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 小鼠的微小病毒（MVM）是parvoviridae家族中原子谷病毒属的成员， 在最小的已知病毒中，其异常线性的单链DNA基因组长，长度约为5kb， 侧面是小结构发夹端粒，并包装成坚固的二十面体（t = 1）蛋白 衣壳，直径约为280Å。小红细胞的衣壳已经发展为可稳定性，正在经历 有限的会议班次有限，该班次是由它在进入期间遇到的各种信号引起的 过程，例如依恋因素和受体参与。 Capsid动力学的几个方面已经 被发现，并且可能对成功的细胞进入至关重要的这些capsid功能很可能 取决于病毒成分与特定产物之间的亲密相互作用的结合 宿主细胞基因。 细小病毒显示两种的偏向主义，一种用于天然宿主物种中不同组织类型，一种， 另一种将病毒感染限制在一个或少数宿主物种的细胞中。而列表 人类细节病毒继续扩展，现在包含多个原病毒属的成员， 长期以来，啮齿动物的原病毒一直无法感染和在正常的人类细胞中生长，但是 可以在人类癌细胞中这样做，该特性已导致当前的临床试验，以溶瘤的有效性 针对胶质母细胞瘤和胰腺癌的病毒。 为了探索小鼠细胞中病毒进入所需的宿主基因，我们进行了CRISPR-CAS9 在鼠BV2-CAS9细胞中的屏幕，我们发现它易受免疫抑制菌株的影响 MVMI高度多样性。鉴定了十二个宿主基因，其基因敲除具有对 MVMI，其中十个显然参与唾液酸代谢和蛋白质糖基化。剩下的 据报道，二，一种参与高尔基管内的囊泡蛋白运输，另一个参与 小鼠是细胞表面糖蛋白生长因子受体。我们将通过将它们淘汰来验证这些命中 使用CRISPR-CAS9方法的鼠宿主细胞，然后分析进入病毒粒子进入的步骤 使用在我们的实验室中开发的几种有据可查的测定法，用于分析病毒进入突变体。 平行CRISPR-CAS9屏幕将在新型的鼠和人类宿主细胞系中进行，以识别鼠标 控制鼠成纤维细胞感染的组织向性，细胞进入和建立的基因：淋巴细胞 杂种细胞通过原毛病毒MVM的原纤维和淋巴样变异体，并逐步识别 转化的人类细胞，宿主基因，限制了啮齿动物原子谷病毒在 永生的人类成纤维细胞，其激活或沉默的基于 啮齿动物病毒。