SETD2 knockdown affects processing of late viral RNAs.

(A) Schematic of the HPV31 genome showing 5’ splice donor (SD) (closed circles) and 3’ splice acceptor (SA) (open circles) sites. Open reading frames are indicated by the shaded boxes, and promoters are indicated by the arrows. The early (pAE) and late (pAL) polyadenylation sites are indicated at the end of the E5 and L1 ORFs, respectively. (B) CIN612 cells were transiently transduced with lentivirus expressing either a control shRNA (shScram) or SETD2 shRNA #2. 72hr post-transduction, cells were harvested as a T0 (undifferentiated) or differentiated in high calcium medium for 72hr. At each time point, RNA and protein were harvested. (B) End-point PCR amplification was performed (35 cycles) using a 5’ primer in the E7 ORF (nt 766) and a 3’ primer in the L1 ORF (nt 6595), as well as primers specific to GAPDH. Primer sequences are listed in S1 Table. PCR products were gel purified and sequenced. Shown is a graphical representation of the identified products. Relative levels of L1a and L1b were determined by densitometry using ImageJ software. Values shown represent the ratios of L1a/L1b at each time point. (C) PCR amplification was performed for 25 cycles using the indicated primers to the early region as well as primers specific to GAPDH. Primer sequences are listed in S1 Table. PCR products generated using the 121F/3373R (E4R) primer pair were gel purified and sequenced. Shown is a graphical representation of the identified products. Relative levels of E1^E4 and E6E7 were determined by densitometry using ImageJ software and normalized to GAPDH. Shown is the fold difference relative to shScram T0, which is set to 1. (D) Western blot analysis was performed using antibodies specific to SETD2 and GAPDH. Shown are representative images of three independent experiments. Ca = calcium. WB = western blot.