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.