Genetic Viral and Host Adaptations to Breach Species Barriers of HCV

项目摘要 丙型肝炎病毒（HCV）是一种重要的传染病，在约71 全球百万人。CDC估计，美国每年约有30，000例新的HCV病例， 每年约有20，000人死亡，使HCV比其他60种传染病的总和更致命，包括 艾滋病。然而，导致慢性HCV感染后出现终末期肝病的潜在机制 我们对此知之甚少。虽然慢性丙型肝炎现在可以有效地治疗直接作用的抗病毒药物 由于治疗费用极高，预防传播的疫苗仍然是高度优先事项 （12周课程80，000美元以上）。此外，一旦感染，个人仍然处于肝病的高风险之中 甚至是治疗后。建议的工作将以我们的大量研究结果为基础，有系统地继续进行。 分析造成HCV种间传播障碍的机制。 目的1：确定不同物种中支持HCV复制的已知宿主因子的机制。 HCV依赖于多种宿主因子在肝细胞中建立复制。我们的初步数据显示 对于所有测试的类人猿，肽基脯氨酰异构酶A（也称为亲环素A（CypA））的直系同源物支持 HCV RNA复制。然而，远亲物种的CypA，如新大陆和旧大陆的猴子， 老鼠的效率要低得多。我们的目标是机械地定义这个主机和其他主机的潜在不兼容性 这些因子与HCV复制机制的病毒编码成分有关。 目的2：研究灵长类动物HCV感染及其免疫应答网络 和啮齿动物种类。我们将研究丙型肝炎病毒是否可以感染和复制在一组新的干细胞衍生的细胞， 肝细胞样细胞来自一系列进化上不同的物种，包括大猿，选择新的和旧的 世界上的猴子和啮齿动物。在第二步中，我们将使用高通量单细胞RNA测序来获得 与HCV复制相关的物种特异性转录组反应网络。 目的3：研究CD 302和Cr 1 L在体内抑制HCV感染的作用。人为进入因素 先天免疫功能减弱的转基因小鼠只支持低水平的病毒复制，这表明小鼠 限制因子可以抑制病毒在体内的复制。我们的初步数据表明，小鼠CD 302和 补体成分（3b/4 b）受体1样（Cr 1 L）在体外限制HCV复制。在这里，我们将评估如果损失- 在我们的HCV进入因子敲入小鼠中，mCD 302和mCr 1 L的功能缺失增强了HCV感染。 这项工作将为研究HCV的种属嗜性提供新的思路。Ploss实验室已经做出了许多 开创性的贡献，该领域，并将在这一重要工作的帮助下，我们的长期合作者博士。 Schwartz（Weill Cornell）、Shalek（MIT）和Pietschmann（Twincore，德国）。我们的工作将推动这一领域 通过在开发适合研究HCV的小动物模型方面取得进展， 感染和免疫反应，这是改善治疗和开发疫苗的必要前提。

Project summary Hepatitis C virus (HCV) is an important and underreported infectious disease, causing chronic infection in ~71 million people worldwide. The CDC estimates there are ~30,000 new cases of HCV every year in the US and about 20,000 deaths annually, making HCV more deadly than 60 other infectious diseases combined, including HIV. However, the underlying mechanisms that lead to chronic HCV infection followed by end-stage liver disease are poorly understood. Although chronic hepatitis C can now be effectively treated with direct-acting antivirals (DAAs), a vaccine to prevent transmission remains a high priority due to extremely high treatment costs ($80,000+ for a 12-week course). Furthermore, once infected, individuals remain at high risk for liver disease even post-treatment. The proposed work will build on our substantial research findings to continue systematically analyzing the mechanisms that create barriers to interspecies HCV transmission. Aim 1: Define mechanisms of known host factors from diverse species that support HCV replication. HCV relies on a variety of host factors to establish replication in hepatocytes. Our preliminary data demonstrate that for all great apes tested, orthologs of peptidylprolyl isomerase A, also known as cyclophilin A (CypA), support HCV RNA replication. However, CypA of distantly related species, such as New and Old World monkeys and mice, is far less efficient. We aim to define mechanistically the underlying incompatibility of this and other host factors with the virally encoded components of the HCV replication machinery. Aim 2: Characterize HCV infection and immune response networks following infection across primate and rodent species. We will study whether HCV can infect and replicate in a novel set of stem cell-derived hepatocyte-like cells from a range of evolutionarily diverse species, including great apes, selected New and Old World monkeys and rodents. In a second step, we will use high-throughput single-cell RNA sequencing to derive species-specific transcriptomic response networks associated with HCV replication. Aim 3: Characterize the impact of CD302 and Cr1L on restricting HCV infection in vivo. Human entry factor transgenic mice with blunted innate immunity only support low-level viral replication, suggesting murine restriction factors may suppress viral replication in vivo. Our preliminary data indicate mouse CD302 and complement component (3b/4b) receptor 1-like (Cr1L) limit HCV replication in vitro. Here, we will assess if loss- of-function of mCD302 and mCr1L augments HCV infection in our HCV entry factor knock-in mice. The proposed work will provide new insights into the species tropism of HCV. The Ploss lab has made many seminal contributions to the field and will be aided in this important work by our long-standing collaborators Drs. Schwartz (Weill Cornell), Shalek (MIT) and Pietschmann (Twincore, Germany). Our work will advance the field of HCV research by making progress in the development of small animal models suitable for studying HCV infection and immune responses, a necessary precursor to improving treatment and developing vaccines.