Mechanism of Hepatitis E Virus Replication and Pathogenesis

戊型肝炎病毒复制和发病机制

• 批准号: 8689884
• 负责人: XIANG-JIN MENG
• 金额: $ 40.25万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8689884
• 关键词: 3' Untranslated Regions; Animals; Antiviral Agents; Attenuated; Calpain; Cell Culture Techniques; Cells; Cerebrospinal Fluid; Chronic; Chronic Hepatitis; Complementarity Determining Regions; DNA Insertion Elements; Developed Countries; Developing Countries; Event; Family suidae; Genes; Genetic Determinism; Genetic Recombination; Genome; Genotype; Goals; Hepatitis E; Hepatitis E virus; Hepatocyte; Host Defense; Human; Immunity; Infection; Interferons; Mediating; Modeling; Neurologic; Oryctolagus cuniculus; Pathogenesis; Pathogenicity; Patients; Play; Prevention strategy; Production; Protease Domain; Proteins; Public Health; RNA replication; Replicon; Reporting; Role; Serum; Symptoms; System; Therapeutic; Tropism; Ubiquitin; Ubiquitin Like Proteins; United States; Variant; Viral; Viral Pathogenesis; Virulence; Virulent; Virus; Virus Diseases; Virus Replication; influenzavirus; insight; multicatalytic endopeptidase complex; neurotropic; novel; overexpression; pathogen; public health relevance; recombinant virus; response; ribosomal protein S19; treatment strategy; viral RNA; virus genetics; virus pathogenesis; virus tropism

DESCRIPTION (provided by applicant): Hepatitis E virus (HEV) is an important but extremely understudied pathogen. Genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 infect humans and other animals. The long-term goals of this project are to delineate the mechanisms of HEV cross-species infection and identify host and viral determinants of host range and pathogenesis. In specific aim 1, we hypothesize that the hypervariable region (HVR) in ORF1 and the inserted host gene sequences in HVR determine HEV host range. Our preliminary studies suggested that HVR and its adjacent region are involved in HEV tropism. The recent findings that host S17 and S19 gene sequences were inserted into the HVR of HEV that resulted in recombinant viruses with expanded cell tropism of different animal origins lends further credence to our hypothesis. We will determine (a) if a genotype 1 human HEV (infecting humans only) will acquire the ability to infect pigs and rabbits if its HVR is replaced with that o a zoonotic genotype 3 HEV, (b) if the genotype 1 human HEV with an inserted host S17 sequence in the HVR will acquire the ability to infect pigs and rabbits, and (c) if the adjacent X domain in ORF1 plays a role, together with HVR, in determining the host range. In specific aim 2, we hypothesize that the genotype- specific HVR sequences and/or inserted host sequences contribute to HEV pathogenicity. Increased pathogenicity by recombination events between host gene sequence and virus genomes has been reported for other viruses. The fact that the neurological and chronic cases of hepatitis E are all caused by the zoonotic genotype 3 HEV and that quasispecies compartmentalization in CSF and serum of clonal HEV sequences within the HVR region was identified from neurological cases suggested that the HVR plays a role in virus pathogenicity and emergence of neurotropic variants. We will determine (a) if the genotype 3 virus with the S17 insertion in HVR is more virulent than the virus without the insertion, (b) if the genotype 3 virus with a S17 insertion establishes chronic infection in pigs, and (c) if the HVR and its adjacent regions evolve during chronic HEV infection in pigs to produce neurotropic variants. In specific aim 3, we hypothesize that interferon stimulated gene 15 (ISG15) plays a role in HEV infection and host anti-HEV response. Our preliminary studies showed that HEV replication requires an active ubiquitin-proteasome system and that overexpression of ISG15 inhibited HEV replication in a HEV replicon system. The findings that the papain-like cysteine protease (PCP) domain in HEV ORF1 has deubiquitinating activity and carries out deISGylation of ISG15-conjugated proteins support our hypothesis. By using an efficient HEV infection cell culture model with genotype 3 HEV Kernow C- 1 strain, we will determine (a) if overexpression of ISG15 will inhibit genotype 3 HEV infection, and if so, what is the mechanism of inhibition, (b) if HEV counteracts ISG15 production and ISGylation, and if so, if the PCP domain plays a role in antagonizing ISG15 function. By completing this project, we expect to identify the viral determinant(s) for HEV host range and pathogenicity and delineate the role of ISG15 in anti-HEV response.

描述（由申请人提供）：戊型肝炎病毒（HEV）是一种重要但研究非常不足的病原体。基因型1和2仅限于人类，而基因型3和4感染人类和其他动物。本项目的长期目标是阐明HEV跨种感染的机制，并确定宿主和病毒的宿主范围和发病机制的决定因素。在具体目标1中，我们假设ORF 1中的高变区（HVR）和HVR中插入的宿主基因序列决定了HEV的宿主范围。我们的初步研究表明，HVR及其邻近区域参与了HEV的嗜性。最近的研究发现，宿主S17和S19基因序列插入到HEV的HVR中，导致重组病毒具有不同动物来源的扩增细胞嗜性，这进一步证实了我们的假设。我们将确定（a）如果基因型1人HEV（仅感染人）的HVR被人畜共患基因型3 HEV的HVR替换，其是否将获得感染猪和兔的能力，（B）在HVR中插入宿主S17序列的基因型1人HEV是否将获得感染猪和兔的能力，和（c）在HVR中插入宿主S17序列的基因型1人HEV中的相邻X结构域是否将获得感染猪和兔的能力。 ORF 1与HVR一起在确定宿主范围中起作用。在具体目标2中，我们假设基因型特异性HVR序列和/或插入的宿主序列有助于HEV致病性。据报道，其他病毒通过宿主基因序列和病毒基因组之间的重组事件增加了致病性。戊型肝炎的神经和慢性病例均由人畜共患基因型3 HEV引起，并且从神经病例中鉴定出HVR区域内克隆HEV序列的CSF和血清中的准种区室化，这一事实表明HVR在病毒致病性和嗜神经性变体的出现中起作用。我们将确定（a）在HVR中具有S17插入的基因型3病毒是否比没有插入的病毒毒力更强，（B）如果 插入S17的基因型3病毒在猪中建立慢性感染，和（c）如果HVR 其邻近区域在猪慢性HEV感染期间进化以产生嗜神经性变体。在具体目标3中，我们假设干扰素刺激基因15（ISG 15）在HEV感染和宿主抗HEV应答中起作用。我们的初步研究表明，戊型肝炎病毒的复制需要一个活跃的泛素-蛋白酶体系统和ISG 15的过表达抑制戊型肝炎病毒复制的复制子系统。HEV ORF 1中的木瓜蛋白酶样半胱氨酸蛋白酶（PCP）结构域具有去泛素化活性，并对ISG 15结合蛋白进行去ISG化，这一发现支持了我们的假设。通过使用基因型3 HEV Kernow C- 1株的有效HEV感染细胞培养模型，我们将确定（a）ISG 15的过表达是否会抑制基因型3 HEV感染，如果是，抑制的机制是什么，（B） 如果HEV抵消ISG 15的产生和ISG化，如果是这样，PCP结构域是否在拮抗ISG 15功能中起作用。通过完成这个项目，我们期望确定HEV宿主范围和致病性的病毒决定因素，并描述ISG 15在抗HEV应答中的作用。