State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, National Medical Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Baruch S. Blumberg Institute, Doylestown, PA, USA.
Methods Mol Biol. 2024;2837:89-97. doi: 10.1007/978-1-0716-4027-2_8.
Chronic hepatitis B virus (HBV) infection is due to the failure of host immune system to resolve the viral infection. Accordingly, restoration or reconstitution of a functional antiviral immune response to HBV is essential to achieve durable control of HBV replication leading to a functional cure of chronic hepatitis B (CHB). Noninfectious subviral particles (SVPs), comprised of HBV surface antigen (HBsAg), are the predominant viral products secreted by HBV-infected hepatocytes. The high levels of SVPs in the circulation induce immune tolerance and contribute to the establishment of chronic HBV infection. The current standard-of-care medications for CHB efficiently suppress HBV replication but fail to reduce the levels of HBsAg in majority of treated patients. Further understanding the mechanisms underlying SVP morphogenesis, secretion and regulation by viral and host cellular factors are critical for the discovery of therapeutics that can inhibit SVP production and/or induce the degradation of HBV envelope proteins. We describe herein a protocol for intracellular SVP detection by a native agarose gel electrophoresis-based particle gel assy. The method is suitable for quantitative detection of intracellular HBV SVPs and can be applied in dissecting the molecular mechanism of SVP morphogenesis and the discovery of antiviral agents targeting SVP formation in hepatocytes.
慢性乙型肝炎病毒（HBV）感染是由于宿主免疫系统未能清除病毒感染。因此，恢复或重建对 HBV 的抗病毒免疫应答对于实现 HBV 复制的持久控制，从而实现慢性乙型肝炎（CHB）的功能性治愈至关重要。由乙型肝炎表面抗原（HBsAg）组成的非感染性亚病毒颗粒（SVP）是 HBV 感染的肝细胞分泌的主要病毒产物。循环中高水平的 SVP 诱导免疫耐受，并有助于慢性 HBV 感染的建立。目前 CHB 的标准治疗药物可有效抑制 HBV 复制，但不能降低大多数治疗患者的 HBsAg 水平。进一步了解 SVP 形态发生、分泌以及病毒和宿主细胞因子调节的机制对于发现能够抑制 SVP 产生和/或诱导 HBV 包膜蛋白降解的治疗方法至关重要。我们在此描述了一种基于天然琼脂糖凝胶电泳的粒子凝胶分析检测细胞内 SVP 的方案。该方法适用于定量检测细胞内 HBV SVP，可用于剖析 SVP 形态发生的分子机制，并发现针对肝细胞中 SVP 形成的抗病毒药物。
