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

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.