Small Extracellular Vesicles Promote HBV Replication via METTL3–IGF2BP2-Mediated m6A Modification

Abstract

Background:

The roles of small extracellular vesicles (sEVs) and mRNA modifications in regulating hepatitis B virus (HBV) transmission, replication, and related disease progression have received considerable attention. However, the mechanisms through which methyltransferase-like 3 (METTL3) and insulin-like growth factor 2 (IGF2BP2), key genes that mediate m6A modifications, regulate HBV replication in sEVs remain poorly understood. Therefore, this study investigated the molecular mechanisms through which the key molecules (METTL3 and IGF2BP2) in sEVs mediate m6A epigenetic modification to regulate HBV replication.

Methods:

Small extracellular vesicles were extracted from the supernatants of HepG2.2.15 and HepG2 cells via ultracentrifugation, followed by purification with hepatitis B virus surface antigen (HepBsAg) immunomagnetic beads. The sEVs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), and Western blotting (WB). Methylation enrichment in the two types of sEVs was analyzed by dot blotting and quantitative reverse transcription-PCR (RT-qPCR). The cells were treated with HepG2.2.15–sEVs transfected with either the METTL3 plasmid, METTL3 siRNA, the IGF2BP2 plasmid, or the IGF2BP2 siRNA. After 48 h, the expression of METTL3, IGF2BP2, and HBV DNA expressions were assessed via dot blotting, quantitative-PCR (qPCR), RT-qPCR, and WB. Co-immunoprecipitation (co-IP) was performed to investigate the interactions between METTL3 and IGF2BP2.

Results:

By conducting TEM, DLS, and WB analyses, we confirmed that the isolated sEVs exhibited typical characteristics. HepG2.2.15-derived sEVs presented elevated levels of m6A modifications, with increased METTL3 and IGF2BP2 mRNA and protein expression levels, respectively (p < 0.05). In the overexpression (OE)-METTL3 group, the expression levels of HBV pregenomic RNA (HBV pgRNA), HBV DNA, HBV relaxed circular DNA (HBV rcDNA), HBV covalently closed circular DNA (HBV cccDNA), HBsAg, hepatitis B virus core antigen (HBcAg), and hepatitis B virus e antigen (HBeAg) were significantly elevated compared to those in the control group (p < 0.01). In contrast, results for the small interfering (SI)-METTL3 group were the opposite. Similarly, in the OE-IGF2BP2 group, HBV pgRNA, HBV DNA, HBV rcDNA, HBV cccDNA, HBsAg, HBcAg, and HBeAg expression were greater than in the control group (p < 0.05), whereas the opposite results were recorded in the SI-IGF2BP2 group. Co-immunoprecipitation confirmed that METTL3 and IGF2BP2 interact synergistically.

Conclusion:

Small extracellular vesicles increase METTL3 and IGF2BP2 expression, synergistically promoting HBV replication by regulating m6A modification levels.

Graphical abstract

Keywords

HBV / small extracellular vesicles / N6-methyladenosine modification / METTL3 / IGF2BP2

1. Introduction

Hepatitis B virus (HBV) possesses a 3.2 kb partial double-stranded relaxed circular DNA (rcDNA) genome, which can be repaired in the nucleus to covalently closed circular DNA (cccDNA) [1, 2, 3]. The cccDNA can be transcribed to generate new pregenomic RNA (pgRNA), which can produce new rcDNA via reverse transcription. This rcDNA can be secreted into the extracellular environment via small extracellular vesicles (sEVs), thus facilitating the infection of additional hepatocytes [4, 5, 6]. The primary factors contributing to persistent HBV infection include the chromosomal integration of cccDNA into host cells and infection mediated by sEVs [7]. However, the precise regulatory mechanisms underlying these processes remain poorly understood.

Small extracellular vesicles (sEVs) are bilayer vesicle structures characterized by a cup-shaped morphology, with particle sizes between 30 and 200 nm [8, 9]. They are crucial in facilitating the transmission of HBV, disease progression, and the development of targeted therapy [10, 11, 12]. The study has indicated that interfering with extracellular vesicles significantly reduces HBV replication [13]. Moreover, engineered sEVs containing clustered regularly interspaced short palindromic repeats (Cas9/gR) and vesicular stomatitis virus-glycoprotein (VSV-G) can decrease HBV replication and cccDNA levels [14]. These findings highlight the close association between sEVs and the loading and dissemination of HBV viral particles, as sEVs contain various components, including HBV DNA, HBV RNA, and proteins [15, 16].

Studies have shown that the m6A sites in the hepatitis C virus (HCV) can regulate the maturation of the virus in complex and undetermined ways [17, 18]. Some researchers found that N6-methyladenosine binding protein (YTHDC2) mediates m6A modifications to regulate HCV internal ribosome entry site (IRES)-dependent translation [19]. The conserved m6A motifs located in the ϵ stem-loop structures at the 3^′ end of the HBV mRNA and the 5^′ and 3^′ ends of the pgRNA represent critical sites for m6A modification [20]. Additionally, m6A modification increases pgRNA reverse transcription and increases HBV viral DNA levels, suggesting a significant role for m6A in the epigenetic regulation of HBV replication [1]. Methylation of the N6 position of adenine (m6A) is a prevalent post-transcriptional modification that influences biological processes by regulating the rates of splicing, translation, and decay of mRNAs [21, 22, 23, 24]. M6A methylation is a dynamic and reversible epigenetic modification that is orchestrated by “readers”, “writers”, and “erasers” [25, 26], and affects viral gene expression by recruiting various m6A-binding proteins throughout the life cycle of HBV [25]. These proteins play crucial roles in viral replication, the immune response, and oncogenesis [27].

Study has found that the proteins methyltransferase-like 3 (METTL3) and insulin-like growth factor 2 (IGF2BP2) that act as a writer and a reader, respectively, function as coregulators of m6A modification during HBV infection, with METTL3 facilitating m6A enrichment in an IGF2BP2-dependent manner [28]. However, the precise mechanism by which sEVs mediate m6A epigenetic modifications to regulate HBV replication during viral packaging into sEVs for transmission to uninfected cells remains to be elucidated.

In our previous study, we found that METTL3 synergizes with IGF2BP2 to regulate HBV replication at the cellular level [29]. To further elucidate the specific molecular mechanisms through which sEVs mediate m6A modification and promote HBV replication via METTL3-IGF2BP2 synergy, we conducted this study. Unlike previous studies that focused on the cellular level, we regulated m6A modifications at the microscopic level in sEVs. We hypothesized that sEVs enhance HBV replication by modulating m6A levels through interactions between METTL3 and IGF2BP2. We examined changes in HBV replication markers by overexpressing or knocking down key m6A regulatory proteins, specifically the “writer” methyltransferase METTL3 and the “reader” protein IGF2BP2, in sEVs. We also characterized the mode of action of these proteins via co-immunoprecipitation (co-IP) to elucidate the mechanism by which sEVs mediate m6A modification to regulate HBV replication. We aimed to provide a framework to comprehensively elucidate the mechanisms underlying HBV replication and contribute to the discovery of novel molecular targets for treating HBV infection.

2. Materials and Methods

2.1 Cell Culture

HepG2.2.15 cells (iCell-h093, Shanghai ICell Bioscience Inc., Ltd., Shanghai, China) stably replicating HBV and their parental HepG2 cells (iCell-h092, Shanghai ICell Bioscience Inc., Ltd., Shanghai, China) were cultured in a medium consisting of 8% exosome-free fetal bovine serum (FBS) (Cat #EXO-FBS-50A-1, SBI, Palo Alto, CA, USA) supplemented with 1% penicillin streptomycin (Cat. NO. 15140122, Lot. 238344, Grand Island, NY, USA). To ensure stable HBV replication, HepG2.2.15 medium was supplemented with G418 solution at a concentration of 400 µg/mL (R20001, Lot.NO71R19494A, Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China). All cell lines were validated via short tandem repeat (STR) profiling, were tested, and were found to be negative for mycoplasma (Supplementary materials).

2.2 Extraction of sEVs

After culturing HepG2 and HepG2.2.15 cells for 48–72 h, to remove impurities such as cells, the cell supernatants were collected and centrifuged at 3000 \timesg for 30 min. Next, the small molecules were separated by centrifugation at 12,000 \timesg for 45 min; subsequently, the supernatant was filtered through a 0.22 µm filter (REF.SLGPR33RE, Lot.R1DB07580, Merck Millipore, Billerica, MA, USA). Following this, sEVs were isolated via ultracentrifugation (MFG.NO. O11284, Hitachi himac-CP80NX, Tokyo, Japan) at 100,000 \timesg for 120 min. The precipitated sEVs were resuspended in 1 mL of precooled 1\timesPBS (Cat. NO. P1022, Solarbio Science & Technology Co., Ltd., Beijing, China).

2.3 Purification of sEVs

Initially, 50 µL of HepBsAg (S38: sc-52412, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to the sEVs following ultracentrifugation. Then, they were transferred to a shaker and placed at 4 °C for 12 h to facilitate thorough rotation and mixing. Next, 20 µL of Protein A/G PLUS-Agarose (sc-22003, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added, and the sample was incubated for 8 h at 4 °C with continuous mixing. After incubation, the mixture was centrifuged at 3000 \timesg for 5 min to collect the supernatant, followed by ultracentrifugation at 100,000 \timesg for 120 min. Finally, 100 µL of precooled 1\times PBS was added to dissolve the pellet, resulting in the purification of sEVs with the removal of HBV virus particles.

2.4 Transmission Electron Microscope (TEM)

Purified sEVs from HepG2.2.15 cell culture supernatant (HepG2.2.15-sEVs) and HepG2 cell culture supernatant (HepG2-sEVs) were deposited onto a carbon-coated copper mesh, with 10 µL of each sample added to the grid. After the excess liquid was aspirated from the edges, the samples were dried for 5 min. Next, 10 µL of 2% phosphotungstic acid (Cat# G1870, Lot No. 2400001, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was added, and the mixture was stained for 1 min. Excess staining solution was removed, and the samples were dried overnight at room temperature. The morphology of the sEVs was observed and photographed using a transmission electron microscope (Hitachi HT7700, Tokyo, Japan).

2.5 Dynamic Light Scattering (DLS)

A 10 µL sample of ultraisolated purified sEVs was added to 1 mL of 1\times PBS, which was filtered through a 0.22 µm filter, and the contents were thoroughly mixed. A zeta particle size analyzer (Malvern Zetasizer ZS90, Malvern, Worcestershire, UK) was used to measure the concentration and size distribution of the sEVs. Next, the number of particles and their diameters were plotted using Origin (Version Pro2021, 64-bit; Origin Lab Inc., Northampton, MA, USA).

2.6 Small Extracellular Vesicle Transfection

A 150 µL transfection system for sEVs was prepared using the Exo-Fect™ Exosome Transfection Reagent Kit (Cat#EXFT20A-1, Lot.230616-001, SBI, Palo Alto, CA, USA) as follows: 10 µL of Exo-Fect, 20 µL of nucleic acids (20 pmol siRNA or 5 µg plasmid DNA), 50 µL of purified HepG2.2.15-sEVs, and 70 µL of sterile 1\times PBS. The components were mixed by gently inverting the EP tubes six times without vortexing. The prepared mixture was placed in a shaker at 37 °C for 10 min and then transferred to ice. Next, 30 µL of Exo Quick-TC was added to the mixture, and the contents were mixed by inverting the EP tube six times without vortexing to halt the transfection reaction. The transfected sEVs were maintained on ice (or 4 °C) for 30–40 min.

The sample suspension was centrifuged at 14,000 rpm for 3 min, and the precipitates were retained. At least 300 µL of sterile 1\times PBS was added to the precipitate, which was subsequently resuspended. The sequence of small interfering METTL3 (si-METTL3) was 5′-CCUGCAAGUAUGUUCACUATT-3′, and that of small interfering IGF2BP2 (si-IGF2BP2) was 5′-GCATATACAACCCGGAAAGAA-3′ (Cat.NO. M01, Guangzhou RiboBio Co., Ltd., Guangzhou, China).

Stably grown HepG2 cells were selected and seeded in six-well plates at a density of 2 \times 10⁵ cells/mL. Transfection was performed when the cells reached 60–80% confluence, about 12 h after seeding. Before transfection, the serum-free medium was removed. The mixture was subsequently incubated for 1 h with serum-free DMEM. Next, at least 150 µL of transfected HepG2.2.15-sEVs (containing siRNA or plasmid as described above) was added to the cells. After 4–6 h, the mixed medium was changed to nonresistant DMEM containing 8% exosome-free FBS. The cells were allowed to grow for 48 h before being collected for subsequent experiments.

2.7 Quantitative Reverse Transcription-PCR (RT-qPCR)

Initially, RNA was extracted using the TRIzol (REF.15596026, Lot.257408, Thermo Fisher Scientific, Waltham, MA, USA) method. After determining its concentration, the RNA was reverse-transcribed to template cDNA in two steps using an RNA reverse transcription kit (REF.1622, Lot.3059833, Thermo Fisher Scientific, Waltham, MA, USA). In step 1, 1 µg of RNA and 1 µL of random primer were added, followed by the addition of ddH₂O to obtain a mixture of 12 µL. The prepared system was reacted at 65 °C for 5 min. In step 2, 4 µL of 5\times buffer, 2 µL of 10 mM dNTPs, 1 µL of RNase inhibitor, and 1 µL of reverse transcriptase were added sequentially. The reaction proceeded at 25 °C for 5 min, 42 °C for 60 min, and 70 °C for 5 min. The resulting cDNA served as a template for quantitative-PCR (qPCR) amplification using a fluorescent quantitative PCR detection kit (Cat.NO. CW0957, Beijing ComWin Biotech Co., Ltd., Beijing, China). Target gene expression was calculated using the 2^{-Δ⁢Δ⁢CT} method. All mRNA sequences of primers used were as follows [30, 31, 32] (Sangon Biotech Shanghai Co., Ltd., Shanghai, China): \beta-ACTIN F: 5^′-CTCCATCCTGGCCTCGCTGT-3^′, R: 5^′-GCTGTCACCTTCACCGTTCC-3^′; METTL3 F: 5^′-TTGTCTCCAACCTTCCGTAGT-3^′, R: 5^′-CCAGATCAGAGAGGTGGTGTAG-3^′; IGF2BP2 F: 5^′-AGCTAAGCGGGCATCAGTTTG-3^′, R: 5^′-CCGCAGCGGGAAATCAATCT-3^′; pgRNA F: 5^′-CTCAATCTCGGGAATCTCAATGT-3^′, R: 5^′-TGGATAAAACCTAGGAGGCATAAT-3^′; hepatitis B virus surface antigen (HBsAg) F: 5^′-TCACAATACCGCAGAGTC-3^′, R: 5^′-ACATCCAGCGATAACCAG-3^′; hepatitis B virus core antigen (HBcAg) F: 5^′-CTGGGTGGGTGTTAATTTGG-3^′, R: 5^′-TAAGCTGGAGGAGTGCGAAT-3^′; hepatitis B virus e antigen (HBeAg) F: 5^′-GATTCGCACTCCTCCAGTCT-3^′, R: 5^′-AGTTCTTCTTCTAGGGGACCTG-3^′.

2.8 m6A Dot Blotting

After extracting RNA using the TRIzol method and determining its concentration, the RNA samples were serially diluted to 200 ng/µL, 100 ng/µL, and 50 ng/µL. The samples were subsequently denatured in a metal bath at 95 °C for 5 min and then cooled immediately on ice. Next, 2 µL of each RNA sample was added dropwise onto a nylon (NC) membrane, which had been cut to the appropriate size. After the RNA was allowed to diffuse naturally and dry, the membrane was cross-linked under UV light for 2 h. Following the cross-linking step, the sealing solution was closed for 1 h.

The NC membrane was incubated at 4 °C for 12–16 h with the primary m6A antibody (dilution 1:1000, Cat. NO. 517924, Lot. NO. KK0512, Chengdu ZEN-BIOSCIENCE Co., Ltd., Chengdu, China). The HRP-labeled goat anti-rabbit IgG secondary antibody (1:5000, Cat.NO. FNSA-0004, Chengdu ZEN-BIOSCIENCE Co., Ltd., Chengdu, China) was added, and the mixture was shaken gently at room temperature for 1 h. The signal was developed using an ECL detection system after washing the film.

2.9 HBV DNA Extraction and Characterization

An animal tissue/cellular genomic DNA extraction kit was used to extract cellular HBV DNA (Cat# D1700, Lot No. 2311002, Solarbio Science & Technology Co., Ltd., Beijing China), and its concentration was subsequently determined. To digest the HBV DNA, the sample was incubated with T5 nucleic acid exonuclease (D7082S-1, Shanghai Biyuntian Biotechnology Co., Ltd., Shanghai, China) for 30 min at 37 °C. The samples were terminated by adding EDTA and then heat-inactivated at 95 °C for 5 min.

PCR amplification was first performed using primers for HBV DNA, HBV rcDNA, and HBV cccDNA, and then the copy number of the DNA was calculated based on a standard curve. The sequences of primers used were as follows (Sangon Biotech Shanghai Co., Ltd., Shanghai, China): HBV DNA F: 5^′-ACCGACCTTGAGGCATACTT-3^′, R: 5^′-GCCTACAGCCTCCTAGTACA-3^′; HBV rcDNA F: 5^′-TTTCACCTCTGCCTAATCATCTCT-3^′, R: 5^′-CTTTATAAGGGTCGATGCCATGC-3^′; and HBV cccDNA F: 5^′-GCCTATTGATTGATTGGAAAGTATGT-3^′, R: 5^′-AGCTGAGGCGGTATCTA-3^′.

2.10 Western Blotting

The cells and sEVs were lysed in RIPA buffer containing 1% PMSF (ST507, Shanghai Biyuntian Biotechnology Co., Ltd., Shanghai, China) to extract total protein. The samples were lysed on ice for 30 min and centrifuged at 12,000 \timesg for 30 min at 4 °C to collect the supernatant. The protein concentration was estimated by the BCA method. (Cat# PC0021, Solarbio Science & Technology Co., Ltd., Beijing, China).

After normalizing the protein concentrations, equal amounts of protein were separated by SDS-PAGE and transferred to 0.45 µm PVDF membranes. Next, 5% skim milk powder was added for 1 h, after which the appropriate primary antibody (METTL3, IGF2BP2 primary antibody dilution of 1:1000; Chengdu ZEN-BIOSCIENCE Co., Ltd., Chengdu, China) was added, and the membrane was incubated at 4 °C for 12–16 h. After washing three times with TBST, the membrane was incubated with an HRP-conjugated secondary antibody (1:5000, Cat.NO. FNSA-0004, Chengdu ZEN-BIOSCIENCE Co. Ltd., Chengdu, China) at room temperature for 1 h. After the film was washed, the protein bands were detected using an ECL detection system. The ImageJ software (Version 1.54v, Java1.8.0, 64-bit; Media Cybernetics, Silver Springs, MD, USA) was used to analyze the Western blotting bands in grayscale, and the results were normalized by dividing the target protein grayscale value by an internal reference grayscale value. The primary antibodies used were as follows: \beta-ACTIN (1:10,000, AC004, ABclonal Technology, Wuhan, China), Alix (1:1000, NO.2215, ABclonal Technology, Wuhan, China), CD63 (1:1000, A19023, ABclonal Technology, Wuhan, China), CD9 (1:1000, A19027, ABclonal Technology, Wuhan, China), GAPDH (R380646, 1:5000, Chengdu ZEN-BIOSCIENCE Co., Ltd., Chengdu, China), METTL3 (R508370, 1:1000, Chengdu ZEN-BIOSCIENCE Co., Wuhan, China), IGF2BP2 (R389232, 1:1000, Chengdu ZEN-BIOSCIENCE Co., Ltd., Chengdu, China).

2.11 Co-Immunoprecipitation

After the cells were transfected with sEVs containing the METTL3 plasmid, they were harvested 48 h post-transfection to prepare protein samples. Following the protocol, 20 µL of protein A+G magnetic beads (Cat. NO. P2179S, Lot NO.091322230131, Shanghai Biyuntian Biotechnology Co., Ltd., Shanghai, China) were mixed with 2 µg of either METTL3 primary antibody or normal rabbit IgG and incubated at room temperature for 4 h. Next, protein samples of equal mass were added, and the mixture was rotated overnight at 4 °C.

After incubation, the beads and supernatant were separated using a magnetic rack for 1 min. Next, 500 µL of a lysis buffer supplemented with protease inhibitors was used to wash the beads three times. After washing, 50 µL of 1\times SDS-PAGE protein loading buffer was added to the beads, and the mixture was heated at 95 °C for 10 min to elute the proteins. The supernatant was then collected after the magnetic beads were separated using a magnetic rack. Finally, the samples were analyzed by SDS-PAGE.

2.12 Statistical Analysis

All data were presented as the mean \pm standard deviation (SD) derived from three independent experiments. Data were analyzed and graphs were made using GraphPad Prism (version 10.1.2, GraphPad Software, Inc., CA, USA). An independent samples t-test was conducted for pairwise comparisons, and a one-way analysis of variance (ANOVA) followed by Šídák’s multiple comparison test was conducted for multiple group comparisons. All differences among and between groups were considered to be statistically significant at p 0.05.

3. Results

3.1 Identification of sEVs

Small extracellular vesicles derived from HepG2 cells (HepG2-sEVs) and HepG2.2.15 cells (HepG2.2.15-sEVs) were characterized following purification by ultracentrifugation. DLS analysis revealed that 82.4% of the HepG2-sEV particles were 30–200 nm in size, and the peak particle size and concentration were 122.4 nm and 2.30 \times 10⁶ particles/mL, respectively. In contrast, 98.7% of the HepG2.2.15-sEV particles were also 30–200 nm in size and exhibited peak sizes and concentrations of 78.82 nm and 7.22 \times 10⁶ particles/mL, respectively (Fig. 1A). TEM demonstrated that both types of sEVs had a double-layered membranous structure with an average diameter of about 100 nm (Fig. 1B). Additionally, the WB analysis confirmed that the characteristic sEV proteins, including Alix, CD63, and CD9, were expressed (Fig. 1C).

3.2 Enrichment of m6A Methylation in sEVs Derived From the HBV Stably Replicating the HepG2.2.15 Cell Line

Compared to HepG2-sEVs, HepG2.2.15-sEVs presented significantly greater levels of m6A methylation (Fig. 2A). The expression levels of METTL3 and IGF2BP2 mRNAs in HepG2.2.15-sEVs were significantly elevated, as determined by RT-qPCR analysis (Fig. 2B; p 0.05). The results of the Western blotting analysis revealed a corresponding increase in protein levels (Fig. 2C,D; p 0.05). These findings suggested that an increase in m6A methylation levels in HepG2.2.15-sEVs is associated with the replication of HBV.

3.3 HepG2.2.15-sEVs Promote HBV Replication Through Upregulation of the Methyltransferase METTL3

METTL3 was overexpressed or knocked down in HepG2.2.15-sEVs, resulting in corresponding alterations in METTL3. In the overexpressing METTL3 (OE-METTL3) group, the METTL3 mRNA and protein levels increased. Conversely, in the SI-METTL3 group, METTL3 mRNA and protein levels were lower than those in the control group (Fig. 3A–C; p 0.01), confirming that METTL3 expression was modulated. Moreover, the levels of m6A modification exhibited a corresponding increase or decrease (Fig. 3D).

Compared to that in parental HepG2 control cells treated with untransfected HepG2.2.15-sEVs, the mRNA level of HBV pgRNA, a marker of HBV replication, was significantly greater in the OE-METTL3 group (Fig. 3E; p 0.05). The HBV DNA, HBV rcDNA, and HBV cccDNA copy numbers also increased significantly (Fig. 3F; p 0.05), whereas the HBV rcDNA conversion rate increased from 3.51% to 9.99% (Fig. 3H; p 0.01). Moreover, the HBsAg, HBcAg, and HBeAg mRNA expression levels increased (Fig. 3J; p 0.001).

In contrast, the SI-METTL3 group presented a decrease in viral replication indicators: HBV pgRNA expression decreased (Fig. 3E; p 0.01), and the copy numbers of HBV DNA, HBV rcDNA, and HBV cccDNA decreased (Fig. 3G; p 0.01). The HBV rcDNA conversion rate decreased from 1.45% to 0.38% (Fig. 3I; p 0.001), and the mRNA levels of HBsAg, HBcAg, and HBeAg decreased (Fig. 3J; p 0.01).

These results suggested that HepG2.2.15-sEVs facilitated an increase in m6A methylation through the upregulation of METTL3, which promoted the conversion of rcDNA to cccDNA, thereby regulating the replication of HBV. Inhibition of METTL3 expression decreased HBV DNA levels in the treatment of HBV-associated diseases, and the detection of METTL3 in sEVs may be used to predict HBV-associated diseases and monitor the prognosis of the disease caused by HBV.

3.4 HepG2.2.15-sEVs Promote HBV Replication Through the Reader Protein IGF2BP2

The IGF2BP2 mRNA expression levels increased or decreased (Fig. 4A; p 0.05) following the overexpression or knockdown of IGF2BP2 in HepG2.2.15-sEVs, as determined by RT-qPCR analysis. The results of the Western blotting assays demonstrated an increase or decrease in the IGF2BP2 protein expression level (Fig. 4B,C; p 0.05), confirming that IGF2BP2 was successfully manipulated in sEVs. Concurrently, the level of m6A modification was also altered (Fig. 4D).

Compared to the parental HepG2 control cells treated with untransfected HepG2.2.15-sEVs, the expression level of HBV pgRNA, a marker for detecting HBV replication, increased or decreased (Fig. 4E; p 0.01). The HBsAg, HBcAg, and HBeAg mRNA levels increased or decreased, respectively (Fig. 4J; p 0.01).

The copy numbers of HBV DNA, HBV rcDNA, and HBV cccDNA increased (Fig. 4F; p 0.05), with the HBV rcDNA conversion rate increasing from 6.14% to 31.34% (Fig. 4H; p 0.01). In contrast, these values decreased (Fig. 4G; p 0.01), with the HBV rcDNA conversion rate decreasing from 31.25% to 2.23% (Fig. 4I; p 0.05).

These results suggested that HepG2.2.15-sEVs, via the reader protein IGF2BP2, can increase m6A levels, promote the conversion of rcDNA to cccDNA, and consequently regulate the replication of HBV. A significant reason for the prolonged treatment of HBV disease is the difficulty in eliminating cccDNA. These experimental results suggested that the rcDNA conversion rate may be reduced by inhibiting the expression of IGF2BP2, thus decreasing the generation of cccDNA and halting the progression of the disease.

3.5 Methyltransferase METTL3 Synergizes With the Reader Protein IGF2BP2 in HepG2.2.15-sEVs

After the cells were transfected with HepG2.2.15-sEVs containing the METTL3 plasmid, IGF2BP2 was detected via immunoprecipitation (IP) using an anti-METTL3 antibody. The immunoprecipitation results indicated a synergistic interaction between METTL3 and IGF2BP2 (Fig. 5A).

After the cells were transfected with HepG2.2.15-sEVs with either overexpression or knockdown of METTL3, the IGF2BP2 mRNA expression level increased, as determined by RT-qPCR (Fig. 5B; p 0.01). The results of the Western blotting analysis revealed that the METTL3 and IGF2BP2 protein levels increased and decreased, respectively (Fig. 5C,D; p 0.01).

After the cells were transfected with HepG2.2.15-sEVs with either overexpression or knockdown of IGF2BP2, the METTL3 mRNA expression levels increased or decreased, respectively (Fig. 5E; p 0.05). Similarly, the METTL3 and IGF2BP2 protein levels increased or decreased, respectively (Fig. 5F,G; p 0.001). These results confirmed the synergistic relationship between METTL3 and IGF2BP2.

4. Discussion

We previously reported that sEVs carry HBV components that evade immune surveillance, allowing the virus to spread to uninfected cells and promote the replication of HBV [33, 34]. These sEVs possess a stable phospholipid bilayer structure that effectively prevents the degradation of HBV components, and their membrane composition closely resembles that of cellular membranes, facilitating efficient cellular uptake. Among the membrane markers of sEVs, CD63 plays a key role in the packaging, transport, and release of nascent HBV [35]. These findings indicate the importance of sEVs as significant participants and signaling mediators in HBV infection [36, 37].

METTL3-mediated and IGF2BP2-mediated m6A modifications have attracted considerable attention. One study showed that the METTL3-IGF2BP2 complex can promote inflammation through m6A modification, providing important information for the treatment of sepsis [38]. Another study revealed that METTL3 and IGF2BP2 influence the progression of colorectal cancer in a m6A modification-dependent manner [39]. Several studies support the notion that METTL3 and IGF2BP2-mediated m6A modifications play broad roles in various pathogenic mechanisms. Studies have also shown that m6A modifications in HBV RNA may affect its stability and translation, subsequently affecting viral replication [20, 33, 40]. However, the mechanisms by which sEVs mediate epigenetic modifications remain poorly understood. One study indicated that the expression of the methyltransferase METTL3 was significantly higher in the liver tissues of patients with HBV-associated hepatocellular carcinoma compared to normal tissues [41]. This study also suggested that METTL3-mediated m6A modification is crucial for maintaining mRNA stability in an IGF2BP2-dependent manner [42].

Based on these findings, we hypothesized that sEVs mediate m6A epigenetic modifications to promote HBV replication through the METTL3-IGF2BP2 pathway. Our results indicated that METTL3 and IGF2BP2 expression levels were higher in the HBV-stably replicating HepG2.2.15 cell line compared to their expression levels in HepG2 cells [29]. Additionally, m6A modification was significantly enriched in HepG2.2.15-sEVs, suggesting a close association between m6A modification in sEVs and HBV replication.

To elucidate the specific mechanism by which METTL3 and IGF2BP2 co-mediate m6A modification in sEVs to regulate HBV replication, the METTL3 gene, a key enzyme, was either overexpressed or knocked down in HepG2.2.15-sEVs, which were subsequently used to transfect HepG2 cells. The results indicated a corresponding increase or decrease in m6A methylation, along with alterations in HBV DNA copy number and HBV pgRNA expression. These findings suggested that METTL3 facilitates HBV DNA replication by increasing the m6A modification of HBV mRNAs in sEVs and increasing pgRNA expression levels. A probable mechanism may be as follows: METTL3 regulates HBV replication by targeting m6A modification sites located in the \epsilon-stem-loop structure at the 3^′ end of the HBV mRNA and the 5^′ and 3^′ ends of pgRNA [20].

Transfection of HepG2 cells with HepG2.2.15-sEVs that either overexpress or knock down METTL3 resulted in a considerable increase or decrease in the mRNA levels of HBsAg, HBcAg, and HBeAg. These findings suggested that METTL3 within sEVs influences the transcription of HBV core proteins and cytosolic proteins, thus leading to an increase in mRNA expression levels. This regulation affects HBV viral packaging and promotes viral replication. A similar observation was reported in a related study by Cheng et al. [43]. Furthermore, an examination of the HBV viral genomic rcDNA and cccDNA revealed that the copy numbers of rcDNA and cccDNA changed simultaneously, accompanied by a significant alteration in the rcDNA to cccDNA conversion rate. These findings indicated that METTL3 in sEVs enhances HBV replication by increasing the expression of HBV rcDNA and cccDNA, as well as by facilitating the conversion of rcDNA to cccDNA, which in turn supports the replication of HBV [1, 2, 44].

We found that sEVs-mediated METTL3 enhances m6A methylation of HBV mRNA and pgRNA, resulting in an increase in the levels of HBV DNA and promoting the conversion of rcDNA to cccDNA. This process serves as a template that enhances the transcription of core viral proteins (HBsAg, HBcAg, and HBeAg), ultimately regulating the replication and transcription of HBV.

Further experiments involving the overexpression or knockdown of IGF2BP2 in sEVs revealed a significant increase or decrease in the copy number of HBV DNA, indicating that IGF2BP2 also plays a role in regulating HBV replication within sEVs. This finding aligned with observations at the cellular level that a targeted reduction in IGF2BP2 inhibits the proliferation of HepG2.215 cells and viral replication [45]. To elucidate the mechanism underlying the role of IGF2BP2 in sEVs, the copy numbers of rcDNA and cccDNA were also examined. The results indicated that overexpressing or knocking down IGF2BP2 in sEVs increased or decreased rcDNA and cccDNA copy numbers in transfected HepG2 cells, along with a corresponding increase or decrease in the rcDNA to cccDNA conversion rate. These findings suggested that IGF2BP2 enhances the expression of rcDNA and cccDNA and promotes the conversion of rcDNA to cccDNA, thereby facilitating the replication of HBV.

The overexpression or knockdown of IGF2BP2 in sEVs resulted in an increase or decrease in the mRNA expression levels of pgRNA, HBsAg, HBcAg, and HBeAg, respectively, along with corresponding alterations in m6A methylation levels in transfected HepG2 cells. These findings indicated that IGF2BP2 in sEVs mediates m6A modifications to positively regulate the transcription of HBV RNA. IGF2BPs can recognize GGm6AC motifs, thus promoting mRNA translation and enhancing mRNA stability [46, 47, 48]. Based on these findings, we speculated that IGF2BP2 in sEVs binds to HBV mRNA, enhancing its stability and promoting translation. This interaction increases mRNA expression of HBV core proteins and cytosolic proteins and also elevates viral DNA levels. This process also facilitates the conversion of rcDNA to cccDNA, thereby regulating the replication and transcription of HBV. Although functionally similar to METTL3, the underlying mechanisms probably differ.

Therefore, the mechanism of action of METTL3 and IGF2BP2 is critical. After METTL3 and IGF2BP2 in sEVs were overexpressed or knocked down, we found a corresponding increase or decrease in the expression levels of IGF2BP2 and METTL3 in the transfected cells. Co-IP experiments showed a synergistic interaction between METTL3 and IGF2BP2. These findings suggested that sEVs regulate HBV replication through the collaborative interaction of METTL3 and IGF2BP2, which mediates m6A methylation. Some studies have also shown that the METTL3-IGF2BP2 axis can interact to regulate m6A methylation [21, 49, 50]. The methyltransferase METTL3 and the reading protein IGF2BP2 work synergistically to form a complex; the process involved is intricate and involves multiple molecules. Among these molecules, the most notable players include IGF2BP1 and IGF2BP3 from the IGF2BP family, which frequently bind to IGF2BP2 [21]. Therefore, we hypothesized that they may also play a role in the synergistic action of METTL3 and IGF2BP2. However, this hypothesis needs to be experimentally verified.

In this study, we investigated the mechanisms regulating HBV replication, beginning at the molecular level of sEVs, while integrating the contemporary research interest in m6A methylation (Fig. 6). This approach offers a relatively novel perspective; however, other genetic components and regulatory pathways may also be involved. For example, IGF2BP2 can directly bind to the structure-specific nuclease Flap Structure-Specific Endonuclease 1 (FEN-1) mRNA, thereby influencing the stability of FEN-1, which presents an additional avenue for future research. However, our study had some limitations, including the absence of certain in vivo experimental data. Therefore, further investigations are needed, and we aim to assess these avenues in our future study.

5. Conclusion

To summarize, sEVs enhance m6A epigenetic modification through the synergistic interaction of METTL3 and IGF2BP2, promoting the conversion of HBV rcDNA to cccDNA and increasing the mRNA transcript levels of HBV core genes, including pgRNA, HBsAg, HBcAg, and HBeAg. This process regulates HBV replication and transcription. In this study, we investigated the molecular mechanism by which METTL3 and IGF2BP2 collaborate to mediate m6A methylation in sEVs, thereby regulating the replication of HBV. However, the effect of sEVs and their contents on HBV regulation necessitates further validation through additional studies. These findings may serve as a basis for developing new therapeutic targets and pathways for treating HBV infection.

Availability of Data and Materials

The datasets used during the current study are available from the corresponding author on reasonable request.

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Funding

Special Project of Strategic Cooperation in Science and Technology of Nanchong City and Colleges and Universities(22SXQT0318)