CLINICAL SIGNIFICANCE AND MOLECULAR PATHOGENESIS OF HEPATITIS B VIRUS MUTANTS

乙肝病毒感染的结局是多种病毒-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，但这一过程往往依赖于各种病毒适应机制。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乙肝病毒株的不同生物学行为相关的特定病毒突变。在与重型肝炎相关的乙肝病毒株中发现了两个HBVC启动子突变，这是由于病毒将前基因组RNA包裹到核心颗粒中而导致复制高度增强的结果。我们最近发表的文章表明，影响一种新的遗传元件的自然发生的突变可能会通过共转录或转录后机制影响病毒的包膜，从而增强核心合成和病毒复制。作为这个项目的第二个方面，我们已经开始研究磨玻璃肝细胞在乙肝病毒感染中的发病机制。磨玻璃肝细胞是慢性乙型肝炎病毒感染的独特组织学特征。这些肝细胞与抗HBs和抗PreS1呈强阳性反应。EM研究表明，大量的HBV膜蛋白积聚在扩张的小泡中，推测是来自内质网。在转基因小鼠中，大表面蛋白的过度表达导致了磨玻璃状肝细胞的形成。大表面蛋白的前-S1区已被证明调节乙肝表面抗原的组装、加工和分泌。因此，我们假设Pre-S1的突变形式影响这一正常的分泌途径，并与毛玻璃肝细胞有关。为了研究这个问题，我们从肝活检中有明显磨玻璃肝细胞的乙肝病毒感染者身上检测了跨越前-S1区的乙肝病毒序列。为了分析单个毛玻璃肝细胞的病毒群，我们使用激光捕获显微切割技术从活检标本中分离单个肝细胞。分别取抗-HBs染色阳性的毛玻璃肝细胞和外观正常的肝细胞进行HBVDNA测序。对一名患者的初步分析显示，来自毛玻璃肝细胞的大多数病毒分离株含有独特的前S1突变序列。相反，对照肝细胞只有WT序列。其他研究正在大量患者中进行，并研究这些前S1突变的功能影响。明确与自然发生的乙肝病毒变异株感染相关的各种肝病表现的分子基础可能有助于进一步了解乙肝病毒感染的发病机制。-暴发型肝炎/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