Potential conflict of interest: Dr. Levrero consults for Roche and Assembly. He advises Gilead, Sanofi, and Galapagos. He received grants from Bristol‐Myers Squibb.
Supported by the “Twelfth Five‐Year” National Key Technology Research and Development Programs of China (2012ZX10002007), the National Natural Science Foundation of China (81461130019), and the German Research Foundation (SFB/Transregio TRR60).
Hepatitis B virus (HBV) is a hepatotropic, partially double‐stranded DNA virus. HBV infection remains a serious global health problem, with more than 240 million people chronically infected. Upon infection, relaxed circular DNA in the HBV virion is delivered into the nucleus and converted into covalently closed circular DNA (cccDNA), which then serves as a template to transcribe pregenomic RNA (pgRNA) and other subgenomic viral RNAs. The 5′‐ɛ signal within pgRNA is recognized and bound by HBV polymerase (Pol), which is required for subsequent pgRNA encapsidation into a newly forming capsid with HBV core protein (HBc) in the cytoplasm. Mature nucleocapsids with HBV relaxed circular DNA which is reverse‐transcribed from pgRNA are either released from the cell after being enveloped with HBV surface proteins or recycled to the cccDNA reservoir in the nucleus. The cccDNA is crucial for persistent HBV infection and recurrence upon the discontinuation of treatment. Current therapies successfully suppress viral replication, but their inhibitory effects on stably existing cccDNA are limited. Identification of the mechanisms and factors involved in silencing or elimination of cccDNA could allow the development of more precisely targeted therapeutic strategies.1
The cccDNA organizes into a minichromosome with histone 3 (H3) and H4 proteins and nonhistone proteins, such as HBc, hepatitis B x protein (HBx), and host transcription factors.2 5 4 7 10
Here, by screening a series of methyltransferases and demethylases, we identified protein arginine methyltransferase 5 (PRMT5) as a restrictor of HBV replication that can preferentially catalyze the symmetric dimethylation of arginine 3 on H4 (H4R3me2s) on cccDNA through its methyltransferase activity to epigenetically silence cccDNA transcription and, in a methyltransferase‐independent manner, interferes with HBV pgRNA encapsidation and thus inhibits HBV DNA production.
Materials and Methods
CELL CULTURE, VIRUSES, AND INFECTION
The HBV‐producing stable cell line HepG2.117 and the HepG2‐NTCP cell line were kindly provided by Prof. Mengji Lu (University Hospital Essen) and Prof. Stephan Urban (University Hospital Heidelberg), respectively. HepaRG cells and primary human hepatocytes (PHHs) were purchased from Biopredic International (France) and RIDL (China), respectively. Huh7 and HEK293 cells were obtained from the Cell Bank of the Chinese Academy of Sciences. Cell cultures are described in the Supporting Information Supporting Information 13
PLASMIDS AND REAGENTS
Plasmids used for transfection are listed in Supporting Table S3 Supporting Information Supporting Table S4 Supporting Table S5
PATIENTS
The clinical and virological characteristics of the patients enrolled in this study are summarized in Supporting Table S2
HBV cccDNA AND HBV CORE PARTICLE DNA EXTRACTION AND QUANTITATION BY QUANTITATIVE PCR
HBV cccDNA was extracted according to the method described, with minor modifications.8
HBV CORE PARTICLE RNA EXTRACTION
The HBV Core particle was purified as described in the Supporting Information
ANALYSIS OF CORE CAPSIDS
Cytoplasmic cell lysates containing HBV capsids were directly resolved on a 1% agarose gel in 0.5 × trishydroxymethylaminomethane–borate–ethylene diamine tetraacetic acid buffer and transferred to nitrocellulose membranes. Proteins were then detected by western blot using anti‐HBc antigen antibody (Dako).13
SOUTHERN BLOT/NORTHERN BLOT
Extracted DNA and extracted RNA were loaded on a 1.2% agarose gel in 1 × trishydroxymethylaminomethane–acetic acid–ethylene diamine tetraacetic acid buffer and on formaldehyde denaturing agarose gel in 1× MOPS buffer, respectively, and then transferred onto a positively charged nylon membrane (Roche, Switzerland) using a vacuum blotter (Bio‐Rad). HBV DNA and RNA were hybridized and detected using a DIG Northern Starter Kit (Roche) according to the manufacturer's instructions.14
CHROMATIN IMMUNOPRECIPITATION ASSAY
Chromatin immunoprecipitation (ChIP) assays were performed using the method described, with minor modifications.8 Supporting Fig. S4
REAL‐TIME REVERSE‐TRANSCRIPTION PCR
Total RNA was extracted using TRIzol reagent and reverse‐transcribed using ReverTra Ace qPCR RT Master Mix with the gDNA Remover kit, followed by quantitative PCR using the Thunderbird SYBR qPCR Mix (both from Toyobo, Japan). The primers used are listed in Supporting Table S6
COIMMUNOPRECIPITATION AND WESTERN BLOT
Cells were lysed on ice in coimmunoprecipitation (CoIP) lysis buffer. After centrifugation, the supernatant was precleared with protein A/G‐agarose beads. Protein complexes were then immunoprecipitated with the indicated antibodies and analyzed by western blot using the specific antibodies (Supporting Table S4
IMMUNOFLUORESCENCE ASSAY
The immunofluorescence assay was performed as described.15
RNA IMMUNOPRECIPITATION ASSAY
The RNA immunoprecipitation (RIP) assay was performed using the method described, with minor modifications.16
STATISTICS
The data are presented as the mean ± standard error of the mean. Statistical analyses were performed using the Student t test or Mann‐Whitney U test. P < 0.05 was considered statistically significant.
Results
IDENTIFICATION OF PRMT5 AS AN ANTI‐HBV HOST FACTOR THAT RESTRICTS cccDNA TRANSCRIPTION AND CORE PARTICLE DNA PRODUCTION
To examine whether HBV transcription and replication are associated with histone methylation and to identify the host factors involved, we applied the RNA interference–based approach to screen a panel of the known histone methyltransferases or demethylases, including SUV39H, G9a, EZH2, PRMT5, LSD1, KDM5A, and KDM2A (Supporting Fig. S1A and Table S1 17 4 1 A) and PRMT5 inhibits the production of pgRNA and core particle DNA in a dose‐dependent manner (Supporting Fig. S1B‐D 1 B), respectively, and regulated the cccDNA transcriptional activity (Fig. 1 C), which was calculated as the ratio of pgRNA to cccDNA. Considering that the efficacy of HBV infection in dHepaRG cells was relatively low, which might lead to an underestimated anti‐HBV effect of PRMT5 when we analyzed at the whole‐cell lysate level, we further evaluated the effect at the single‐cell level by immunofluorescence assay (Fig. 1 D; Supporting Fig. S1F 1 E) and PHHs (Supporting Fig. S1E
Figure 1: PRMT5 inhibits HBV transcription and core particle DNA production. (A) The indicated small interfering RNAs were cotransfected with monomeric linear HBV DNA into Huh7 cells. Culture supernatants were collected to detect HBeAg and HBsAg by enzyme‐linked immunosorbent assay. (B‐D) PRMT5 was transduced into HBV‐infected dHepaRG cells for 6 days, and shPRMT5 was transduced for 5 days. Expression of PRMT5 was detected by western blot. Levels of HBV antigens in the supernatant were detected by enzyme‐linked immunosorbent assay. HBV pgRNA, core particle DNA, and cccDNA were analyzed by quantitative PCR. Levels of pgRNA and cccDNA were used to calculate the ratio of pgRNA/cccDNA (B,C). HBV core protein and endogenous PRMT5 were observed by immunofluorescence assay using the specific antibodies (D). (E) PRMT5 expression plasmids or 100 nM of siPRMT5 were transfected into HBV‐infected HepG2‐NTCP cells. Cells were collected to examine the level of HBV antigens, core particle DNA, and pgRNA/cccDNA at day 4 posttransfection. * P < 0.05, ** P < 0.01. Abbreviations: Ad, adenovirus; DAPI, 4′,6‐diamidino‐2‐phenylindole; IB, immunoblot; N.S., not significant; si, small interfering.
Considering that PRMT5, a member of the type II protein arginine methyltransferases, epigenetically regulates gene expression mainly through methylating histone and nonhistone targets, we next examined whether the enzymatic activity of PRMT5 is essential for its inhibitory effect on cccDNA transcription and HBV DNA production. The mutation of the methyltransferase domain of PRMT5 (PRMT5Δ; Fig. 2 A; Supporting Fig. S2A 2 B‐D; Supporting Fig. S1G 2 E; Supporting Fig. S2B
Figure 2: Effects of PRMT5Δ on HBV transcription and core particle DNA production. (A) A schematic representation of the catalytically inactive form of human PRMT5 (PRMT5Δ) and the deleted amino acids in PRMT5Δ are shown. (B‐E) PRMT5Δ was transduced into HBV‐infected dHepaRG cells for 6 days. (B) Expression of PRMT5Δ was detected by western blot. (C) Culture supernatants were collected to detect HBeAg and HBsAg by enzyme‐linked immunosorbent assay. (D) HBV pgRNA and cccDNA were extracted and quantified by quantitative PCR for calculating the ratio of pgRNA/cccDNA. (E) The level of core particle DNA was analyzed by quantitative PCR. ** P < 0.01. Abbreviations: Ad, adenovirus; IB, immunoblot; N.S., not significant.
PRMT5 INCREASES THE SYMMETRIC DIMETHYLATION OF cccDNA‐BOUND H4R3 TO INHIBIT cccDNA TRANSCRIPTION IN A METHYLTRANSFERASE ACTIVITY–DEPENDENT MANNER
Because H3R8me2s and H4R3me2s are reported to be catalyzed by PRMT5,17 3 A; Supporting Fig. S3A,B 3 B, red line and black line), whereas H3R8me2s was almost undetectable on the HBV minichromosome throughout the period of viral infection (Supporting Fig. S3C 3 B, blue line). We repeated this experiment three times with sera from different chronic HBV patients. Although the kinetics of cccDNA transcriptional activity varied a bit in the three experiments, they were always negatively associated with the status of H4R3me2s on the HBV minichromosome.
Figure 3: cccDNA transcriptional activity was negatively correlated with cccDNA‐bound H4R3me2s. (A,B) HBV‐infected dHepaRG cells in 100‐mm dishes were harvested by trypsinization on the indicated days. One eighth of the cells were used to extract HBV cccDNA, and one sixteenth of the cells were used to extract HBV pgRNA, followed by quantitative PCR analysis. (A) The ratio of pgRNA to cccDNA at 4 dpi was set as 1. (B) Three quarters of HBV‐infected dHepaRG cells harvested at 7, 13, and 19 dpi were used for ChIP assays with the indicated antibody. Levels of the specific modified histones on HBV cccDNA or the host gene GAPDH were analyzed by quantitative PCR. The ratio of pgRNA to cccDNA at 7 dpi was set as 1. (C,D) Each liver biopsy was used to conduct RNA extraction and ChIP assay with the indicated antibody. Relative HBV pgRNA/cccDNA was calculated as the ratio of normalized HBV pgRNA to cccDNA. Levels of H4R3me2s on HBV cccDNA or the host GAPDH DNA were examined by quantitative PCR. * P < 0.05, ** P < 0.01. Abbreviations: dpi, days postinfection; IC, inactive carrier; IT, immune‐tolerant; N.S., not significant.
To further validate the effect of cccDNA‐bound H4R3me2s on HBV transcription in vivo , we collected liver biopsies from 10 chronic HBV patients including 5 HBeAg‐positive patients in immune‐tolerant phase and 5 HBeAg‐negative inactive carriers to analyze the association of the status of cccDNA‐bound H4R3me2s with the levels of cccDNA transcriptional activity. The level of pgRNA/cccDNA in inactive carrier patients was, as expected, significantly lower than that in immune‐tolerant patients (Fig. 3 C), whereas the average level of cccDNA‐bound H4R3me2s was about 3‐fold higher in inactive carriers than in immune‐tolerant patients. Moreover, the level of H4R3me2s on cccDNA varied more than that on the host GAPDH DNA (Fig. 3 D). The level of H3 acetylation on cccDNA, which was reported to be involved in the regulation of HBV replication in the liver tissue,7 Supporting Fig. S5A
We further examined whether alteration of PRMT5 expression affects the status of cccDNA‐bound H4R3me2s. Overexpression or knockdown of PRMT5 led to increased or reduced binding of PRMT5 to cccDNA and accordingly increased or reduced the level of H4R3me2s on the minichromosome and cccDNA transcription activity, respectively (Fig. 4 A,B). In addition, although the level of cccDNA‐bound PRMT5Δ was comparable with that of cccDNA‐bound WT PRMT5, neither the status of H4R3me2s nor the cccDNA transcriptional activity was affected by the increased expression of PRMT5Δ (Fig. 4 C). Together, these results indicate that cccDNA‐bound H4R3me2s is a repressive marker of cccDNA transcription and that PRMT5 can be recruited to cccDNA and, in a methyltransferase‐dependent manner, catalyze H4R3me2s modification on cccDNA to repress cccDNA transcription.
Figure 4: PRMT5 increases the symmetric dimethylation of cccDNA‐bound H4R3 to inhibit cccDNA transcription. (A,C) PRMT5 or PRMT5Δ was transduced into HBV‐infected dHepaRG cells in 100‐mm dishes for 6 days. (B) shPRMT5 was transduced into HBV‐infected dHepaRG cells in 100‐mm dishes for 5 days. One eighth of the cells were used to extract HBV cccDNA, and one sixteenth of the cells were used to extract total RNA, followed by quantitative PCR analysis. Three quarters of the cells were harvested and used for ChIP assays with the indicated antibodies. Levels of the specific proteins on HBV cccDNA were analyzed by quantitative PCR. * P < 0.05, ** P < 0.01. Abbreviation: N.S., not significant.
PRMT5 PREFERENTIALLY REGULATES H4R3me2s ON cccDNA AND INTERACTS WITH HBc
Because H4R3me2s was present not only on the cccDNA minichromosome but also on the host genome, we next compared the effect of PRMT5 on cccDNA‐bound or host gene–bound H4R3me2s. Histone–DNA complexes were immunoprecipitated using antibodies specifically against H4R3me2s, followed by quantitative PCR analysis with primers designed to detect cccDNA and three host genes—RPL30, MYOD1, and GAPDH. The results showed that overexpression or knockdown of PRMT5 significantly elevated or reduced the level of H4R3me2s as well as PRMT5 on HBV cccDNA minichromosome, respectively, but had much less impact on the host genes studied here (Fig. 5 A,B), implying that PRMT5 can, to some extent, preferentially bind to cccDNA and catalyze cccDNA‐bound H4R3me2s.
Figure 5: PRMT5 preferentially regulates H4R3me2s on cccDNA and interacts with HBc. (A,B) PRMT5 was transduced into HBV‐infected dHepaRG cells in 100‐mm dishes for 6 days. shPRMT5 was transduced into HBV‐infected dHepaRG cells in 100‐mm dishes for 5 days. Three quarters of the cells were used for ChIP assays with the indicated antibodies. Levels of H4R3me2s or PRMT5 on HBV cccDNA or the host RPL30, MYOD1, and GAPDH genes were analyzed by quantitative PCR. (C) Huh7 cells were cotransfected as indicated, and the cells were subjected to CoIP assay with the indicated antibody. Expression of the indicated proteins was analyzed by western blot (β‐actin as a loading control). (D) Huh7 cells were cotransfected with plasmids encoding PRMT5 and 1.3‐fold HBV genome. Cells were harvested for the immunofluorescence assay. PRMT5 and HBc were detected by the specific antibodies. (E) dHepaRG cells in 100‐mm dishes were infected by HBV WT and HBc protein–deficient virus (HBV‐ΔHBc). Three quarters of the cells were used for ChIP assays with the anti‐PRMT5antibody. The rest of the cells were subjected to analysis of the expression levels of HBc and PRMT5 by western blot. * P < 0.05, ** P < 0.01. Abbreviations: Ad, adenovirus; DAPI, 4′,6‐diamidino‐2‐phenylindole; IB, immunoblot; IP, immunoprecipitation; N.S., not significant; WCL, whole‐cell lysate.
The HBc and HBx proteins have been associated with the HBV minichromosome3 Supporting Fig. S8A Supporting Fig. S8B 5 C; Supporting Fig. S8C‐F 5 D). To further examine the role of HBc in the binding of PRMT5 to cccDNA, we constructed an HBc‐deficient virus (HBV‐ΔHBc) and found that less PRMT5 was bound to the cccDNA of HBV‐ΔHBc compared to that of HBV‐WT. Moreover, overexpression of PRMT5 resulted in significant up‐regulation of the level of cccDNA‐bound PRMT5 in the HBV‐WT group but not in the HBV‐ΔHBc group (Fig. 5 E). These results provide a possibility that PRMT5 is targeted to HBV cccDNA through its interaction with the viral core protein.
PRMT5‐MEDIATED SUPPRESSION OF cccDNA TRANSCRIPTION INVOLVES THE Brg1‐BASED HUMAN SWI/SNF CHROMATIN REMODELER AND RNA POL II
It has been reported that PRMT5 acts as a part of a variety of complexes to regulate gene transcription and cell signal transduction.22 23 24 6 A). Unlike Brg1, the binding of hBrm to cccDNA did not significantly change with the alteration of PRMT5 expression (Supporting Fig. S9 6 B), which confirms the role of Brg1 in PRMT5‐mediated repression of HBV cccDNA transcription.
Figure 6: Effects of PRMT5 on the binding of Brg1 and RNA Pol II to cccDNA. (A,C) PRMT5 was transduced into HBV‐infected dHepaRG cells in 100‐mm dishes for 6 days. shPRMT5 was transduced into HBV‐infected dHepaRG cells in 100‐mm dishes for 5 days. Three quarters of the cells were used for ChIP assay with the indicated antibodies. Levels of Brg1 or Pol II on the indicated genes were analyzed by quantitative PCR. (B) PRMT5 was transduced into HBV‐infected dHepaRG cells at day 7 post–HBV infection. Adeno‐shBrg1 was added 1 day later, and the cells were cultured for another 5 days before being subjected to quantitative PCR analysis. Expression of PRMT5 and Brg1 was detected by western blot. HBV pgRNA and cccDNA were quantified by quantitative PCR for calculating the ratio of pgRNA/cccDNA. * P < 0.05, ** P < 0.01. Abbreviations: Ad, adenovirus; IB, immunoblot; KD, knockdown.
Because cccDNA is transcribed by the host RNA Pol II,25 26 6 C). Together, the above data suggest that PRMT5 regulates the binding of the host Brg1‐based human SWI/SNF chromatin remodeler and RNA Pol II to cccDNA, which may contribute to the PRMT5‐mediated epigenetic suppression of cccDNA transcription.
PRMT5 INHIBITS HBV CORE PARTICLE DNA PRODUCTION IN A METHYLTRANSFERASE ACTIVITY–INDEPENDENT MANNER
As indicated above, PRMT5Δ maintained the activity of inhibiting the production of core particle DNA but did not affect cccDNA transcription (Fig. 2 ). To confirm this, we further tested the levels of HBV core capsids, RNAs, and core particle DNA in an HBV‐replicating cell model, in which the viral pgRNA is transcribed under the CMV promoter. Southern blot results showed that PRMT5Δ expression or knockdown of endogenous PRMT5 decreased or increased the level of core particle DNA in a dose‐dependent manner, respectively, while it did not markedly affect the levels of HBV total RNA and core capsids (Fig. 7 A; Supporting Fig. S10A 7 B). To further clarify the step at which PRMT5 inhibits HBV core particle DNA production, we constructed cytoplasm‐localized or nucleus‐localized PRMT5Δ. Expression of both NES‐PRMT5Δ and NLS‐PRMT5Δ in CMV‐HBV‐replicating Huh7 cells did not reduce the levels of HBV RNAs and core capsids; however, the cytoplasm‐localized, but not the nucleus‐localized, PRMT5Δ significantly decreased the core particle DNA levels (Fig. 7 C). In HBV‐infected dHepaRG cells, the cytoplasm‐localized PRMT5 decreased the level of viral core particle DNA but did not affect the production of viral antigens and pgRNA and the level of cccDNA‐bound H4R3me2s and PRMT5 proteins (Fig. 7 D, E). These results suggest that PRMT5 does not affect the HBV RNA transcription or the formation of the core capsid itself but inhibits HBV core particle DNA production at the cytoplasmic level in a methyltransferase activity–independent manner.
Figure 7: Cytoplasmic PRMT5Δ reduces HBV core particle DNA production. (A) PRMT5Δ expression plasmids were cotransfected with pCMV‐HBV into Huh7 cells. The pcDNA3.1a vector was used as a negative control. Cells were harvested at 72 hours posttransfection. (B) PRMT5Δ was transduced into HepG2.117 cells, and the cells were harvested at 5 days after transduction. Core particle DNAs were analyzed by Southern blot. The positions of HBV relaxed circular DNA and single‐stranded DNA are indicated. Numbers below the lanes indicate the intensity of viral DNA bands relative to the vector group. HBV RNA was analyzed by northern blot. The 28S and 18S ribosomal RNAs served as loading controls. The positions of the 3.5‐kb and 2.4/2.1‐kb HBV RNAs are indicated. An aliquot of cell lysates was loaded onto an agarose gel to detect cytoplasmic core capsids. Overexpression of PRMT5Δ, NES‐PRMT5Δ, and NLS‐PRMT5Δ was detected by western blot; and β‐actin was used as a loading control. (C) Huh7 cells transfected with NES‐PRMT5Δ and NLS‐PRMT5Δ, followed by an immunofluorescence assay with anti‐PRMT5 antibody. (D,E) NES‐PRMT5 was transduced into HBV‐infected dHepaRG cells in 100‐mm dishes for 6 days. Expression of NES‐PRMT5 was detected by western blot. Levels of HBV antigens in the supernatant were detected by enzyme‐linked immunosorbent assay. HBV pgRNA, core particle DNA, and cccDNA were analyzed by quantitative PCR. Three quarters of the cells were harvested and used for ChIP assays with the indicated antibodies. Levels of the specific proteins on HBV cccDNA were analyzed by quantitative PCR. ** P < 0.01. Abbreviations: Ad, adenovirus; DAPI, 4′,6‐diamidino‐2‐phenylindole; IB, immunoblot; N.S., not significant; RC, relaxed circular DNA; rRNA, ribosomal RNA; SS, single‐stranded DNA; WCL, whole‐cell lysate.
PRMT5 INTERFERES WITH pgRNA ENCAPSIDATION BY COUNTERACTING THE INTERACTION BETWEEN pgRNA AND HBV POL THROUGH BINDING TO THE REVERSE TRANSCRIPTASE–RIBONUCLEASE H REGION OF POL
Because pgRNA encapsidation is an essential step before core particle DNA synthesis, we further evaluated whether PRMT5 prevents pgRNA encapsidation to block viral DNA synthesis. To address this, we transfected Huh7 cells with the pCMV‐HBV YMHD vector, which lacks the Pol catalytic active site and thus leads to the accumulation of RNA‐containing capsids in the cells. Northern blot results showed that while the levels of HBV total RNA and capsids were not affected by PRMT5Δ, it dramatically reduced the level of encapsidated RNA in a dose‐dependent manner (Fig. 8 A), indicating that PRMT5Δ inhibits pgRNA encapsidation rather than directly targeting the cytoplasmic viral pgRNA for degradation.
Figure 8: PRMT5 interferes with pgRNA encapsidation by counteracting the interaction between pgRNA and HBV Pol through binding to the RT‐RH region of Pol. (A) PRMT5Δ was cotransfected with pCMV‐HBV YMHD into Huh7 cells. Cells were harvested at 72 hours posttransfection. Core particle RNAs were analyzed by northern blot. Numbers below the lanes indicate the intensity of the core particle RNA bands relative to the vector group. Total HBV RNA was analyzed by northern blot. An aliquot of cell lysates was loaded onto an agarose gel to detect the cytoplasmic core capsids. Overexpression of PRMT5Δ was detected by western blot. (B) Huh7 cells were cotransfected as indicated, and pCMV‐HBV‐ΔPol was transfected in all groups to transcribe pgRNA. At 36 hours posttransfection, cells were treated with 10 μM of MG132 for 9 hours and harvested for RIP assay. (C) PRMT5 was cotransfected with hemagglutinin Pol into Huh7 cells. After 48 hours, cells were harvested for an immunofluorescence assay. Anti‐PRMT5 and anti‐HA antibodies were used. (D) Huh7 cells were cotransfected as indicated. At 36 hours posttransfection, cells were treated with 10 μM of MG132 for 9 hours and then harvested for CoIP assay with anti‐Flag antibody. (E) Schematic diagram of the full‐length and truncated constructs of HBV Pol (upper panel). Huh7 cells were cotransfected with Flag‐PRMT5 and the indicated Myc‐tagged HBV Pol constructs or the negative control, Myc‐Rluc. At 36 hours posttransfection, cells were treated with 10 μM of MG132 for 9 hours and then harvested for CoIP assay with anti‐Flag antibody (lower panel). * P < 0.05, ** P < 0.01. Abbreviations: aa, amino acid; DAPI, 4′,6‐diamidino‐2‐phenylindole; HA, hemagglutinin; IB, immunoblot; IP, immunoprecipitation; si, small interfering; SP, spacer; TP, terminal protein; WCL, whole‐cell lysate.
Because HBc, Pol and pgRNA are the three key factors involved in the process of pgRNA encapsidation and we found that PRMT5 interacts with the HBc, we speculated whether PRMT5 affects the associations of HBc with Pol and with pgRNA. The CoIP assay suggested that the interaction between HBc and Pol was not affected by PRMT5Δ (Supporting Fig. S10B Supporting Fig. S10C 27 8 B). Thus, we further investigated how PRMT5 counteracted the Pol–pgRNA interaction. While there was no direct interaction between PRMT5 and pgRNA (Supporting Fig. S10D 8 C) and a physical interaction between HBV Pol and endogenous or ectopic PRMT5 or PRMT5Δ (Fig. 8 D) were observed. Further studies indicated that PRMT5 interacted with the reverse transcriptase–ribonuclease H (RT‐RH) region of Pol (Fig. 8 E), which has been recognized as the domain that is responsible for the Pol–pgRNA interaction.28
Discussion
Here, we identified PRMT5 as a potent host restrictor of HBV replication. We demonstrate that PRMT5, in a methyltransferase‐dependent manner, epigenetically silences cccDNA transcription by triggering H4R3me2s on the cccDNA minichromosome, which involved the regulation of the binding of the Brg1‐based human SWI/SNF chromatin remodeler and RNA Pol II to cccDNA. Importantly, the negative correlation between the cccDNA‐bound H4R3me2s and cccDNA transcription was validated in vitro and in vivo . In addition, PRMT5, in a methyltransferase‐independent manner, disrupted pgRNA encapsidation by competitively binding to the RT‐RH region of HBV Pol, which further inhibited HBV DNA production and may affect cccDNA replenishment (Supporting Fig. S11
In recent years, a number of epigenetic markers on the HBV cccDNA minichromosome associated with viral transcription have been identified. While high levels of posttranscriptional modifications of the cccDNA‐bound histones associated with active transcription are observed across the HBV genome and the role of acetylation of cccDNA‐bound histones in the regulation of HBV transcription has been recognized,7 26 in vitro and in vivo , cccDNA‐bound H4R3me2s could be detected, was significantly associated with repressed cccDNA transcription, and could be manipulated by the alteration of PRMT5 expression. Because the previous study with a ChIP‐sequencing approach did not include H4R3me2s, it will be interesting to map its distribution on cccDNA in the future. In addition to the present study, the role of methyltransferases including PRMT1 and SETDB1 in regulating HBV transcription has been reported.29 19 Supporting Fig. S3C 30 – ),26 Supporting Fig. S3C
Previous studies have suggested that PRMT5 methylates histones H4R3 and H3R8 as a part of the Brg1‐based or hBrm‐based SWI/SNF chromatin remodeling complex23 31 20
Although both the host chromosomes and the HBV cccDNA minichromosome are organized with the host histone proteins, we showed that the status of H4R3me2s varied more on cccDNA than on the host GAPDH gene during HBV infection and that the alteration of the expression of PRMT5 significantly changed the level of cccDNA‐bound, but not host gene–bound, H4R3me2s and PRMT5, implying that PRMT5 can, to some extent, specifically bind to cccDNA and catalyze cccDNA‐bound H4R3me2s to repress cccDNA transcription. Based on the evidence that PRMT5 interacted and colocalized with HBc in the nucleus and knockdown of HBc decreased the binding of PRMT5 to cccDNA, we proposed that HBc plays a role in targeting PRMT5 to cccDNA. The factors that have been reported to mediate the binding of PRMT5 to host chromosomes, such as the histone‐binding protein COPR5, may also be involved in the binding of PRMT5 to cccDNA, which remains to be further studied. The preference of PRMT5 could also be attributed to the differences in chromatin protein composition and the chromosome structure between cccDNA and host genes,3
Until now, a number of host restrictors that target different steps of HBV replication have been identified, including the APOBEC cytidine deaminases,33 14 35 16 28
Besides histones, PRMT5 can target a series of other host proteins and has been implicated in various cellular processes, including pluripotency, tumorigenesis,37 38 Supporting Fig. S7 39 Supporting Figs. S5B and S6 Supporting Fig. S5C,D in vitro and in vivo .
In conclusion, the present study revealed a two‐part mechanism of the regulation of HBV gene transcription and viral replication by PRMT5, which promotes the understanding of the epigenetic modulation of cccDNA transcription and the control of HBV replication and may provide new insight into noncytolytic mechanisms of cccDNA elimination.
Author names in bold designate shared co‐first authorship.
Acknowledgment
We thank Prof. Ningshao Xia (Xiamen University) for providing mouse antibodies against HBc; Prof. Jiming Zhang (Huashan Hospital affiliated to Fudan University), Dr. Zhanqing Zhang (Shanghai Public Health Clinical Center affiliated to Fudan University), and Prof. Jian Zhou (Zhongshan Hospital affiliated to Fudan University) for patient recruitment and providing clinical samples; Dr. Yinghui Liu for preparing several of the truncations used; Jin Li, Cong Wang, and Ke Qiao for technical assistance; and Dr. Yuchen Xia for critical reading of the manuscript.
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