HBx interacts with the host YBX3 protein and up-regulates its expression to mediate efficient Hepatitis B viral replication

Background Chronic Hepatitis B (CHB) remains a global health challenge, and identifying the precise viral-host interactions that sustain the viral life cycle is essential for developing next-generation therapeutic strategies. In this study, we identify the host factor YBX3 (Y-box binding protein 3) as a critical requirement for efficient Hepatitis B Virus (HBV) replication. Methods Using immunoprecipitation-coupled mass spectrometry, we identified YBX3 as a novel interactor of the HBV regulatory protein, HBx. This interaction was validated via co-immunoprecipitation. The functional role of YBX3 was characterized using CRISPR/Cas9-mediated knockout in HepG2-hNTCP cells, followed by genetic rescue experiments. Viral fitness was assessed by quantifying cccDNA, pgRNA, HBsAg, HBeAg, and extracellular HBV DNA. Subcellular fractionation was employed to determine the distribution of YBX3 and HBx. Results YBX3 interacts specifically with HBx via its C-terminal domain. Genetic ablation of YBX3 drastically impaired HBV replication, resulting in a significant reduction in viral DNA, HBeAg, and HBsAg, a phenotype significantly reversed by YBX3 re-expression. Subcellular fractionation confirmed that both proteins are predominantly nuclear and that HBx expression upregulates YBX3 abundance in both nuclear and cytoplasmic fractions. YBX3 mRNA and protein levels were significantly upregulated during active HBV infection, suggesting a proviral feedback loop. Conclusion YBX3 is a novel proviral host factor that drives efficient HBV replication by binding to HBx. This interaction likely supports the crucial nuclear functions of HBx that are vital for efficient viral replication. These findings establish YBX3 as a compelling new target for antiviral therapeutic development.