New antiviral agents are needed for the treatment of hepatitis B virus (HBV) infection because currently available drugs do not completely eradicate chronic HBV in patients. Phosphorylation dynamics of the HBV core protein (HBc) regulate several processes in the HBV life cycle, including nucleocapsid formation, cell trafficking, and virus uncoating after entry. In this study, the SRPK inhibitors SPHINX31, SRPIN340, and SRPKIN-1 showed concentration-dependent anti-HBV activity. Detailed analysis of the effects of SRPKIN-1, which exhibited the strongest inhibitory activity, on the HBV replication process showed that it inhibits the formation of infectious particles by inhibiting pregenomic RNA packaging into capsids and nucleocapsid envelopment. Mass spectrometry analysis combined with cell-free translation system experiments revealed that hyperphosphorylation of the C-terminal domain of HBc is inhibited by SRPKIN-1. Further, SRPKIN-1 exhibited concentration-dependent inhibition of HBV infection not only in HepG2-hNTCP-C4 cells but also in fresh human hepatocytes (PXB cells) and in the single-round infection system. Treatment with SRPKIN-1 at the time of infection reduced the nuclease sensitivity of HBV DNA in the nuclear fraction. These results suggest that SRPKIN-1 has the potential to not only inhibit the HBV particle formation process but also impair the early stages of viral infection.

治疗乙型肝炎病毒（HBV）感染需要新的抗病毒药物，因为目前可用的药物不能完全根除患者的慢性乙型肝炎。HBV 核心蛋白 (HBc) 的磷酸化动态调节 HBV 生命周期中的多个过程，包括核衣壳形成、细胞运输和进入后病毒脱壳。在这项研究中，SRPK 抑制剂 SPHINX31、SRPIN340 和 SRPKIN-1 显示出浓度依赖性的抗 HBV 活性。对表现出最强抑制活性的SRPKIN-1对HBV复制过程的影响的详细分析表明，它通过抑制前基因组RNA包装成衣壳和核衣壳包封来抑制感染性颗粒的形成。质谱分析结合无细胞翻译系统实验表明，SRPKIN-1 抑制 HBc C 端结构域的过度磷酸化。此外，SRPKIN-1不仅在HepG2-hNTCP-C4细胞中，而且在新鲜人肝细胞（PXB细胞）和单轮感染系统中都表现出浓度依赖性的HBV感染抑制作用。感染时用 SRPKIN-1 治疗降低了核部分中 HBV DNA 的核酸酶敏感性。这些结果表明 SRPKIN-1 不仅有可能抑制 HBV 颗粒形成过程，而且还可以损害病毒感染的早期阶段。