title: Mutational analysis identifies leucine-rich repeat insertions crucial for pigeon toll-like receptor 7 recognition and signaling.
abstract: Toll-like receptor 7 (TLR7) is responsible for recognizing viral single-stranded RNA and antiviral imidazoquinoline compounds, leading to the activation of the innate immune response. In this study, mutated pigeon TLR7 fragments, in which the insertion at position 10 of leucine-rich repeat 10 (LRR10) or at position 15 of LRR2/11/13/14 was deleted, were amplified with an overlap-PCR method, and inserted into the expression vector pCMV. The immune functions of the TLR7 mutants were determined with an NF-κB luciferase assay of transfected cells. The deletion of the insertions absolutely abolished TLR7-NF-κB signaling. With quantitative real-time PCR and sandwich enzyme-linked immunosorbent assay, we observed that stimulation with R848 failed to induce the expression of interleukin 8 (IL-8) in any of the mutant-TLR7-transfected cells, consistent with their lack of NF-κB activity. However, the expression of interferon α (IFN-α) and tumor necrosis factor α (TNF-α) was significantly upregulated in the Del10IN10 and Del14IN15 groups. Remarkably, the levels of pigeon TLR7 expression were significantly increased in all the TLR7-mutated groups. Therefore, we speculate that another part of the deficient TLR7 mediates the induction of IFN-α and TNF-α by increasing the expression of TLR7 as compensation. However, the increased expression of TLR7 in the Del11IN15 group failed to induce the production of IFN-α, IL-8, or TNF-α, indicating that a false compensation occurred when the crucial LRR insertion was deleted.
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, Jiangsu 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, China. Electronic address: xiongdan_yzu@163.com.
authors.forename: Dan
authors.initials: D
authors.lastname: Xiong
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, Jiangsu 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, China. Electronic address: songli_yzu@163.com.
authors.forename: Li
authors.initials: L
authors.lastname: Song
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, Jiangsu 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, China. Electronic address: 983899312@qq.com.
authors.forename: Yang
authors.initials: Y
authors.lastname: Jiao
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, Jiangsu 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, China. Electronic address: 455157409@qq.com.
authors.forename: Xilong
authors.initials: X
authors.lastname: Kang
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, Jiangsu 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, China. Electronic address: chenxiang@yzu.edu.cn.
authors.forename: Xiang
authors.initials: X
authors.lastname: Chen
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, Jiangsu 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, China. Electronic address: gszhong@yzu.edu.cn.
authors.forename: Shizhong
authors.initials: S
authors.lastname: Geng
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, Jiangsu 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, China. Electronic address: zmpan@yzu.edu.cn.
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Pan
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, Jiangsu 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, China. Electronic address: jiao@yzu.edu.cn.
authors.forename: Xinan
authors.initials: X
authors.lastname: Jiao
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D010856:Columbidae / Q000235:genetics* / Q000276:immunology*; D004252:DNA Mutational Analysis; D057809:HEK293 Cells; D006801:Humans; D007113:Immunity, Innate / Q000235:genetics; D007371:Interferon-gamma / Q000235:genetics; D016209:Interleukin-8 / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051199:Toll-Like Receptor 7 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: D030161:Avian Proteins; C508606:CXCL8 protein, human; D016209:Interleukin-8; D012333:RNA, Messenger; D011994:Recombinant Proteins; D051199:Toll-Like Receptor 7; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma
keywords: Innate immunity; LRR insertion; Mutational analysis; Pigeon; TLR7