Mechanism of Hepatitis E Virus Replication and Pathogenesis

描述(申请人提供)：戊型肝炎病毒(HEV)是一种重要但研究极少的病原体。基因1和2仅限于人类，而基因3和4可感染人类和其他动物。该项目的长期目标是描述HEV跨物种感染的机制，并确定宿主和病毒决定宿主范围和致病的因素。在特定的目标1中，我们假设ORF1的高变区(HVR)和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，HEV的趋向性与HVR及其邻近区域有关。最近发现宿主S17和S19基因序列被插入到HEV的HVR中，从而产生了具有不同动物来源的扩增细胞嗜性的重组病毒，这进一步支持了我们的假设。我们将确定(A)如果用人畜共患病的3型HEV取代1型人类HEV(仅感染人类)，它是否会获得感染猪和兔的能力；(B)如果在HVR中插入宿主S17序列的1型人HEV将获得感染猪和兔的能力，以及(C)如果相邻的X结构域在 ORF1与HVR一起在决定寄主范围方面发挥作用。在特定的目标2中，我们假设特定于基因型的HVR序列和/或插入的宿主序列与HEV的致病性有关。在其他病毒中，已有报道通过宿主基因序列和病毒基因组之间的重组事件来增强致病性。神经科和慢性戊型肝炎都是由人畜共患的3型HEV引起的，并且从神经科病例中发现了HEV区克隆HEV序列在脑脊液和血清中的准种区划，提示HVR在病毒致病和嗜神经性变异株的出现中起作用。我们将确定(A)在HVR中插入S17的3型病毒是否比没有插入的病毒更强，(B)是否 带有S17插入的3型病毒建立了猪的慢性感染，以及(C)如果HVR 在猪的慢性HEV感染过程中，其邻近区域发生进化，产生嗜神经的变异体。在特定的目标3中，我们假设干扰素刺激基因15(ISG15)在HEV感染和宿主抗HEV反应中起作用。我们的初步研究表明，HEV的复制需要一个活跃的泛素-蛋白酶体系统，而ISG15的过表达抑制了HEV复制子系统中的HEV复制。HEV ORF1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ISG15结合蛋白进行去ISG化，这一发现支持我们的假说。通过使用基因3型HEV Kernow C-1毒株的有效HEV感染细胞培养模型，我们将确定(A)过表达ISG15是否会抑制基因3HEV感染，如果是，抑制机制是什么，(B) 如果HEV抵消了ISG15的产生和ISG化，如果是的话，如果PCP结构域在拮抗ISG15功能方面发挥了作用。通过完成这个项目，我们希望确定决定HEV宿主范围和致病性的病毒决定因素(S)，并描述ISG15在抗HEV应答中的作用。

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.