Human cytomegalovirus RNA2.7 inhibits RNA polymerase II (Pol II) Serine-2 phosphorylation by reducing the interaction between Pol II and phosphorylated cyclin-dependent kinase 9 (pCDK9)

Abstract

Human cytomegalovirus (HCMV) is a ubiquitous pathogen belongs to betaherpesvirus subfamily. RNA2.7 is a highly conserved long non-coding RNA accounting for more than 20% of total viral transcripts. In our study, functions of HCMV RNA2.7 were investigated by comparison of host cellular transcriptomes between cells infected with HCMV clinical strain and RNA2.7 deleted mutant. It was demonstrated that RNA polymerase II (Pol II)-dependent host gene transcriptions were significantly activated when RNA2.7 was removed during infection. A 145 ​nt-in-length motif within RNA2.7 was identified to inhibit the phosphorylation of Pol II Serine-2 (Pol II S2) by reducing the interaction between Pol II and phosphorylated cyclin-dependent kinase 9 (pCDK9). Due to the loss of Pol II S2 phosphorylation, cellular DNA pre-replication complex (pre-RC) factors, including Cdt1 and Cdc6, were significantly decreased, which prevented more cells from entering into S phase and facilitated viral DNA replication. Our results provide new insights of HCMV RNA2.7 functions in regulation of host cellular transcription.

Keywords: Cyclin-dependent kinase 9 (CDK9); Human cytomegalovirus (HCMV); Phosphorylation; RNA polymerase II (Pol II); RNA2.7.

Fig. 1

Repression of cellular Pol II-dependent transcription by HCMV RNA2.7. A General alteration of genome transcription in cells infected with HAN or HANΔRNA2.7 (MOI: 1.0). Cellular transcripts changed by infection are sorted in Venn diagram. B Comparison results on diseases and biofunctions of HAN or HANΔRNA2.7 altered transcripts. Their effects are indicated with Z-score: an active effect is indicated with Z-score > 2.0; a suppressive effect is indicated with Z-score < −2.0. C Venn diagram showing genes involved in host gene activation. HANΔRNA2.7 specific genes were analyzed and categorized into transcription from Pol II promoter. D Activation of Pol II-dependent regulators. Activities of transcriptional regulators were modified by RNA2.7. The effects are indicated with Z-score: Z-score > 2.0 indicates a significantly transcriptional activation of downstream genes; 2.0 ​> ​Z-score > 0 indicates a transcriptional activation of downstream genes; 0 ​> ​Z-score indicates a transcriptional repression of downstream genes. E Western blot analysis for pCDK9, Pol II and its two phosphorylated forms (Pol II S2 and Pol II S5) in cells infected with HAN or HANΔRNA2.7 ​at different time points. IE1 and GAPDH were measured as controls. Data are presented as mean ​± ​SEM. Statistical analysis was performed by Student's t-test. ∗∗∗P ​< ​0.0001.

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.

Fig. 3

Regulation of host cell cycle by HCMV RNA2.7. A Block of host cells entry into S phase by RNA2.7. HELF cells were infected with HAN or HANΔRNA2.7 (MOI ​= ​1.0). DRB was added 4 ​h before infection with a final concentration of 200 ​nmol/L and was used as a positive control. Cells were stained and analyzed by flow cytometry. Percentages of cells in the G0/G1 phases of the cell cycle were calculated and presented as mean ​± ​SEM. B Model of pre-RC formation for cellular DNA replication. Components involved in pre-RC formation were up-regulated in HANΔRNA2.7 infected cells and were facilitated cellular DNA replication. Gene that mRNA fold change more than 2 is indicated with red color. Gene that mRNA fold change less than −2 is indicated with green color. C Effects of RNA2.7 on genes facilitating pre-RC formation. Transcriptions of MCM2, MCM4, MCM5, Cdt1 and Cdc6 were increased in HANΔRNA2.7 infected cells, and could be decreased by inhibiting Pol II S2 phosphorylation. Data are presented as mean ​± ​SEM. D Effects of RNA2.7 on expressions of Cdt1 and Cdc6. Protein levels of Cdt1 and Cdc6 were increased in HANΔRNA2.7 infected cells, and could be decreased by inhibiting Pol II S2 phosphorylation. Data are presented as mean ​± ​SEM. Statistical analysis was performed by Student's t-test. ∗P ​< ​0.05; ∗∗P ​< ​0.01; ∗∗∗P ​< ​0.0001. DRB, 5,6-dichloro-1-b-D-ribofuranosyl benzimidazole; pre-RC, pre-replication complex.

Fig. 4

Effects of RNA2.7 on viral DNA replication. A Growth curves of HAN and HANΔRNA2.7 in HELF cells. Growth curves were performed in quiescent HELF cells infected with HAN or HANΔRNA2.7 ​at an MOI of 0.1 and 3. Supernatant samples were collected at 1, 3, 6 and 9 dpi. Titers were determined by TCID₅₀. HAN is indicated with solid lines, and HANΔRNA2.7 is indicated with dashed lines. Data are averages from three independent experiments, and standard errors are indicated. B Up-regulation of viral replication by Cdt1 and Cdc6 knockdown. Evaluated siRNA specific to Cdt1 and Cdc6 were transfected into HELF cells. Cells transfected with siRNAs specific to Cdt1 or Cdc6 were infected with HANΔRNA2.7 (MOI ​= ​1.0). Cells transfected with siRNA negative control were infected with HAN or HANΔRNA2.7. Viral DNA levels were quantified at 2, 24 and 96 hpi. Relative viral DNA levels are presented as mean ​± ​SEM. Statistical analysis was performed by Student's t-test. ∗P ​< ​0.05; ∗∗P ​< ​0.01. TCID₅₀, standard median tissue culture infective dose.

Fig. 5

Model for the mechanism of HCMV RNA2.7 in cell cycle control. RNA2.7 binds to Pol II and inhibits Pol II S2 phosphorylation by blocking interactions between pCDK9 and Pol II. The inhibition of Pol II S2 phosphorylation decreases the levels of MCMs, Cdt1 and Cdc6, and disturbs the formation of pre-RC which leads to host cell cycle arrest at G0/G1 phase and facilitates viral DNA replication.