FoxK1 and FoxK2 cooperate with ORF45 to promote late lytic replication of Kaposi's sarcoma-associated herpesvirus

Abstract

Lytic replication is essential for persistent infection of Kaposi's sarcoma-associated herpesvirus (KSHV) and the pathogenesis of related diseases, and many cellular pathways are hijacked by KSHV proteins to initiate and control the lytic replication of this virus. However, the mechanism involved in KSHV lytic replication from the early to the late phases remains largely undetermined. We previously revealed that KSHV open reading frame 45 (ORF45) plays important roles in late transcription and translation. In the present study, we revealed that the Forkhead box proteins FoxK1 and FoxK2 are ORF45-binding proteins and are essential for KSHV lytic gene expression and virion production, and that depletion of FoxK1 or FoxK2 significantly suppresses the expression of many late viral genes. FoxK1 and FoxK2 directly bind to the promoters of several late viral genes, ORF45 augments the promoter binding and transcriptional activity of FoxK1 and FoxK2, and then FoxK1 or FoxK2 cooperates with ORF45 to promote late viral gene expression. Our findings suggest that ORF45 interacts with FoxK1 and FoxK2 and promotes their occupancy on a cluster of late viral promoters and their subsequent transcriptional activity; consequently, FoxK1 and FoxK2 promote late viral gene expression to facilitate KSHV lytic replication.IMPORTANCEThe forkhead box proteins FoxK1 and FoxK2 can act as transcriptional inhibitors or activators to regulate several important processes, including aerobic glycolysis, metabolism, autophagy, and antiviral responses. However, the subversion and functions of FoxK1 and FoxK2 during KSHV infection and the pathogenesis of related diseases remain unknown. Here, we revealed that ORF45 binds to FoxK1 and FoxK2 and increases their transcriptional activity during KSHV lytic replication; consequently, FoxK1 and FoxK2 bind to late viral promoters and cooperate with ORF45 to promote late lytic gene expression. Our findings reveal two new ORF45 partners and a new function of ORF45 in which it utilizes FoxK1 and FoxK2 to promote transcription during late KSHV lytic replication.

Keywords: FoxK1; FoxK2; Kaposi’s sarcoma-associated herpesvirus; ORF45; lytic replication.

Fig 1

ORF45 interacts with FoxK1/K2 and induces their nuclear translocation during lytic replication. (A and B) HEK293T cells were cotransfected with the GST-ORF45 and/or Flag-FoxK1 (A) or with the GST-ORF45 and/or Flag-FoxK2 (B) expression plasmids. After 48 h, the cells were harvested, and whole-cell extracts were subjected to co-IP assays using GST beads. The input and immunoprecipitated samples were analyzed by Western blotting as indicated. (C) Purified ORF45, FoxK1, and FoxK2 proteins were prepared by affinity purification, and the same amounts of proteins were mixed and subjected to pull-down assay. The samples were analyzed by Western blots as indicated. (D) iSLK.Bac16 and iSLK.STOP45 cells were left untreated or induced with NaB+Dox for 72 h. The cells were harvested, the cell extracts were subjected to co-IP with an anti-ORF45, anti-FoxK1 or anti-FoxK2 antibody, the immunoprecipitates were then analyzed by Western blotting as indicated, and anti-GAPDH antibody was used as the loading and nonspecific binding controls. (E) HEK293 cells were transfected with the mCherry-FoxK1 or mCherry-FoxK2 and GFP empty vector or GFP-ORF45 expression plasmids for 24 h, after which the cells were fixed and stained with DAPI solution. Images were acquired using confocal microscopy, and the representative images from three independent experiments are shown and analyzed. (F) BCBL1 cells were induced by TPA for 48 h and then subjected to immunofluorescence staining with anti-ORF45 and anti-FoxK1 or anti-FoxK2 antibodies. The representative images from three independent experiments are shown and analyzed. (G) The percentages of ORF45-FoxK1/K2 colocalization and the cell numbers analyzed in (E) and (F) are calculated and shown. (H) HEK293 cells were transfected with empty vector or ORF45 expression plasmids for 48 h, and then, the cells were collected, and the nuclear and cytoplasmic fractions were isolated. The whole-cell extracts and subcellular fractions were analyzed by Western blots as indicated. (I) iSLK.Bac16 and iSLK.STOP45 cells were left untreated or induced with NaB+Dox for 72 h. After the cells were harvested, the nuclear fraction and cytoplasmic fraction were isolated and then analyzed by Western blotting as indicated.

Fig 2

Mapping of the binding regions required for the ORF45-FoxK1/K2 interaction. (A) Schematic diagrams of the wild-type and truncated ORF45 constructs. (B) HEK293T cells were cotransfected with Flag-FoxK1 and the GST-tagged full-length or truncated ORF45 expression plasmid. Then, 48 h post-transfection, the cells were harvested, whole-cell extracts were subjected to co-IP using GST beads, and the samples were analyzed by Western blotting as indicated. (C) Flag-FoxK2 and the GST-tagged full-length or truncated ORF45 plasmids were cotransfected into HEK293T cells, and their interactions were analyzed as described above. (D) Schematic diagrams of the FoxK1 and FoxK2 truncation constructs. (E and F) HEK293T cells were cotransfected with Flag-ORF45 and the full-length or truncated GST-FoxK1 (E) or GST-FoxK2 (F) expression plasmid. After 48 h, the whole-cell extracts were subjected to co-IP using GST beads and analyzed as described above.

Fig 3

FoxK1 and FoxK2 facilitate KSHV lytic replication and virion production. (A and B) BCBL1 cells were lentivirally transduced with a scrambled shRNA (SC), shFoxK1, or shFoxK2 for 24 h. (A) The cells were left untreated or induced TPA for 72 h, after which the cells were harvested, and the whole-cell extracts were subjected to Western blot analysis to evaluate viral protein expression. (B) Cells were left untreated or induced with TPA for 96 h, the supernatants were collected, and virion DNA was isolated and analyzed by real-time PCR to evaluate virion production. The results are calculated from three independent experiments in triplicate and presented as the mean ± the standard deviation (SD). ****, P < 0.001, t test. (C–F) iSLK.Bac16 and iSLK.STOP45 cells were transduced with the scrambled shRNA, shFoxK1, or shFoxK2 for 24 h. (C and D) The cells were left untreated or induced with NaB+Dox for 72 h, after which the cells were harvested, and whole-cell extracts were subjected to Western blot analysis to evaluate viral gene expression. (E) After being left untreated or induced with NaB+Dox for 72 h, intracellular viral and cellular genomic DNAs were isolated, and the amounts of viral DNA genome were analyzed by real-time PCR. (F) Cells were left untreated or induced for 96 h, the supernatants were collected, and virion production was analyzed by real-time PCR as described above. ****, P < 0.001, t test. (G and H) iSLK.Bac16 and iSLK.STOP45 cells were lentivirally transduced with empty vector or the FoxK1 or FoxK2 overexpressing vector for 24 h. (G) The cells were left untreated or induced with NaB+Dox for an additional 72 h, and the whole-cell extracts were subjected to Western blot analysis to evaluate viral gene expression. (H) The cells were left untreated or induced for 96 h, and virion production was analyzed as described above.

Fig 4

Depletion of FoxK1 or FoxK2 suppresses late gene expression during the KSHV lytic life cycle. iSLK.Bac16 cells were infected with a scramble-, shFoxK1-, or shFoxK2-expressing lentiviruses for 24 h and were then induced with NaB+Dox for 72 h. (A) Total RNA was extracted and subjected to deep RNA sequencing analysis. The viral gene expression data were filtered, and expression levels were calculated and are shown in a heatmap. The viral genes were classified as latent, immediate–early (IE), delayed–early (DE), or late genes according to KSHV2.0 (7). *, P < 0.05, t test. (B–F) Total RNA was extracted, reverse transcribed, and subjected to real-time PCR analysis. The expression levels of ORF45-dependent (B) and ORF45-independent (C) late genes, IE genes (D), latent genes (E), and two negative cellular genes (F) were measured in three independent experiments in triplicate, and the relative levels were calculated and are shown as the mean ± SD values. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, t test.

Fig 5

FoxK1 and FoxK2 bind to viral late promoters in an ORF45-dependent and an ORF45-independent manner. (A) The binding motifs of FoxK1/K2 in viral promoters with the 3 kb fragment upstream ORF regions were identified in the Bac16 genome, and their positions upstream of the ATG codon and specific sequences are shown. (B–D) iSLK.Bac16 and iSLK.STOP45 cells were left untreated or induced with NaB+Dox for 72 h, and the cells were then fixed, harvested, and sonicated. The samples were subjected to ChIP assays with control IgG, anti-FoxK1 antibody, or anti-FoxK2 antibody. DNA was extracted from the input and enriched samples and analyzed by real-time PCR. Three independent experiments were performed in triplicate, the enrichment of RTA and LANA promoter DNA (B) and late viral promoter DNA (C and D) was determined by normalization to the input, and the percentages are shown as the mean ± SD values. ns, no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, t test.

Fig 6

FoxK1/K2 and ORF45 promote the activity of KSHV late promoters. (A and B) One of the firefly luciferase-based promoter reporters of late genes ORF25, ORF30, ORF33, and ORF54 (A) and IE genes ORF45 and ORF50 (B) was cotransfected into HEK293T cells with the empty vector, FoxK1 expression plasmid and/or FoxK2 expression plasmid and the control vector or ORF45 expression plasmid for 48 h, and the Renilla luciferase expression plasmid pRL-TK was also co-transfected as an internal control. Whole-cell extracts were prepared, and the relative activity of the promoters was measured in triplicate in three independent experiments. The values are shown as the means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001, t test. (C) iSLK-cells harboring Bac16 or STOP45 were transfected with the scrambled shRNA, shFoxK1 alone, shFoxK2 alone, or double along with the firefly luciferase reporter of ORF25, ORF30, ORF33, or ORF54 promoters plus the Renilla luciferase expression plasmid pRL-TK. The cells were left untreated or induced with NaB+Dox for 72 h, and the relative activity of the promoters was determined in triplicate in three independent experiments and is shown as described above. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, t test.