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: 10193407
• 负责人: Peter J. Tattersall
• 金额: $ 25.13万
• 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 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10193407
• 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.

项目摘要 小鼠微小病毒（Minute Virus of Mice，MVM）是细小病毒科原细小病毒属（Protovarvovirus）的成员， 是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5 kb， 两侧是小的结构发夹端粒，并包装成一个粗糙的二十面体（T=1）蛋白质 衣壳直径约280 μ m。细小病毒衣壳已经进化为亚稳定的，经历了一个程序， 有限的构象变化，推测是由它在进入过程中遇到的各种信号引起的 过程，如附着因子和受体接合。衣壳动力学的几个方面已经被 未被发现，并且很可能这些衣壳功能中的一些对于成功进入细胞是必不可少的， 这取决于病毒成分和特定病毒的产物之间的密切相互作用的组合。 宿主细胞基因 细小病毒表现出两种嗜性，一种是对天然宿主物种内不同组织类型的嗜性， 另一种是将病毒感染限制在一种或少数宿主物种的细胞中。虽然名单 人细小病毒继续扩大，现在包含原细小病毒属的几个成员， 啮齿动物原细小病毒长期以来被认为不能感染正常的人类细胞并在其中生长，但是 可以在人类癌细胞中这样做，这一特性导致了目前的临床试验， 针对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基因测序。 在鼠BV 2-Cas9细胞中筛选，我们发现其对免疫抑制菌株敏感 MVMi在高多样性。鉴定了12个宿主基因，其敲除赋予显著的抗性， MVMi，其中10个明显参与唾液酸代谢和蛋白质糖基化。剩余 两个，据报道，一个是参与囊泡蛋白运输的高尔基体内腔，和其他，在 小鼠，是细胞表面糖蛋白生长因子受体。我们将通过在第一时间内 使用CRISPR-Cas9方法对鼠宿主细胞进行病毒粒子进入，然后分析病毒粒子进入的步骤。 使用我们实验室开发的用于分析病毒进入突变体的几种有据可查的测定方法进行阻断。 将在新的鼠和人宿主细胞系中进行平行的CRISPR-Cas9筛选，以鉴定小鼠肿瘤细胞。 控制鼠成纤维细胞：淋巴细胞的组织嗜性、细胞进入和感染建立的基因 通过原细小病毒MVM的亲纤维和亲淋巴变体的杂交细胞，并逐步鉴定 转化的人细胞，限制啮齿动物原细小病毒感染建立的宿主基因， 永生化的人成纤维细胞，并且其激活或沉默是永生化的人成纤维细胞的固有嗜瘤性的基础。 啮齿类病毒