Host genes controlling rodent protoparvovirus tissue and species tropism

项目总结 小鼠微小病毒(MVM)是细小病毒科原细小病毒属(ProtoparvoVirus)的一员，属于细小病毒科。 在已知最小的病毒中，其不同寻常的线性单链DNA基因组，约5kb长， 侧翼是有结构的小发夹端粒，并包装成一个粗糙的二十面体(T=1)蛋白质 衣壳，直径约280？细小病毒衣壳已经进化成亚稳定的，正在经历一项 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，如依附因子和受体参与。衣壳动力学的几个方面已经被 很可能这些衣壳功能中的一些对成功进入细胞至关重要， 依赖于病毒成分和特定病毒产物之间的亲密相互作用的组合 宿主细胞基因。 细小病毒表现出两种嗜性，一种是针对自然寄主物种内的不同组织类型， 另一种是将病毒感染限制在一个或少数宿主物种的细胞上。而名单上的 人类细小病毒继续扩张，现在包含原细小病毒属的几个成员， 长期以来，人们一直知道啮齿动物原病毒不能在正常的人类细胞中感染和生长，但 在人类癌细胞中可以这样做，这一特性导致了目前对其作为溶瘤药物的疗效的临床试验 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 我们发现对免疫抑制株敏感的小鼠BV2-Cas9细胞的筛选 MVMi具有较高的多样性。鉴定了12个寄主基因，它们的敲除对白粉病具有显著的抗性。 MVMi，其中10个明显参与唾液酸代谢和蛋白质糖基化。剩下的 据报道，一个参与高尔基体腔内的囊泡蛋白运输，另一个参与高尔基体腔内的囊泡蛋白运输。 小鼠，是一种细胞表面糖蛋白生长因子受体。我们将通过击倒他们来验证这些命中 小鼠宿主细胞使用CRISPR-Cas9方法，随后分析病毒粒子进入的步骤 使用我们实验室开发的几种有良好文件记录的分析方法来阻止病毒进入突变。 将在新的小鼠和人类宿主细胞系中进行平行CRISPR-Cas9筛选以识别小鼠 控制小鼠成纤维细胞组织趋向性、细胞进入和感染建立的基因：淋巴细胞 通过亲纤维和亲淋巴变异体的原弓状病毒MVM杂交细胞，并逐步鉴定 转化的人类细胞，宿主基因，限制建立由啮齿动物原病毒感染 永生化的人成纤维细胞，其激活或沉默是固有的促癌作用的基础 啮齿动物病毒。

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.