Fig. 6. CAD and RelA deamidation for KSHV pathogenesis.
A HOKs depleted of RelA were reconstituted with RelA-DD or RelA-64A by lentiviral transduction, then infected with KSHV (MOI = 30) for 72 h. RT-qPCR analysis of viral mRNAs was performed. B The viral titers in the media of the cells in (A) at the indicated hours. C TIME depleted of RelA were reconstituted with RelA-DD or RelA-64A by lentiviral transduction, then infected with KSHV (MOI = 3) for 48 h. RT-qPCR analysis of LANA mRNAs was performed. D, E RT-qPCR analysis of viral mRNAs in HOKs depleted of CAD by CRISPR-Cas9/sgRNA and infected with KSHV (MOI = 30) for 72 h (D). Viral titers in the media were determined (E). F, G Viral titers in the media of HOKs depleted of CAD by CAD-specific shRNA and infected with KSHV (MOI = 30) for 72 h (F). Cell numbers were counted and normalized (G). H Viral titers in the media of LECs depleted of CAD and infected with KSHV (MOI = 10) for 72 h. I TIME cells depleted of RelA were reconstituted with RelA-WT or RelA-64A, then infected with KSHV (MOI = 3) for 48 h. Lactate in the media was determined at 20 h post medium replacement. J Lactate in the media at 24 h post medium replacement in HOKs depleted of CAD and infected with KSHV for 48 h. K Viability of mock and KSHV-infected TIME cells in the presence of N-phosphonacetyl-l-aspartate (PALA). L Proliferation of BCBL-1 depleted of CAD by CRISPR-Cas9/sgCAD. M Single clone of CAD+/+ and CAD-/- BCBL-1-luciferase was amplified. 5 ×106 of cells were engrafted into NOD-SCID mice and IVIS imaging was performed. N Proliferation of JSC-1 depleted of CAD by CRISPR-Cas9/sgCAD. O Single clone of CAD+/+ and CAD-/- JSC-1-luciferase was amplified. 1 ×107 of cells were engrafted into NOD-SCID mice and solid tumors were extracted and weighed at day 35. Data are presented as mean ± SD of n = 3 biological replicates (6A-6L, and 6 N). Significance was calculated using two-tailed, unpaired Student’s t-test. Source data are provided as a source data file. See also Fig. S6.