Fig 2: KSHV vCyclin interacts with CAD and drives RelA deamidation.A 293 T cells were transfected with plasmids expressing Flag-CAD and Myc-tagged KSHV latent proteins. WCLs were precipitated with anti-Flag. Precipitated proteins and WCLs were analyzed by immunoblotting with the indicated antibodies. B 293 T cells were infected with KSHV (MOI = 5) for 48 h. WCLs were precipitated with IgG or anti-CAD. Precipitated proteins and WCLs were analyzed by immunoblotting with the indicated antibodies. C, D 293 T cells were transfected with plasmids expressing vCyclin and Flag-tagged CAD truncation mutants. WCLs were precipitated with anti-Flag. Precipitated proteins and WCLs were analyzed by immunoblotting with the indicated antibodies (C) and the results were summarized in (D). E HOKs were infected with lentivirus containing Flag-tagged vCyclin. WCLs were prepared at 72 h and analyzed by 2DGE and immunoblotting. F 293 T cells were transfected with plasmids expressing Myc-vCyclin. WCLs were prepared at 48 h and analyzed by 2DGE and immunoblotting. G 293 T cells were transfected with plasmids expressing CAD with or without vCyclin for 48 h. CAD was then purified and analyzed by Coomassie staining (right panel). In vitro deamidation was performed with purified GST-RelA for 15 min, which was analyzed by 2DGE (left panel). H 293 T cells were transfected with the NF-κB reporter cocktail, a plasmid containing RelA, and increasing amounts of a plasmid containing vCyclin or CDK6. NF-κB activation was determined by luciferase assay at 30 h. I HOKs were infected with lentivirus containing vCyclin. Intracellular metabolites were extracted, and lactate was quantified by mass spectrometry. J 293 T cells were depleted of CAD by CAD-specific shRNA. The stable cells were then infected with lentivirus containing vCyclin. The culturing media were collected to determine lactate concentration at 16 h post medium replacement. K HOKs were infected with lentivirus containing vector control or vCyclin. Extracellular acidification rate (ECAR) was measured by Seahorse analyzer. Data are presented as mean ± SD of n = 3 biological replicates (2H-2K). Blots were representative of at least two independent experiments (2A-2C, 2E-2G). Significance was calculated using two-tailed, unpaired Student’s t-test. Source data are provided as a source data file. See also Fig. S2.

Fig 3: CDK6 and CAD inhibitors block KSHV pathogenesis.A HOKs infected with KSHV (MOI = 30) for 48 h were treated with vehicle or Palbociclib for 4 h. WCLs were precipitated with anti-CAD. Precipitated proteins were analyzed by immunoblotting with the indicated antibodies. B BCBL-1 cells were treated with Palbociclib (50 nM). WCLs were precipitated with anti-CAD. Precipitated proteins were analyzed by immunoblotting with the indicated antibodies. C HOKs infected with KSHV (MOI = 30) for 48 h were treated with vehicle or Palbociclib for 4 h. WCLs were analyzed by 2DGE. D LECs infected with KSHV (MOI = 10) for 24 h were treated with DON (30 µM) or Palbociclib (2 µM) for 48 h. 72 h post infection, RT-qPCR analysis of the indicated mRNAs was performed. E LECs infected with KSHV (MOI = 10) for 24 h were treated with DON (30 µM) or Palbociclib (2 µM). Lactate in the media was determined at the indicated hours. F RT-qPCR analysis of viral mRNAs in LECs infected with KSHV (MOI = 10) for 24 h and then treated with Palbociclib (L: 0.4 µM, H: 2 µM) for 24 h. ND: undetected. G Viral titers in the media of the infected LECs in (E). H Viability of BC-3, BCBL-1, BJAB, and JSC-1 cells in the presence of DON. I Viability of BC-3, BCBL-1, BJAB, and JSC-1 cells in the presence of Palbociclib. J BJAB cells were infected with KSHV and selected with hygromycin (500 µg/mL). Viability of BJAB and BJAB-KSHV was determined in the presence of DON or PALA. K IVIS imaging of NOD-SCID mice engrafted with 2.5 ×106 BCBL-1-luciferase and treated with vehicle control or JHU083. L Survival curve of the 1 ×107 BC-3-luciferase-engrafted NOD-SCID mice treated with vehicle control, JHU083, or Palbociclib. Solid tumors extracted (M) and weighed (N) from the 1 ×107 JSC-1-luciferase-engrafted NOD-SCID mice treated with vehicle control, JHU083, or Palbociclib. Data are presented as mean ± SD of n = 3 biological replicates (7D-7J). Significance was calculated using two-tailed, unpaired Student’s t-test. Source data are provided as a source data file. See also Figs. S7 and S8.

Fig 4: CDK6 phosphorylates CAD at S1900.A 293 T cells were transfected with plasmids expressing Flag-CDK6 and V5-CAD for 48 h. WCLs were precipitated with anti-Flag (CDK6). Precipitated proteins and WCLs were analyzed by immunoblotting with the indicated antibodies. B 293 T cells were transfected with plasmids expressing Flag-CDK6 with or without vCyclin for 48 h. WCLs were precipitated with anti-Flag (CDK6). Precipitated proteins and WCLs were analyzed by immunoblotting with the indicated antibodies. C 293 T cells were transfected with a plasmid expressing Flag-CDK6 for 48 h. WCLs were prepared and analyzed by 2DGE and immunoblotting. D In vitro [γ-32P]-ATP kinase assay was performed on CAD with purified CDK4 or CDK6 and visualized by autoradiography (Top). The membrane was then stained with Ponceau S (Bottom). Palbociclib: a CDK4/CDK6 inhibitor. E The m/z spectra of the peptide containing S1900 phosphorylation was shown (Left), with its abundance in 293 T cells ectopically expressing an empty vector, vCyclin, or CDK6 (Right). F In vitro ATP kinase assay was performed on CAD wild-type (WT) and S1900A mutant with purified CDK6, which was then analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. G 293 T cells were transfected with a plasmid expressing Flag-CAD and then infected with KSHV-wild-type (WT) or KSHV-vCyclin.STOP (∆Cyclin) (MOI = 5) for 24 h. WCLs were precipitated with anti-Flag (CAD). Precipitated proteins and WCLs were processed in parallel and analyzed by immunoblotting with the indicated antibodies. H 293 T cells were depleted of CDK6 by CDK6-speific shRNA and then infected with KSHV (MOI = 5) for 24 h. WCLs were precipitated with anti-CAD. Precipitated proteins and WCLs were processed in parallel and analyzed by immunoblotting with the indicated antibodies. Data are presented as mean ± SD of n = 4 biological replicates (4E). Blots were representative of at least two independent experiments (4A-4D, 4F-4H). Source data are provided as a source data file. See also Fig. S4.

Fig 5: CAD S1900 phosphorylation promotes metabolic reprogramming.A In vitro deamidation was performed on purified GST-RelA with purified CAD wild-type (WT), CAD S1900D mutant (S1900D), or CAD S1900A mutant (S1900A) (Right), and analyzed by 2DGE with anti-GST antibody (Left). B CAD WT, S1900D, S1900A, and S1859E were purified from ectopically expressed 293 T cells. In vitro enzymatic (glutaminase) assay was performed. Glutamate concentration was quantified at 565 nm. C CAD-/- 293 T cells were reconstituted with CAD WT, S1900D, or S1900A by transient transfection for 48 h. Mass spectrometry analysis of 15N-labeled-dihydroorotic acid was performed at 15 min after labeling the cells with [Amide-15N] glutamine. D Mass spectrometry analysis of intracellular dihydroorotic acid was performed on the reconstituted CAD-/- 293 T cells as shown in (C). E WCLs were prepared from the reconstituted CAD-/- 293 T cells as shown in (C) and analyzed by 2DGE and immunoblotting. F 293 T cells were depleted of CAD by CAD-specific shRNA targeting 3’UTR. The stable cells were then transfected with plasmids expressing CAD WT, S1900D, or S1900A. The culturing media were collected from 1 ×106 cells to determine lactate concentration at 20 h post medium replacement. G 1 ×104 of the reconstituted 293 T cells as shown in (F) were seeded and cultured for a continuous 4 days and cell numbers were counted. H HOKs were depleted of CAD by CAD-specific shRNA targeting 3’UTR. The stable cells were then transfected with plasmids expressing CAD WT, S1900D, or S1900A, before infection with KSHV (MOI = 30). The viral titers in the culturing medium were determined 72 h post infection. Data are presented as mean ± SD of n = 3 biological replicates (5 C, 5D, 5F-5H) and mean ± SEM of n = 8 biological replicates (5B). Blots were representative of at least two independent experiments (5 A and 5E). Significance was calculated using two-tailed, unpaired Student’s t-test (paired t-test for 5B). Source data are provided as a source data file. See also Fig. S5.