CLINICAL SIGNIFICANCE AND MOLECULAR PATHOGENESIS OF HEPATITIS B VIRUS MUTANTS

乙型肝炎病毒（HBV）感染的结果是多种病毒与宿主相互作用的复杂相互作用的结果。虽然宿主反应可能起主要作用，但这一过程往往取决于多种病毒适应机制。通常，病毒基因组关键区域的病毒突变是这些适应机制的结果。我们的实验室已经确定并表征了与某些HBV毒株变异生物学行为相关的特定病毒突变。在与暴发性肝炎相关的HBV毒株中发现了HBV核心启动子的两个突变，由于基因组前RNA被病毒封装到核心颗粒中，导致复制高度增强。我们最近的出版物表明，影响新遗传元件的自然发生的突变可能通过共同或转录后机制影响病毒的衣壳化，从而增强核心合成和病毒复制。作为该项目的第二个方面，我们已经开始研究磨玻璃肝细胞在HBV感染中的发病机制。磨玻璃肝细胞是慢性乙型肝炎病毒（HBV）感染的独特组织学特征。这些肝细胞被抗hbs和抗pres1强烈染色。EM研究显示，大量HBV包膜蛋白积聚在可能来自内质网的扩张囊泡内。在转基因小鼠中，大表面蛋白的过度表达导致磨玻璃肝细胞。大表面蛋白的前s1区已被证明调节HBsAg的组装、加工和分泌。因此，我们假设前s1的突变形式影响这种正常的分泌途径，并负责磨砂玻璃肝细胞。为了研究这个问题，我们检测了HBV感染患者的前s1区HBV序列，肝活检显示患者有明显的磨玻璃肝细胞。为了分析单个磨玻璃肝细胞的病毒群，我们使用激光捕获显微解剖技术从活检标本中分离单个肝细胞。分别收获抗- hbs染色阳性的磨玻璃肝细胞和正常肝细胞，并对其HBV DNA进行序列分析。对一名患者的初步分析显示，大多数从磨砂玻璃肝细胞分离的病毒含有独特的前s1突变序列。相比之下，对照肝细胞只有WT序列。目前正在对大量患者进行进一步的研究，以研究这些前s1突变的功能影响。通过自然发生的HBV突变体确定与感染相关的肝脏疾病的变异表现的分子基础，可能有助于进一步了解HBV感染的发病机制。-暴发性肝炎/复制/转录/细胞损伤-人类受试者

The outcome of hepatitis B virus (HBV) infection results from complicated interplays among a variety of virus-host interactions. Although host responses likely play a major role, this process often depends on a variety of viral adaptive mechanisms. Frequently, viral mutations in critical regions of viral genome are the results of these adaptive mechanisms. Our laboratory has identified and characterized specific viral mutations associated with variant biological behaviors of certain HBV strains. Two mutations in the HBV core promotor were identified in a HBV strain associated with fulminant hepatitis leading to highly enhanced replication as a result of increased viral encapsidation of pregenomic RNA into the core particles. Our recent publications suggest that naturally occurring mutations affecting a novel genetic element may influence viral encapsidation by a co- or post-transcriptional mechanism resulting in enhanced core synthesis and viral replication. As a second aspect of this project, we have initiated studies into the pathogenesis of ground glass hepatocytes in HBV infection. Ground glass hepatocytes are an unique histological feature of chronic hepatitis B virus (HBV) infection. These hepatocytes are stained strongly with anti-HBs and anti-preS1. EM studies show that large amounts of HBV envelope proteins accumulate within dilated vesicles presumably derived from the endoplasmic reticulum. In transgenic mice, overexpression of large surface protein leads to ground glass hepatocytes. The pre-S1 region of the large surface protein has been shown to regulate assembly, processing and secretion of HBsAg. We therefore hypothesize that a mutant form of pre-S1 affects this normal secretory pathway and is responsible for ground glass hepatocytes. To investigate this question, we examined HBV sequences spanning the pre-S1 region from HBV infected patients with evident ground glass hepatocytes on liver biopsy. To analyze the viral population of single ground glass hepatocytes, we used the technique of laser capture microdissection to isolate individual hepatocytes from the biopsy specimen. Ground glass hepatocytes that stained positively with anti-HBs as well as normal appearing hepatocytes were harvested individually and their HBV DNA subjected to sequence analysis. Preliminary analysis of one patient revealed that the majority of viral isolates from the ground glass hepatocytes contained a unique pre-S1 mutant sequence. In contrast, the control hepatocytes had exclusively WT sequence. Additional studies are being pursued in a large number of patients and to study the functional effect of these pre-S1 mutations. Defining the molecular basis of variant manifestations of liver disease associated with infection by naturally occurring HBV mutants may contribute to further understanding of the pathogenesis of HBV infection. - Fulminant Hepatitis/Replication/Transcription/Cell Injury - Human Subjects