Construction of TRIF-knockout NA and BHK-21 cell lines and effect on rabies virus proliferation efficiency
-
摘要:目的
通过CRISPR/Cas9技术构建β干扰素TIR结构域衔接蛋白(TIR-domain-containing adaptor-inducing interferon β,TRIF)基因敲除小鼠NA细胞系与仓鼠BHK-21细胞系,评估狂犬病病毒(Rabies virus,RABV)在敲除细胞系中的增殖能力,探究TRIF在RABV感染过程中的功能。
方法首先,设计单向导RNA(Single guide RNA,sgRNA),构建用于TRIF基因敲除的重组质粒pSpCas9-NA-TRIF-KO和pSpCas9-BHK-21-TRIF-KO,将重组质粒转染野生型NA与BHK-21细胞,通过嘌呤霉素初步筛选阳性细胞,并使用有限稀释法获得单细胞亚克隆。随后,对筛选的细胞扩大培养,并通过PCR扩增、测序以及Western blot鉴定,最终筛选TRIF敲除NA与BHK-21细胞系。最后,将RABV标准攻击毒株CVS-11与疫苗毒株BNSP-SAD分别接种TRIF敲除细胞系与野生型细胞系,通过Western blot检测病毒N蛋白的表达水平,并利用TCID50检测病毒滴度,以评估RABV在不同细胞系中的增殖能力。
结果成功构建了用于TRIF基因敲除的重组质粒pSpCas9-NA-TRIF-KO与pSpCas9-BHK-21-TRIF-KO,并筛选鉴定出TRIF基因敲除NA细胞系与BHK-21细胞系。与野生型细胞系相比,2种TRIF敲除细胞系接种RABV CVS-11与BNSP-SAD后,N蛋白的表达量均在感染24 h后明显升高,病毒滴度同样均在感染24 h后显著上升。
结论相较于野生型细胞系,RABV在TRIF基因敲除细胞系中的增殖能力更优,TRIF可能具有抑制病毒复制的能力。研究结果为深入研究TRIF功能提供了有力支持,也为RABV的培养与研究提供了新的细胞模型。
-
关键词:
- CRISPR/Cas9 /
- β干扰素TIR结构域衔接蛋白基因 /
- 基因敲除 /
- 狂犬病病毒
Abstract:ObjectiveTo construct TIR-domain-containing adaptor-inducing interferon β (TRIF) gene knockout mouse NA cell line and hamster BHK-21 cell line using CRISPR/Cas9 technology, evaluate the proliferative capacity of rabies virus (RABV) in these knockout cell lines, and investigate TRIF function during RABV infection.
MethodFirst, single guide RNA (sgRNA) was designed, and recombinant plasmids pSpCas9-NA-TRIF-KO and pSpCas9-BHK-21-TRIF-KO were constructed for TRIF gene knockout. After plasmid transfection, positive cells were preliminarily screened using puromycin followed by single-cell subclone isolation through limited dilution. The expanded cell populations were subsequently validated by PCR amplification, sequencing, and Western blot identification, TRIF-knockout NA and BHK-21 cell lines were established. Standard challenge strain CVS-11 and vaccine strain BNSP-SAD of RABV were then inoculated into both knockout and wild-type cells. Viral N protein expression levels were detected by Western blot, and viral titers were determined through TCID50 assay to evaluate rabies virus proliferation efficiency in different cell lines.
ResultThe recombinant plasmids pSpCas9-NA-TRIF-KO and pSpCas9-BHK-21-TRIF-KO for TRIF-knockout were successfully constructed, the TRIF-knockout NA and BHK-21 cell lines were screened and identified. Compared with the wild-type cell line, the expressions of N proteins in two TRIF-knockout cell lines inoculated with RABV CVS-11 and BNSP-SAD were obviously elevated after 24 h infection, and the virus titer significantly increased after 24 h infection.
ConclusionRABV has superior proliferation capacity in TRIF-knockout cell lines compared to wild-type counterparts, and TRIF may have the ability to inhibit viral replication. The findings not only provide substantial support for further investigation of TRIF function, but also establish novel cellular models for RABV cultivation and research.
-
表 1 单向导RNA序列
Table 1 Sequence of single guide RNA
单向导RNA
Single guide RNA上游引物序列(5′→3′)
Sequence of forward primer下游引物序列(5′→3′)
Sequence of reverse primersgRNA-1(NA-TRIF-KO) CACCggccccgtcaggtaccccga AAACtcggggtacctgacggggcc sgRNA-2(NA-TRIF-KO) CACCGagcttctcccgaatacgta AAACtacgtattcgggagaagctC sgRNA-3(NA-TRIF-KO) CACCGacacgaaattagcgttccag AAACctggaacgctaatttcgtgtC sgRNA-1(BHK-21-TRIF-KO) CACCGtagcttctcccggatacgta AAACtacgtatccgggagaagctaC sgRNA-2(BHK-21-TRIF-KO) CACCgagcacgtggccctacgtatc AAACgatacgtagggccacgtgctc sgRNA-3(BHK-21-TRIF-KO) CACCgccaggcacaccggatacgc AAACgcgtatccggtgtgcctggc 表 2 鉴定测序引物
Table 2 Primers used for identification and sequencing
基因
Gene上游引物序列(5′→3′)
Sequence of forward primer下游引物序列(5′→3′)
Sequence of reverse primerNA-TRIF(NM_174989.5) ATGTAACACACCGCTGGACA TCTGCTCCTTGAGGGTTCTG BHK-21-TRIF(XP_040595166.1) GGCCACCTTCTGTGAGGAAT GCTGAACCATCTGGGCATGA -
[1] AKIRA S, UEMATSU S, TAKEUCHI O. Pathogen recognition and innate immunity[J]. Cell, 2006, 12(4): 783-801.
[2] AKIRA S. Toll-like receptor signaling[J]. Journal of Biological Chemistry, 2003, 278(40): 38105-38108. doi: 10.1074/jbc.R300028200
[3] FANG F, OOKA K, SUN X Y, et al. A synthetic TLR3 ligand mitigates profibrotic fibroblast responses by inducing autocrine IFN signaling[J]. The Journal of Immunology, 2013, 191(6): 2956-2966. doi: 10.4049/jimmunol.1300376
[4] CHEN C Y, SHIH Y C, HUNG Y F, et al. Beyond defense: Regulation of neuronal morphogenesis and brain functions via Toll-like receptors[J]. Journal of Biomedical Science, 2019, 26(1): 90. doi: 10.1186/s12929-019-0584-z.
[5] BUGGE M, BERGSTROM B, EIDE O K, et al. Surface Toll-like receptor 3 expression in metastatic intestinal epithelial cells induces inflammatory cytokine production and promotes invasiveness[J]. Journal of Biological Chemistry, 2017, 292(37): 15408-15425. doi: 10.1074/jbc.M117.784090
[6] OSHIUMI H, MATSUMOTO M, FUNAMI K, et al. TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-β induction[J]. Nature Immunology, 2003, 4(2): 161-167. doi: 10.1038/ni886
[7] YAMAMOTO M, SATO S, MORI K, et al. Cutting edge: A novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-β promoter in the Toll-like receptor signaling[J]. Journal of Immunology, 2002, 169(12): 6668-6672. doi: 10.4049/jimmunol.169.12.6668
[8] MAHITA J, SOWDHAMINI R. Integrative modelling of TIR domain-containing adaptor molecule inducing interferon-β (TRIF) provides insights into its autoinhibited state[J]. Biology Direct, 2017, 12(1): 9. doi: 10.1186/s13062-017-0179-0.
[9] JIANG Z F, MAK T W, SEN G, et al. Toll-like receptor 3-mediated activation of NF-κB and IRF3 diverges at Toll-IL-1 receptor domain-containing adapter inducing IFN-β[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(10): 3533-3538.
[10] NATH P, JENA K K, MEHTO S, et al. IRGM links autoimmunity to autophagy[J]. Autophagy, 2021, 17(2): 578-580. doi: 10.1080/15548627.2020.1810920
[11] KAISER W J, OFFERMANN M K. Apoptosis induced by the toll-like receptor adaptor TRIF is dependent on its receptor interacting protein homotypic interaction motif[J]. Journal of Immunology, 2005, 174(8): 4942-4952. doi: 10.4049/jimmunol.174.8.4942
[12] CHEN Y J, LIN J H, ZHAO Y, et al. Toll-like receptor 3 (TLR3) regulation mechanisms and roles in antiviral innate immune responses[J]. Journal of Zhejiang University: Science B, 2021, 22(8): 609-632. doi: 10.1631/jzus.B2000808
[13] GHITA L, BREITKOPF V, MULENGE F, et al. Sequential MAVS and MyD88/TRIF signaling triggers anti-viral responses of tick-borne encephalitis virus-infected murine astrocytes[J]. Journal of Neuroscience Research, 2021, 99(10): 2478-2492.
[14] ZENG Q, LIU J Q, LI Z Y, et al. Japanese encephalitis virus NS4B inhibits interferon beta production by targeting TLR3 and TRIF[J]. Veterinary Microbiology, 2023, 284: 109849. doi: 10.1016/j.vetmic.2023.109849.
[15] SHINYA K, ITO M, MAKINO A, et al. The TLR4-TRIF pathway protects against H5N1 influenza virus infection[J]. Journal of Virology, 2012, 86(1): 19-24. doi: 10.1128/JVI.06168-11
[16] 肖宇, 吴凡, 张宝石, 等. 狂犬病病毒强、弱毒株糖蛋白调节Ⅰ型干扰素作用的差异分析[J]. 华南农业大学学报, 2024, 45(2): 190-198. doi: 10.7671/j.issn.1001-411X.202211015 [17] DU PONT V, PLEMPER R K, SCHNELL M J. Status of antiviral therapeutics against rabies virus and related emerging lyssaviruses[J]. Current Opinion in Virology, 2019, 35: 1-13. doi: 10.1016/j.coviro.2018.12.009
[18] MA Y W, ZHANG L F, HUANG X X. Genome modification by CRISPR/Cas9[J]. The FEBS Journal, 2014, 281(23): 5186-5193. doi: 10.1111/febs.13110
[19] 张雅玲, 王锌和, 李构思, 等. 新型DNA碱基编辑器的研究进展[J]. 华南农业大学学报, 2022, 43(6): 1-16. doi: 10.7671/j.issn.1001-411X.202208053 [20] DOUDNA J A, CHARPENTIER E. The new frontier of genome engineering with CRISPR-Cas9[J]. Science, 2014, 346(6213): 1258096. doi: 10.1126/science.1258096.
[21] MARCEAU C D, PUSCHNIK A S, MAJZOUB K, et al. Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens[J]. Nature, 2016, 535(7610): 159-163.
[22] HAN J, PEREZ J T, CHEN C, et al. Genome-wide CRISPR/Cas9 screen identifies host factors essential for influenza virus replication[J]. Cell Reports, 2018, 23(2): 596-607. doi: 10.1016/j.celrep.2018.03.045
[23] 郑艳虹, 吴梓琦, 刘谕儒, 等. 狂犬病病毒的起源、传播及遗传演化[J]. 畜牧兽医学报, 2025, 56(6): 2613-2625. [24] ULLAH M O, SWEET M J, MANSELL A, et al. TRIF-dependent TLR signaling, its functions in host defense and inflammation, and its potential as a therapeutic target[J]. Journal of Leukocyte Biology, 2016, 100(1): 27-45. doi: 10.1189/jlb.2RI1115-531R
[25] ULLAH M O, VE T, MANGAN M, et al. The TLR signalling adaptor TRIF/TICAM-1 has an N-terminal helical domain with structural similarity to IFIT proteins[J]. Acta Crystallographica Section D, 2013, 69(12): 2420-2430. doi: 10.1107/S0907444913022385
[26] SATO S, SUGIYAMA M, YAMAMOTO M, et al. Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) associates with TNF receptor-associated factor 6 and TANK-binding kinase 1, and activates two distinct transcription factors, NF-κB and IFN-regulatory factor-3, in the Toll-like receptor signaling[J]. Journal of Immunology, 2003, 171(8): 4304-4310. doi: 10.4049/jimmunol.171.8.4304
[27] BAKER M O D G, SHANMUGAM N, PHAM C L L, et al. The RHIM of the immune adaptor protein TRIF forms hybrid amyloids with other necroptosis-associated proteins[J]. Molecules, 2022, 27(11): 3382. doi: 10.3390/molecules27113382.