The ORF45 Protein of Kaposi Sarcoma-Associated Herpesvirus Is an Inhibitor of p53 Signaling during Viral Reactivation

Abstract

Kaposi sarcoma-associated herpesvirus (KSHV) is a carcinogenic double-stranded DNA virus and the etiological agent of Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). To prevent premature apoptosis and support its replication cycle, KSHV expresses a series of open reading frames (ORFs) that regulate signaling by the p53 tumor suppressor protein. Here, we describe a novel viral inhibitor of p53 encoded by KSHV ORF45 and identify its mechanism of action. ORF45 binds to p53 and prevents its interactions with USP7, a p53 deubiquitinase. This results in decreased p53 accumulation, localization of p53 to the cytoplasm, and diminished transcriptional activity. IMPORTANCE Unlike in other cancers, the tumor suppressor protein p53 is rarely mutated in Kaposi sarcoma (KS). Rather, Kaposi sarcoma-associated herpesvirus (KSHV) inactivates p53 through multiple viral proteins. One possible therapeutic approach to KS is the activation of p53, which would result in apoptosis and tumor regression. In this regard, it is important to understand all the mechanisms used by KSHV to modulate p53 signaling. This work describes a novel inhibitor of p53 signaling and a potential drug target, ORF45, and identifies the mechanisms of its action.

Keywords: HAUSP; KSHV; ORF45; PEL; TP53; USP7; lytic; p53; primary effusion lymphoma; ubiquitination.

FIG 1

p53-luciferase (Luc) screening of virus-encoded open reading frames (ORFs). (A) p53 signaling is activated either by etoposide that binds and inhibits topoisomerase II, which results in the formation of double-strand breaks (DSB) and activates the DNA damage response (DDR), or by nutlin-3, which inhibits MDM2 binding to p53 and results in p53 activation. ATM senses DSB, autophosphorylates, and phosphorylates p53 and HDM2 (an E3 ubiquitin ligase), which breaks the HDM2-USP7-DAXX complex, promotes binding of USP7 to p53, and rescues p53 accumulation. HDM2 self-ubiquitinates and gets degraded via proteasomal pathway. As a result, p53 target genes, including cell cycle kinase (CDK) inhibitor p21, are expressed. The response is measured using a Luc reporter assay and immunoblotting with antibodies (Abs) specific for ATM pSer1981, ATR pThr1989, p53 pSer15, p53, and p21. (B) Cell viability for empty vector (EV) (pCMV-Neo-Bam), p53-273 (pCMV-Neo-Bam-p53R273H), or ORF45-Flag was measured with the CellTiter-Glo luminescent cell viability assay kit following transfection with each vector (18 h), treatment with indicated drugs (6 h), and an additional 24 h-incubation. (C to F) p53-Luc assays for KSHV ORF45-expressing cells, untreated or stimulated with etoposide or nutlin-3. U2OS cells were transfected with either pGL3 control reporter (C) or p53-responsive reporter pGL13 (D to F) and EV or plasmids expressing p53R273 or ORF45-Flag. At 18 h posttransfection (p.t.), the cells were stimulated with 5 μM etoposide (E) or 10 μM nutlin-3 (F) for 6 h or left untreated (C and D) and then incubated for 24 h. (G) p53-Luc assay for KSHV ORF45-expressing SAOS-2 (p53-null) cells. The cells were transiently transfected with p53-Luc reporter alone or with wild-type p53 and either EV, p53R273, or ORF45-Flag expression vectors. Firefly luciferase levels were measured with the One-Glo luciferase assay system. P values were calculated for comparison to the empty vector using Student’s t test (n = 4) and are indicated with an asterisk (*) at the stringent P ≤ 0.001 level to account for multiple comparisons.

FIG 2

ORF45 interacts with USP7. (A) Schematic depiction of ORF45 amino acid sequence. The protein possesses multiple predicted USP7 binding motifs (yellow boxes), a single nuclear localization signal (NLS; green box), and two nuclear export signal (NES; blue boxes) motifs. (B) Endogenous p53 pulldown. U2OS cells were transfected with constructs expressing either wild-type ORF45-Flag or ORF45^E223A-S226A-Flag or left untransfected (UN). At 18 h p.t., the cells were stimulated with 10 μM etoposide for 6 h. ORF45-Flag or ORF45^E223A-S226A-Flag were immunoprecipitated with mouse α-Flag Ab. Lysates and immunoprecipitants were tested with rabbit α-USP7 and mouse α-Flag Abs. (C to F) ORF45 retains USP7 in the cytoplasm. U2OS cells were transfected with constructs expressing either wild-type ORF45-Flag or ORF45^E223A-S226A-Flag. At 18 h p.t., the cells were left untreated or were stimulated with 10 μM etoposide for 1.5 h (D, F), fixed with methanol, and stained with the indicated Abs. The images were taken as Z-stack sections and subjected to a digital deconvolution. Bar, 50 μm. Arrows indicate the cell expressing KSHV ORF45 (wild-type or mutant). (G) ORF45 expression, but not that of the E223A-S226A mutant, increases p53 ubiquitination. U2OS cells were transfected with constructs expressing either wild-type ORF45-Flag or ORF45^E223A-S226A-Flag or were left untransfected. At 18 h p.t., the cells were incubated in the presence or absence of 30 μM MG132 for 6 h. p53 was coimmunoprecipitated with p53-speciifc mouse pAb421 Ab and blotted with rabbit α-ubiquitin and mouse α-p53 DO7 Abs.

FIG 3

Expression of the p53-signaling components in the presence of ORF45. (A) Cells were transfected with empty vector (EV) or constructs expressing either wild-type ORF45-Flag or ORF45^E223A-S226A-Flag or were left untransfected (UN). At 18 h after transfection, the cells were stimulated with 10 μM etoposide for 6 h. Cell lysates were analyzed by SDS-PAGE and immunoblotting with indicated antibodies. (B) Total p53 accumulation tested by cycloheximide chase assay. U2OS cells were transfected with either wild-type ORF45-Flag or ORF45^E223A-S226A-Flag or were left untransfected (UN) were stimulated with 10 μM etoposide for 1.5 h. p53 expression was chased in the presence of 100 μg/ml cycloheximide for the indicated time periods and tested by immunoblotting.

FIG 4

ORF45 inhibits accumulation of phosphorylated p53 and p21 and inhibits p53 transcriptional transactivation independently of USP7. (A to D) Accumulation of phosphorylated p53 and p21 determined by immunofluorescence (IF). U2OS cells were transfected with plasmids expressing ORF45-Flag or ORF45^E223A-S226A-Flag. At 18 h after transfection, the cells were stimulated with 10 μM etoposide for 1.5 h, fixed, and stained with anti-Flag, anti-phospho-p53Ser15, or anti-p21 antibodies. DAPI (4′,6-diamidino-2-phenylindole) was used to delineate the nucleus. The images were subjected to digital deconvolution. Each image represents an individual optical section. Bar, 50 μm. Arrows indicate cells expressing ORF45 (wild type or mutant). (E and F) Protein levels in ORF45-Flag-expressing (E) or ORF45^E223A-S226A-Flag-expressing (F) cells determined by immunofluorescence relative to levels in untransfected cells (%UN) that were calculated based on pixel density measured using ImageJ software. Bars indicate standard error of the mean (SEM) (n ≥ 8). P values were calculated using Student’s t test. ns, P > 0.05; *, P ≤ 0.001. (G) p53-Luc assay for ORF45 (wild type or E223A-S226A mutant)-expressing cells stimulated with etoposide. U2OS cells were transfected with either p53-responsive reporter pGL13 and EV or plasmids expressing p53R273, ORF45-Flag, or ORF45^E223A-S226A-Flag. At 18 h posttransfection (p.t.), the cells were stimulated with 5 μM etoposide and then incubated for 24 h. Firefly luciferase levels were measured with the One-Glo luciferase assay system. P values were calculated using Student’s t test (n = 4). ns, P > 0.05; *, P ≤ 0.001.

FIG 5

ORF45 sequesters p53 within the cytoplasm. (A to D) U2OS cells were transfected with plasmids expressing ORF45-Flag or ORF45^E223A-S226A-Flag. At 18 h p.t., the cells were left untreated (A and C) or were stimulated with 10 μM etoposide for 1.5 h (B and D), fixed with methanol, and stained with the indicated Abs. The images were taken as Z-stack sections and subjected to digital deconvolution. Bar, 50 μm. Arrows indicate cells expressing the ORF45 wild type or the mutant. (E) Number of cells with nuclear (“N”) or cytoplasmic (“C”; the majority of the signal localizes in the cytoplasm, but residual amounts of p53 can be detectable in the nucleus) p53 localization for untransfected and cells expressing either wild-type ORF45-Flag (E) or ORF45^E223A-S226A-Flag (F), determined by immunofluorescence. Boxes indicate the interquantile range and lines the median (n = 3). P values were calculated using nonparametric factorial analysis of variance (ANOVA) following by post hoc pairwise comparisons using Dunnett’s adjustment. *, P ≤ 0.05.

FIG 6

ORF45 binds to p53. (A) Endogenous p53 pulldown. U2OS cells were transfected with plasmids expressing ORF45-Flag or ORF45^E223A-S226A-Flag or were left untransfected (UN). At 18 h p.t., the cells were stimulated with 10 μM etoposide for 6 h. Wild-type or mutant ORF45-Flag fusions were immunoprecipitated with mouse α-Flag Ab. Lysates and immunoprecipitates were tested with mouse α-p53 DO7 and mouse α-Flag Abs. (B and C) ORF45 pulldown using p53-binding Ab. U2OS cells were transfected with plasmids expressing ORF45-Flag or ORF45^E223A-S226A-Flag or were left untransfected (UN). At 18 h p.t., the cells were stimulated with 10 μM etoposide for 6 h. p53 was immunoprecipitated with either pAb421 or the proteins binding to the p53 N or C terminus (VHH Ab). Presence of wild-type or mutant ORF45-Flag, USP7, and p53 in the lysates and immunoprecipitants was tested with mouse α-Flag and mouse α-p53 DO7 Abs. (D) Coimmunoprecipitation of ectopic p53 and ORF45 from p53-null SAOS-2 cells. Cells were transfected with vectors expressing p53-273 or ORF45 (wild type or E223A-S226A mutant). At 24 h p.t, p53 was immunoprecipitated with p53 N-terminal VHH Ab. Presence of wild-type or mutant ORF45-Flag and p53 in the lysates and immunoprecipitants was tested with mouse α-Flag and mouse α-p53 DO7 Abs. (E) p53 and USP7 coimmunoprecipitation with ORF45 from PEL-derived TREx BCBL1-RTA cells treated with 1 μg/ml doxycycline for 16 h to induce the KSHV lytic cycle. ORF45 was immunoprecipitated with α-ORF45 Ab. Presence of ORF45, p53, and USP7 was detected with mouse α-ORF45, mouse α-p53 DO7 Abs, and rabbit α-USP7 Ab, respectively. (F) ORF45 pulldown using p53-binding proteins from PEL-derived TREx BCBL1-RTA cells. The cells were treated with 1 μg/ml doxycycline for 16 h to induce the KSHV lytic cycle. p53 was immunoprecipitated with the proteins binding to the p53 N or C terminus (VHH Ab). The presence of ORF45, p53, and USP7 was detected with mouse α-ORF45, mouse α-p53 DO7 Abs, and rabbit α-USP7 Ab, respectively.

FIG 7

Shuttling of ORF45 between the nucleus and cytoplasm is required for p53 sequestration. (A to F) U2OS cells were transfected with plasmids expressing ORF45^RC-Flag, ORF45^RN1-Flag, or ORF45^RN2-Flag. At 18 h p.t., the cells were left untreated (A, C, E) or were stimulated with 10 μM etoposide for 1.5 h (B, D, F), fixed with methanol, and stained with the indicated Abs. The images were taken as Z-stack sections and subjected to digital deconvolution. Bar, 50 μm. Arrows indicate cells expressing the indicated ORF45 mutants.

FIG 8

Shuttling of ORF45 between the nucleus and cytoplasm is required for sequestration of USP7. (A to F) U2OS cells were transfected with plasmids expressing ORF45^R_C-Flag, ORF45^RN1-Flag, or ORF45^RN2-Flag. At 18 h p.t., the cells were left untreated (A, C, E) or were stimulated with 10 μM etoposide for 1.5 h (B, D, F), fixed with methanol, and stained with the indicated Abs. The images were taken as Z-stack sections and subjected to digital deconvolution. Bar, 50 μm. Arrows indicate cells expressing the indicated ORF45 mutants.

FIG 9

Subcellular localization of ORF45 does not affect its interactions with p53 and USP7, nor its ability to suppress p53-driven Luc expression. (A) ORF45 pulldown using p53-binding Ab. U2OS cells were transfected with plasmids expressing ORF45-Flag, ORF45^RC-Flag, ORF45^RN1-Flag, or ORF45^RN2-Flag or were left untransfected (UN). At 1 8 h p.t., the cells were stimulated with 10 μM etoposide for 6 h. p53 was immunoprecipitated with p53 C terminus-binding VHH Ab. Presence of wild-type or mutant ORF45-Flag fusions and p53 in the lysates and immunoprecipitants was tested with mouse α-Flag and mouse α-p53 DO7 Abs. (B) USP7 pulldown using Flag Ab binding to ORF45-Flag fusion proteins. U2OS cells were transfected with plasmids expressing ORF45-Flag, ORF45^RC-Flag, ORF45^RN1-Flag, or ORF45^RN2-Flag or were left untransfected (UN). Presence of USP7 and wild-type or mutant ORF45-Flag fusions in the lysates and immunoprecipitants was tested with rabbit α-USP7 and mouse α-Flag Abs. (C) p53-Luc assay for cells expressing ORF45 (wild type or the indicated mutants) stimulated with etoposide. U2OS cells were transfected with either p53-responsive reporter pGL13 and EV or with plasmids expressing p53R273, ORF45-Flag, ORF45^RC-Flag, ORF45^RN1-Flag, or ORF45^RN2-Flag. At 18 h posttransfection (p.t.), the cells were stimulated with 5 μM etoposide and then incubated for 24 h. Firefly luciferase levels were measured with the One-Glo luciferase assay system. P values were calculated using Student’s t test (n = 4). ns, P > 0.05; **, P ≤ 0.001.

FIG 10

ORF45 double mutants lack USP7-binding activity and do not alter the subcellular localization of USP7. (A) USP7 pulldown using α-Flag Ab. U2OS cells were transfected with plasmids expressing ORF45-Flag, ORF45^{E223A-S226A/RC}-Flag, or ORF45^{E223A-S226A/RN2}-Flag or were left untransfected (UN). At 18 h p.t., the cells were stimulated with 10 μM etoposide for 6 h. ORF45-Flag fusion proteins were immunoprecipitated with α-Flag Ab. Presence of wild-type or mutant ORF45-Flag fusions and USP7 in the lysates and immunoprecipitated fractions was tested with mouse α-Flag and rabbit α-USP7 Abs. (B to E) Subcellular localization of USP7 in the presence of ORF45 double mutants. U2OS cells were transfected with plasmids expressing ORF45^{E223A-S226A/RC}-Flag or ORF45^{E223A-S226A/RN2}-Flag. At 18 h p.t., the cells were left untreated (A, C) or were stimulated with 10 μM etoposide for 1.5 h (B, D), fixed with methanol, and stained with the indicated Abs. The images were taken as Z-stack sections and subjected to digital deconvolution. Bar, 50 μm.

FIG 11

ORF45 double mutants bind to p53 and inhibit p53 transcriptional activity. (A) ORF45 pulldown using p53-binding Ab. U2OS cells were transfected with plasmids expressing ORF45-Flag, ORF45^{E223A-S226A/RC}-Flag, or ORF45^{E223A-S226A/RN2}-Flag or were left untransfected (UN). At 18 h p.t., the cells were stimulated with 10 μM etoposide for 6 h. p53 was immunoprecipitated with p53 C terminus-binding VHH Ab. Presence of wild-type or mutant ORF45-Flag fusions and p53 in the lysates and immunoprecipitants was tested with mouse α-Flag and mouse α-p53 DO7 Abs. (B) p53-Luc assay for cells expressing ORF45 (wild type or the indicated mutants) stimulated with etoposide. U2OS cells were transfected with either p53-responsive reporter pGL13 and EV or plasmids expressing p53R273, ORF45-Flag, ORF45^{E223A-S226A/RC}-Flag, or ORF45^{E223A-S226A/RN2}-Flag. At 18 h posttransfection (p.t.), the cells were stimulated with 5 μM etoposide and then incubated for 24 h. Firefly luciferase levels were measured with the One-Glo luciferase assay system. P values were calculated using Student’s t test (n = 4). ns, P > 0.05; *, P ≤ 0.001.