SETD2 is necessary for productive viral replication.

(A) CIN612 cells were transiently transduced with either a scramble control shRNA (shScram) or one of two SETD2 shRNAs (shSetd2#1 and shSetd2#2) for 72hrs. At this time, DNA and protein were either harvested as an undifferentiated (T0) sample, or cells were grown in high calcium medium to induce differentiation (72hr). DNA was digested with BamHI, which does not cut the viral genome, and Southern blot analysis was performed using the HPV31 genome as a probe. Lysates harvested at the indicated time points were analyzed by immunoblotting to demonstrate the decrease in SETD2 and H3K36me3 upon shRNA-mediated knockdown. Involucrin and K10 were used as markers of differentiation, and p84 and histone H3.1 (H3.1) served as loading controls. (B) CIN612 cells were transduced with lentivirus expressing either control guide RNAs (sgCTR) or guide RNAs targeting SETD2 (sgSETD2 #1 and sgSETD2 #2) and selected with puromycin. Following selection, DNA and protein were harvested from the heterogenous population of cells. DNA was digested with BamHI (non-cutter) and Southern blot analysis performed using the HPV31 genome as a probe. Western blot analysis was performed to examine the levels of SETD2, involucrin and K10 as differentiation controls, with GAPDH as a loading control. (A, B) Fold change in episome copy number for SETD2 knockdown using shRNAs as well as guide RNAs was determined by performing densitometry of episomal bands from three independent experiments using ImageJ software. Shown is the fold change relative to shScram T0 (A) and sgCTR T0 (B), which are set to one. Error bars represent means +/- standard error. Statistics were assayed using a student’s t test. *p≤ .05. WB = western blot. Ca = calcium.