Fig. 2.

Inhibition of Pol II S2 phosphorylation by HCMV RNA2.7. A Co-immunoprecipitation and Western blot analysis of Pol II binding pCDK9 proteins in cells infected with HAN or HANΔRNA2.7. The relative amounts of pCDK9 binding to Pol II are quantified by densitometry with Pol II as a reference. Data are presented as mean ​± ​SEM. B Interaction between RNA2.7 and Pol II. Pol II binding RNAs were immunopricipitated. Captured RNA was reverse transcribed and amplified using RNA2.7 specific primer. IE1 and GAPDH cDNA was amplified as negative control. M: DNA ladder marker (DL2000 for RNA2.7 and GAPDH; DL500 for IE1). C Effect of RNA2.7C2c on inhibiting Pol II S2 phosphorylation. A series of vectors transcribing RNA2.7 gene with different lengths were constructed and transfected into HEK293 ​cells. By Western blot analysis, RNA2.7C2c was identified to inhibit Pol II S2 phosphorylation functionally. The amounts of Pol II S2 proteins are quantified by densitometry. Data are presented as mean ​± ​SEM. D RNA2.7C2c showing dose-dependent effect on inhibition of Pol II S2 phosphorylation. E Effect of RNA2.7C2c on interaction between Pol II and pCDK9. Vector transcribing RNA2.7C2c was transfected into HEK293 ​cells. Cells transfected with pcDNA3.1 were used as a negative control. By co-immunoprecipitation and Western blot analysis, RNA2.7C2c was verified to block the interaction between pCDK9 and Pol II protein. The relative amounts of pCDK9 binding to Pol II are quantified by densitometry with Pol II as a reference. Data are presented as mean ​± ​SEM. F Confirmation of interaction between RNA2.7C2c and Pol II protein by RNA EMSA. RNA2.7C2c probe and competitive RNA2.7C2c were 145 ​nt in length. RNA2.7C2c probe was labeled with biotin. Pol II CTD proteins were purified from nucleoprotein using anti Pol II CTD antibody. Statistical analysis was performed by Student's t-test. ∗P ​< ​0.05; ∗∗P ​< ​0.01; ∗∗∗P ​< ​0.0001. CTD, C-terminal domain.