Suchergebnisse

Background Previously, we showed that targeted disruption of viral receptor genes in avian leukosis virus (ALV) subgroups using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9))-based genome editing confers resistance to ALV subgroups B and J. Here, we used the same strategy to target the receptor expressed by ALV subgroup A (TVA) and generate chicken cells resistant to infection by this virus. Results CRISPR/Cas9-based disruption of exon 2 within the tva gene of DF-1 fibroblasts conferred resistance to infection by ALV subgroup A regardless of whether frameshift mutations were introduced during editing. Conversely, overexpression of the wild-type TVA receptor (wtTVA) by tva-modified DF-1 clones restored susceptibility to ALV subgroup A. The results confirm that exon 2, which contains the low-density lipoprotein receptor class A domain of TVA, is critical for virus entry. Furthermore, we sequentially modified DF-1 cells by editing the tva, tvb, and Na+/H+ exchange 1 (chNHE1) genes, which are the specific receptors for ALV subgroups A, B, and J, respectively. Conclusions Simultaneous editing of multiple receptors to block infection by different subgroups of ALV confirmed that ALV subgroups A, B, and J do not share host receptors. This strategy could be used to generate cells resistant to multiple viral pathogens that use distinct receptors for cell entry. ; This work was supported by This work was supported by the National Research Foundation of Korea grant funded by the Korea government (MSIP) (NRF2015R1A3A2033826), and "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ0131642018)" Rural Development Administration, Republic of Korea. Also this work was supproted by the Royal Society International Professorships (No.IC160046) and Biotechnology and BiologicalSciences Research Council (BBSRC) Partnership award (BB/M027481/1), UK.

Abbreviations CRDs: cysteine rich domains; ALV: avian leukosis virus; tvb: tumor virus locus B; tva: tumor virus locus A; tvc: tumor virus locus C; W38: tryptophan 38; CRISPR/ Cas9: clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated; DMEM: Dulbecco's minimum essential medium; FBS: fetal bovine serum; ABAM: antibiotic–antimycotic; WL: White Leghorn; CEF: chicken embryonic fbroblast; EDTA: ethylenediaminetetraacetic acid; dpi: days postinfection; SAS: the statistical analysis system; ANOVA: analysis of variance; WT: wild type; GFP: green fuorescent protein; TNFRSF10B: tumor necrosis factor receptor superfamily member 10B; DD: death domain; PAM: protospacer adjacent motif; LTR: long terminal repeat; RCAS: replication-competent ALV LTR with a splice acceptor; HIV: human immunodefciency virus; EIAV: equine infectious anemia virus; FIV: feline immunodefciency virus; RABS: rabies virus; HVEM: herpes virus entry mediator; LDLR: low-density lipoprotein receptor; PGC: primordial germ cell; gRNA: guide RNA. ; Abstract Avian leukosis virus (ALV) is a retrovirus that causes tumors in avian species, and its vertical and horizontal transmission in poultry flocks results in enormous economic losses. Despite the discovery of specific host receptors, there have been few reports on the modulation of viral susceptibility via genetic modification. We therefore engineered acquired resistance to ALV subgroup B using CRISPR/Cas9-mediated genome editing technology in DF-1 chicken fibroblasts. Using this method, we efficiently modified the tumor virus locus B (tvb) gene, encoding the TVB receptor, which is essential for ALV subgroup B entry into host cells. By expanding individual DF-1 clones, we established that artificially generated premature stop codons in the cysteine-rich domain (CRD) of TVB receptor confer resistance to ALV subgroup B. Furthermore, we found that a cysteine residue (C80) of CRD2 plays a crucial role in ALV subgroup B entry. These results suggest that CRISPR/Cas9-mediated genome editing can be used to efficiently modify avian cells and establish novel chicken cell lines with resistance to viral infection. ; This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2015R1A3A2033826). Authors also acknowledge the funding support from the Royal Society International Professorships.