Cell Culture and Virus
Chicken embryonic fibroblast cell line DF1, human 293T cells, and bat TB1 Lu cells were obtained from ATCC and cultured in DMEM supplemented with 10% FBS and cells were incubated at 37°C in a 5% CO2 incubator. Newcastle disease virus (NDV-GFP) was a low virulent strain LaSota named NDV-GFP. Avian influenza virus (AIV) was A/Chicken/Shanghai/010/2008(H9N2) virus (SH010) was isolated from chicken in Shanghai, China, in 2008 and identified as H9N2 avian influenza A virus. The GFP tagged vesicular stomatitis virus (VSV) VSV-GFP were stored in our Laboratory. The viruses were purified, propagated, and stored as described in our previous study[20].
Virus Infection
The TB1Lu cells were plated, washed twice with PBS. Each well was seeded with 1 MOI of NDV-GFP or AIV or VSV-GFP. After 3h, 6h, 12h and 24h of infection, samples were collected for subsequent experiments.
Cloning and Bioinformatics Analysis of Bat IRF7
Based on the Molossus molossus IRF7 sequence (XM_036281692.1) obtained from the National Center for Biotechnology Information (NCBI), the primers batIRF7-F and batIRF7-R (Supplementary Table 1) were designed and used to amplify batIRF7 from TB 1 Lu cells cDNA. The PCR product was ligated into a pTOPO-Blunt vector (Vazyme Biotech co.,ltd) for sequencing, and the positive colonies were sent to the Beijing Genomics Institute (Beijing, China) for sequencing. The amino acid sequence of batIRF7 was aligned with the other animal IRF7 proteins from chicken, ducks, pigs, cattles, dogs, cats, mice, humans, zebrafishes, and salmons using Clustal W and edited with ESPript 3.0 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Sequence homology and phylogenetic analysis of the IRF7 amino acid sequences were conducted using DNASTAR. A phylogenetic tree was constructed based on the IRF7 from 15 different species, including mammals, birds, fish. Different domains in the IRF7 amino acid sequences were predicted using the simple modular architecture research tool (SMART) program (http://smart.embl-heidelberg.de/). Homology modeling for IRF7 was conducted using the online protein-modeling server Swiss Model (https://swissmodel.expasy.org/).
Plasmid construction
pcDNA3.1-bat-IRF7 plasmids were constructed by inserting full- length TbIRF7 into the Hind III, and EcoR I sites pcDNA3.1 of the expression vector using a ClonExpress II one-step cloning kit (Yeasen, Shanghai, China). The primers used in the PCR are listed in Supple-mentary Table 1. The truncated plasmids of bat IRF7, including deleting amino acid 123–223(dAA123-223), amino acid 223–335(dAA223-335), insulin growth factor-binding protein homologues (IB), interferon regulatory factor (IRF), interferon-regulatory factor 3 (IRF-3), and repeat of unknown function (DUF), were con-structed using a modified homologous recombination method and the primers were listed in Supplementary Table 1. The DH5α Chemically Competent Cell (Tsingke Biology Technology, Beijing, China) was used for plasmid transformation. The pGL-IFN-β-Luc plasmid was constructed in our previous study (23).
Cells transfection
293T, DF1and TB1Lu cells were seeded in 12-well or 24-well plates (NEST Biotechnology, Wuxi, China) at 5 × 105/mL or 1 × 106/mL. And the plasmid was transfected 250 ng/well in 24-well or 500 ng/well in 12-well transfected with plasmid were performed with Nulen Plus-Trans™ Transfection Reagent (Nulen, Shanghai, China) according to the manufacturer’s protocol.
Luciferase Reporter Assay
The DF-1, 293T, and TB 1 Lu cells were plated in 24-well plates and were transiently transfected with the reporter plasmid pGL-chIFN-β-Luc or pGL-huIFN-β-Luc or pGL-batIFN-β-Luc (120 ng/well) and the control Renilla luciferase (pRL-TK, 60 ng/well). According to the manufacturer’s instructions, the cells were lysed 24 hours after transfection, and luciferase activity was detected using a Dual-Luciferase Reporter Assay System kit (Promega, Madison,WI). Renilla luciferase activity was used for normalization.
RNA Extraction and Quantitative Real-Time PCR
Cells’ total RNAs were extracted with AG RNAex Pro Reagent (Ac-curate Biology, Hunan, China). mRNA was reverse-transcribed to cDNA using a two-step reverse transcription kit, the first reaction removed genomic DNA by adding gDNA wiper enzyme, and the second step reverse transcribed mRNA to cDNA. The specific operation is carried out according to the instructions provided by the kit (Vazyme, Nanjing, China), and the cDNA was analyzed using the SYBR green PCR mix (Vazyme, Nanjing, China) with the Applied Biosystems machine (ABI 7500; Thermo Fisher Scientific). Relative gene expression was analyzed using the 2−ΔΔCt method. The β-actin was the internal reference when examining the level of genes. The primer sequences for the genes are shown in the Supplementary Table 1.
Western Blot Analysis
The cells’ total proteins were extracted by radioimmunoprecipitation assay (Beyotime, Shanghai, China) containing a protease cocktail (Yeasen, Shanghai, China) and phenylmethylsulfonyl fluoride (PMSF) (Yeasen, Shanghai, China). The lysate was centrifuged at 13,000 rpm for 10 min to obtain the supernatant, and a 5 × SDS loading buffer was added before the lysates were boiled for 10 min. The proteins isolated from the cell lysates were separated via SDS-PAGE and analyzed using Western blot. The antibody included ant-GFP (Yeasen, Shanghai, China) and β-tubulin overnight at 4°C. The membrane was washed 3 times with tris buffered saline andTween-20 (TBST) (Sangon Biotech Co., Ltd, Shanghai, China). Then, the secondary antibody was added for 1 h incubation at 4°C shaker Images were obtained using the Tanon 5200 imaging system (Tanon, Shanghai, China).
Statistical Analysis
Results are expressed as the mean ± SD. GraphPad Prism 8.0 was utilized to graph the results. Data were analyzed by using a two-tailed independent the Student’s t-test. P < 0.05 was considered statistically significant, and P < 0. 0 1 was considered highly statistically significant (*P < 0.05; **P < 0.01).