2152A KI reduce KSHV de novo infection of cell-free virions.
A-B. Filamin WT, S2152A KI or KO HEK293-mCherry cells were transfected with empty vector or ORF45 expressing plasmid for 24 h (A), or left uninfected or infected with Bac16 or STOP45 viral stocks (MOI = 10) for 24 h (B). The cells were collected and whole cell extracted were subjected to Western blotting analysis as indicated. C-E. Filamin WT, S2152A KI or KO HEK293-mCherry cells were left uninfected or infected with iSLK.Bac16 or iSLK.STOP45 viral stocks (MOI = 10). (C) Two hours post infection, the cells were collected by trypsin digestion, and the intracellular viral DNA and cellular DNA genomes were extracted. (D) Eight hours post infection, the cells were collected and nuclear fractions were isolated, and then viral DNA and cellular DNA genome were extracted from nuclear fractions. The viral DNA copy numbers were detected by real-time PCR and GAPDH DNA was used as internal control. (E) Twenty four hours post infection, the total RNA were extracted, reverse-transcribed and detected by real-time PCR. The levels of viral mRNA were normalized to β-actin mRNA.. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, t test.
Fig 6.
Filamin A KO and S2152A KI reduce KSHV de novo infection and cell-contact mediated viral infection.
A. Filamin WT, S2152A KI or KO HEK293-mCherry cells were infected with purified iSLK.Bac16 or iSLK.STOP45 viral stocks (MOI = 10) for 24 h. The cells were harvested and analyzed by FACS flow cytometer. The representative images and the percentages of KSHV-infected HEK293-mCherry cells were calculated and shown in three independent experiments. B. After lytic induction for 72 h, iSLK.Bac16 or iSLK.STOP45 cells were collected and washed twice, and directly added to the monolayer of Filamin WT, S2152A KI or KO HEK293-mCherry cells and co-cultured for additional 24 h. Total cells were harvested and analyzed by FACS flow cytometer. The representative images of mCherry-positive cells are shown and the percentages of KSHV-infected HEK293-mCherry cells were calculated in three independent experiments and shown. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, t test.
Fig 7.
Filamin A KO and S2152A KI reduce cell migration under ORF45 overexpression or during KSHV de novo infection or lytic reactivation.
A-C. Filamin WT, S2152A KI or KO HEK293-mCherry cells were transfected with empty vector or ORF45 expressing plasmid for 24 h (A), or left uninfected or infected with iSLK.Bac16 or iSLK.STOP45 viral stocks (MOI = 10) for 24 h (B). The cells were subjected to a wound healing scratch assay, and representative images are shown. (C) The relative wound healing abilities were calculated using ImageJ software and shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, t test. D-E. Bac16 or STOP45-harboring Filamin WT, S2152A KI or KO HEK293-mCherry cells were induced by TPA + NaB for 48 h, and then subjected to a wound healing scratch assay and representative images are shown (D). The relative wound healing abilities were calculated and shown (E). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, t test.