Transcriptional Elongation Control of Hepatitis B Virus Covalently Closed Circular DNA Transcription by Super Elongation Complex and BRD4

Abstract

Chronic hepatitis B virus (HBV) infection can lead to liver cirrhosis and hepatocellular carcinoma. HBV reactivation during or after chemotherapy is a potentially fatal complication for cancer patients with chronic HBV infection. Transcription of HBV is a critical intermediate step of the HBV life cycle. However, factors controlling HBV transcription remain largely unknown. Here, we found that different P-TEFb complexes are involved in the transcription of the HBV viral genome. Both BRD4 and the super elongation complex (SEC) bind to the HBV genome. The treatment of bromodomain inhibitor JQ1 stimulates HBV transcription and increases the occupancy of BRD4 on the HBV genome, suggesting the bromodomain-independent recruitment of BRD4 to the HBV genome. JQ1 also leads to the increased binding of SEC to the HBV genome, and SEC is required for JQ1-induced HBV transcription. These findings reveal a novel mechanism by which the HBV genome hijacks the host P-TEFb-containing complexes to promote its own transcription. Our findings also point out an important clinical implication, that is, the potential risk of HBV reactivation during therapy with a BRD4 inhibitor, such as JQ1 or its analogues, which are a potential treatment for acute myeloid leukemia.

Keywords: BRD4; hepatitis B virus; super elongation complex; transcriptional elongation.

FIG 1

Different P-TEFb-containing complexes are recruited to the HBV genome. (A) Cartoon model illustrating the position of the primer pairs used for qPCR after ChIP along the HBV genome. (B to E) The SEC subunits AFF4 (B), ELL2 (C), and BRD4 (D) and the P-TEFb kinase module CDK9 (E) are all recruited to the HBV genome, as detected in HepG2.2.15 cells by ChIP-qPCR assays with specific antibodies. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent the standard deviations from three independent measurements.

FIG 2

Bromodomain inhibitor JQ1 activates HBV transcription and replication. (A) Treatment of HepG2.2.15 cells by JQ1 promotes HBV transcription. The levels of HBV pgRNA were assessed by RT-qPCR and normalized to the internal control GAPDH mRNA levels before and after JQ1 exposure. (B) HBV core-associated DNAs were extracted from culture medium treated with DMSO and JQ1. The DNA levels were then quantified by standard curve qPCR analysis. (C) Treatment of HepG2.2.15 cells by I-BET151 promotes HBV transcription. The levels of HBV pgRNA were assessed by RT-qPCR and normalized to the internal control GAPDH mRNA levels before and after JQ1 exposure. (D) JQ1 treatment also upregulates the transcription from the nonintegrated form of the HBV genome. Huh7 cells transiently transfected with HBV were treated with different doses (0, 100, and 250 nM) of JQ1. The levels of HBV pgRNA were then assessed by RT-qPCR and normalized to the internal control GAPDH mRNA levels. Error bars represent standard deviations from three independent measurements. (E) The surface antigen of HBV, HBsAg, is upregulated by JQ1 treatment in Huh7 cells, which were treated as described for panel B. HBsAg levels in cell culture supernatant before and after different doses of JQ1 treatment were quantified by ELISA.

FIG 3

JQ1 treatment increases the occupancy of BRD4 and SEC on the HBV genome. (A) HepG2.2.15 cells were treated with 250 nM JQ1 for 48 h. Nontreated and treated cells were used in ChIP assays with specific antibodies, followed by qPCR amplification using the primer pairs spanning the HBV genome, as illustrated in Fig. 1A. The occupancy of BRD4 is significantly enhanced after JQ1 treatment. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent the standard deviations from three independent measurements. (B to G) BRD2, BRD3, and BRD4 were individually knocked down to examine their effects on HBV transcription upon JQ1 treatment. The levels of HBV pgRNA were then assessed by RT-qPCR and normalized to the internal control GAPDH mRNA levels. Error bars represent the standard deviations from three independent measurements.

FIG 4

SEC is required for JQ1-mediated HBV induction. (A to C) JQ1 treatment slightly enhances the occupancies of SEC subunits CDK9, AFF4, and ELL2. (D) Knockdown of AFF4 in HepG2.2.15 cells by lentivirus-mediated shRNA targeting AFF4. (E) Knockdown of AFF4 in HepG2.2.15 cells inhibits basal and JQ1-induced HBV pgRNA transcription. Control and AFF4-depleted cells were treated with DMSO, for the vehicle control, and JQ1. The expression levels were normalized to the internal control GAPDH mRNA levels. Error bars represent the standard deviations from three independent measurements. (F) The CDK9 inhibitor flavopiridol inhibits basal and JQ1-induced HBV pgRNA transcription. pgRNA levels were measured by RT-qPCR and normalized to GAPDH mRNA levels. Error bars represent the standard deviations from three independent measurements. (G and H) BRD4 and AFF4 enrichment analysis in BRD4-depleted cells. The HepG2.2.15 cells were treated with BRD4- and AFF4-specific shRNAs for 2 days, followed by 48 h of JQ1 treatment. IgG serves as the nonrelated antibody control. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent the standard deviations from three independent measurements.