Host genes controlling rodent protoparvovirus tissue and species tropism

控制啮齿动物原细小病毒组织和种向性的宿主基因

• 批准号: 10385761
• 负责人: Peter J. Tattersall
• 金额: $ 20.94万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-09 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10385761
• 关键词: Affect; Architecture; Binding; Biological Assay; CRISPR library; CRISPR screen; CRISPR/Cas technology; Caliber; Capsid; Capsid Proteins; Cell Line; Cell Nucleus; Cell surface; Cells; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Cytosol; Enzymes; Family; Fibroblast Growth Factor Receptors; Fibroblasts; Genes; Genome; Glioblastoma; Glycoproteins; Golgi Apparatus; Growth; Growth Factor Receptors; Human; Hybrid Cells; Hybrids; In Vitro; Infection; Invertebrates; Knock-out; Lead; Length; Libraries; Lymphocyte; Malignant neoplasm of pancreas; Membrane Glycoproteins; Membrane Protein Traffic; Membrane Proteins; Metabolism; Mice Minute Virus; Molecular Conformation; Mouse Strains; Mus; Nature; Oncolytic; Oncolytic viruses; Parents; Parvoviridae; Parvovirus; Parvovirus Infections; Pathway interactions; Penetration; Phenotype; Phospholipase; Polysaccharides; Process; Production; Property; Protein Glycosylation; Proteins; Reporting; Resistance; Rodent; Role; Sialic Acids; Signal Transduction; Single-Stranded DNA; Structure; Survivors; TP53 gene; Tissues; Tropism; Variant; Viral; Virion; Virus; Virus Diseases; c-myc Genes; cancer cell; cell killing; conformational alteration; gene discovery; gene product; gene synthesis; glycosylation; in vivo; man; member; mutant; novel; oncolytic virotherapy; overexpression; programs; protein transport; receptor; receptor mediated endocytosis; telomere; tissue tropism

PROJECT SUMMARY Minute Virus of Mice (MVM) is a member of genus Protoparvovirus in the family Parvoviridae, which are among the smallest known viruses, and whose unusual linear single-stranded DNA genomes, ~5kb in length, are flanked by small structured hairpin telomeres, and packaged into a rugged icosahedral (T=1) protein capsid, ~280Å in diameter. The parvoviral capsid has evolved to be metastable, undergoing a program of limited conformational shifts, presumed to be induced by various signals that it encounters during the entry process, such as attachment factor and receptor engagement. Several aspects of capsid dynamics have been uncovered, and it is likely that some of these capsid functions that are essential to successful cell entry, depend upon the combination of intimate interactions between viral components and the products of specific host cell genes. The parvoviruses display two kinds of tropism, one for different tissue types within the natural host species, and the other that restricts virus infection to cells of one or a small number of host species. While the list of human parvoviruses continues to expand, and now contains several members of the Protoparvovirus genus, the rodent protoparvoviruses have long been known to be unable to infect and grow in normal human cells, but can do so in human cancer cells, a property that has lead to current clinical trials of their efficacy as oncolytic viruses against glioblastoma and pancreatic cancer. In order to explore the host genes required for viral entry in mouse cells, we have performed a CRISPR-Cas9 screen in murine BV2-Cas9 cells, which we had found to be susceptible to the immunosuppressive strain MVMi at high multiplicity. Twelve host genes were identified whose knockout conferred significant resistance to MVMi, of which ten are clearly involved in sialic acid metabolism and protein glycosylation. Of the remaining two, one is reported to be involved in vesicular protein trafficking within the Golgi lumen, and the other, in the mouse, is a cell surface glycoprotein growth factor receptor. We will verify these hits by knocking them out in murine host cells using a CRISPR-Cas9 approach, followed by analysis for the step at which virion entry is blocked using several well-documented assays developed in our lab for the analysis of viral entry mutants. Parallel CRISPR-Cas9 screens will be performed in novel murine and human host cell lines to identify mouse genes controlling the tissue tropism, cell entry and establishment of infection of murine fibroblast:lymphocyte hybrid cells by fibrotropic and lymphotropic variants of the protoparvovirus MVM, and to identify, in stepwise transformed human cells, host genes that restrict the establishment of infection by rodent protoparvoviruses in immortalized human fibroblasts, and whose activation or silencing underlie the inherent oncotropism of the rodent viruses.

项目概要 小鼠微小病毒（MVM）是细小病毒科原细小病毒属的成员， 已知最小的病毒之一，其不寻常的线性单链 DNA 基因组长度约为 5kb， 两侧是小结构发夹端粒，并包装成崎岖不平的二十面体 (T=1) 蛋白质 衣壳，直径约280Å。细小病毒衣壳已进化为亚稳态，经历了一系列过程 有限的构象转变，推测是由进入过程中遇到的各种信号引起的 过程，例如附着因子和受体接合。衣壳动力学的几个方面已被 未被发现，并且很可能其中一些衣壳功能对于成功进入细胞至关重要， 取决于病毒成分和特定产物之间密切相互作用的组合 宿主细胞基因。 细小病毒表现出两种向性，一种针对自然宿主物种内的不同组织类型， 另一种是限制病毒感染一个或少数宿主物种的细胞。虽然名单 人类细小病毒继续扩大，现在包含原细小病毒属的几个成员， 人们早就知道啮齿动物原细小病毒无法感染正常人体细胞并在其中生长，但是 可以在人类癌细胞中做到这一点，这一特性导致目前对其作为溶瘤药物的功效进行临床试验 抗胶质母细胞瘤和胰腺癌的病毒。 为了探索病毒进入小鼠细胞所需的宿主基因，我们进行了 CRISPR-Cas9 在小鼠 BV2-Cas9 细胞中进行筛选，我们发现该细胞对免疫抑制菌株敏感 高多样性下的 MVMi。鉴定出 12 个宿主基因，其敲除赋予了显着的抗性 MVMi，其中十个明显参与唾液酸代谢和蛋白质糖基化。剩余的 两个，据报道，一个参与高尔基腔内的囊泡蛋白运输，另一个参与高尔基腔内的囊泡蛋白运输。 小鼠，是细胞表面糖蛋白生长因子受体。我们将通过敲除这些命中来验证它们 使用 CRISPR-Cas9 方法对小鼠宿主细胞进行分析，然后分析病毒颗粒进入的步骤 使用我们实验室开发的几种有据可查的分析病毒进入突变体的方法进行阻断。 平行 CRISPR-Cas9 筛选将在新型小鼠和人类宿主细胞系中进行，以鉴定小鼠 控制小鼠成纤维细胞：淋巴细胞的组织向性、细胞进入和感染建立的基因 通过原细小病毒 MVM 的亲纤维性和亲淋巴性变体杂交细胞，并逐步识别 转化的人类细胞，限制啮齿动物原细小病毒感染建立的宿主基因 永生化的人类成纤维细胞，其激活或沉默是其固有的促癌性的基础 啮齿动物病毒。