Dissecting Structural Details of Hepadnavirus Subviral Particles

乙肝病毒感染是一个全球性的公共卫生问题；然而，目前批准的治疗方法很少能完全治愈。乙肝表面抗原(HBs)是乙肝病毒感染的血清学标志。它的特征是三个跨膜蛋白具有相同的C-末端，在N-末端具有可变的框内延伸。自然地，乙肝表面抗原蛋白被整合到病毒的包膜中，或与来自宿主细胞的脂膜组装成无基因组和无衣壳的亚病毒颗粒(SVPs)。最近，一种通过阻断SVPs的组装和释放的新的治疗方法在快速降低慢性乙肝患者的HBs Ag水平方面显示出良好的效果。尽管它具有潜在的临床应用价值，但目前还没有关于乙肝表面抗原的结构信息，但人们已经提出了不同的SVPS模型。在这个方案中，我们将重点放在使用冷冻电子显微镜(Cryo-EM)从不同的模型体系中获得SVP的高分辨率结构。我们将检验这一中心假设，即SVP结构组织的变化将对其分泌途径产生重要影响。在目标1中，我们将确定土拨鼠肝炎病毒(WHV)SVP的结构。土拨鼠感染WHV是研究其发病机制的重要实验模型。在目标2中，我们将从携带者血清和肝癌细胞系中阐明HBVSVP的结构细节。我们将使用抗体在空间上标记乙肝表面抗原的不同区域。最后，根据这一建议确定的所有结构将被比较并用于构建具有详细结构信息的SVP的完整图像。该项目的成功完成将揭示SVP的新的结构信息，并将加深我们对乙肝病毒颗粒形态发生的理解，以促进目前寻找治疗乙肝病毒感染的努力。

Hepatitis B virus (HBV) infection is a global public health concern; however, current approval therapies rarely lead to a complete cure. Hepatitis B surface antigen (HBsAg) is the serological marker for HBV infection. It is characterized by three transmembrane proteins having the same C-termini with variable in-frame extension at the N-terminal ends. Naturally, HBsAg proteins are incorporated in the viral envelope or assembled into genome-free and capsid-free subviral particles (SVPs) with the lipid membrane derived from the host cells. Recently, a new therapeutic approach by blocking assembly and release of SVPs has shown promising results in rapidly reducing HBsAg level in chronic HBV- infected patients. Despite its potential clinically relevant applications, there is no structural information available for HBsAg and yet different models of SVPs have been proposed. In this proposal we focus on obtaining the high resolution structures of SVPs from different model systems using cryo-electron microscope (cryo-EM). We will test the central hypothesis that changes in the structural organizations of SVPs will have important implications on their secretion pathway. In Aim 1, we will determine the structures of SVP from woodchuck hepatitis virus (WHV). Woodchuck infected with WHV is a critical experimental model for studying pathogenesis of HBV infection. In Aim 2, we will elucidate the structural details of HBV SVP from carrier's sera and hepatoma cell line. We will use antibodies to spatially label different regions of HBsAg. Finally, all structures determined from this proposal will be compared and used to build a complete picture of SVP with the detailed structural information of Hepadnavirus surface antigen. Successful completion of the proposed project will reveal new structural information of SVPs and will deepen our understanding of HBV particles morphogenesis to facilitate current efforts in finding a cure for HBV infection.