PRKDC promotes hepatitis B virus transcription through enhancing the binding of RNA Pol II to cccDNA

Abstract

Hepatitis B virus infection remains a major health problem worldwide due to its high risk of liver failure and hepatocellular carcinoma. Covalently closed circular DNA (cccDNA), which is present as an individual minichromosome, serves as the template for transcription of all viral RNAs and pla ays critical role in viral persistence. Therefore, there is an urgent need to gain broader insight into the transcription regulation of cccDNA. Here, we combined a modified Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with an engineered ascorbate peroxidase 2 (APEX2) to identify cccDNA associated proteins systematically in living cells. By functional screening, we verified that protein kinase, DNA-activated, catalytic subunit (PRKDC) was an effective activator of HBV cccDNA transcription in HBV-infected HepG2-NTCP cells and primary human hepatocytes. Mechanismly, PRKDC interacted with POLR2A and POLR2B, the two largest subunits of RNA polymerase II (Pol II) and recruited Pol II to HBV cccDNA minichromosome in a kinase-dependent manner. PRKDC knockdown or inhibitor treatment significantly decreased the enrichment of POLR2A and POLR2B on cccDNA, as well as reducing the levels of cccDNA associated Pol II Ser5 and Ser2 phosphorylation, which eventually inhibited the HBV cccDNA activity. Collectively, these findings give us new insights into cccDNA transcription regulation, thus providing new potential targets for HBV treatment in patients.

Fig. 1. Systematic identification of cccDNA-associated proteins.

A Schematic of the methodology to identify cccDNA-associated proteins. MCP MS2 coat protein. B Western blotting (WB) analysis with antibodies against dCas9. β-actin was used as a loading control. C The level of dCas9 associated with cccDNA was examined by ChIP-qPCR assay. Cross-linked chromatin was immunoprecipitated with specific anti-dCas9 antibody or corresponding IgG, followed by PCR quantification of HBV cccDNA using specific primers. D APEX2-mediated biotinylation of endogenous proteins associated with cccDNA, as detected by WB with streptavidin-HRP (SRP). E Mass spectrometry results were verified by immunoprecipitation and WB assays. HBV-infected cells were collected, lysates were immunoprecipitated with streptavidin agarose, and the products were separated by SDS–PAGE. GAPDH antibody was used as a negative control.

Fig. 2. PRKDC silencing inhibits HBV transcription.

A The knockdown efficiency was verified by WB analysis with antibodies against PRKDC. β-actin was used as a loading control. B, C PRKDC suppression decreased HBeAg (B) and HBsAg (C) secretion, as detected by ELISA kit. D–F The expressions of total HBV RNAs and 3.5-kb pgRNA were down regulated upon PRKDC disruption. HBV RNAs (D) and pgRNA (E) were analyzed by real-time PCR using specific primers and Northern blotting (F). Ribosomal RNAs (28S and 18S) served as loading controls. G HBsAg and HBc were analyzed by immunoblotting analysis. H PRKDC suppression in HBV-infected PHHs decreased total HBV RNAs and 3.5-kb RNA. *P < 0.05; **P < 0.01.

Fig. 3. NU7026 reduced cccDNA transcription.

HBV-infected HepG2-NTCP cells or PHHs were treated with a concentration gradient of NU7026, and the concentrations of HBeAg (A) and HBsAg (B) in cell culture supernatants were detected by ELISA kit. Total HBV RNA (C) and pgRNA (D) transcriptions were analyzed by real-time PCR using specific primers and Northern blotting (E). Ribosomal RNAs (28 S and 18 S) served as loading controls. HBsAg and HBc were detected by immunoblotting analysis (F). G NU7026 treated in HBV-infected PHHs decreased total HBV RNAs and 3.5-kb RNA. *P < 0.05; **P < 0.01.

Fig. 4. PRKDC is dispensable for the formation of cccDNA.

HBV cccDNA was extracted at day 5 post HBV infection using the Hirt method. Real-time PCR analysis was performed using cccDNA-specific primers upon shRNA knockdown (A) or NU7026 treatment (D). Droplet digital PCR (ddPCR) was also carried out to detect the numbers of cccDNA after PRKDC knockdown (B) or inhibitor treatment (E). The decrease in total HBV RNAs or pgRNA expression was calculated as the ratio of normalized HBV RNAs or pgRNA to cccDNA after PRKDC knockdown (C) or inhibitor treatment (F). **P < 0.01; n.s. not significant.

Fig. 5. PRKDC interacts with POLR2A and POLR2B, and associates with cccDNA.

A, B Immunofluorescent staining demonstrates partial colocalization of PRKDC (green) and POLR2A (A) or POLR2B (B) in HBV-infected HepG2-NTCP and HepG2.2.15 cells. Scale bar, 10 μm. C Endogenous co-immunoprecipitation with PRKDC (left), POLR2A (middle), and POLR2B (right) was carried out in HBV-infected HepG2-NTCP and HepG2.2.15 cells using indicated antibodies and blotted with specific antibodies. D–F Cross-linked chromatin from HBV-infected or non-HBV-infected HepG2-NTCP cells was immunoprecipitated with PRKDC (D), POLR2A (E), and POLR2B (F), and the corresponding IgG was used as a control, followed by PCR quantification of HBV cccDNA and the promoter of GAPDH and MYH7 using specific primers. The results are displayed as the percentage of input.

Fig. 6. PRKDC knockdown and NU7026 treatment inhibits the binding of POLR2A and POLR2B to HBV cccDNA.

HBV-infected HepG2-NTCP cells were treated with PRKDC shRNAs or NU7026, and then cross-linked with 4% formaldehyde solution, sonicated, and immunoprecipitated with indicated antibodies. Levels of POLR2A (A) or POLR2B (B) associated with HBV cccDNA or the GAPDH or MYH7 promoter upon PRKDC knockdown were determined by qPCR. The effect of NU7026 treatment on the recruitment of POLR2A (C) or POLR2B (D) to cccDNA or the GAPDH or MYH7 promoter was examined by ChIP-qPCR. The results are displayed as the percentage of input. **P < 0.01.

Fig. 7. cccDNA-associated Pol II CTD Ser2 and Ser5 phosphorylation modifications decrease upon PRKDC depletion or NU7026 treatment.

A Western blot analysis with antibodies against specified proteins. β-actin was used as a loading control. B, C Effects of PRKDC knockdown on the recruitment of Pol II Ser5 or Pol II Ser2 to cccDNA. The level of Pol II Ser5 (B) or Pol II Ser2 (C) associated with HBV cccDNA or the GAPDH or MYH7 promoter was analyzed by ChIP assay with the indicated antibodies. D, E NU7026 treatment in HBV-infected HepG2-NTCP cells correlated with decreased Pol II Ser5 and Pol II Ser2 binding. The level of Pol II Ser5 (D) or Pol II Ser2 (E) associated with HBV cccDNA or the GAPDH or MYH7 promoter was analyzed by ChIP assay. The results are displayed as the percentage of input. **P < 0.01.