Cells and viruses

Huh7 and A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco–Invitrogen) with 10% fatal bovine serum (FBS) (Gibco–Invitrogen) and 1% penicillin/streptomycin (Thermo Fisher). All cells were cultured at 37 °C in an incubator containing 5% CO2. The Huh7 and A549 cell lines were obtained from our laboratory.

The plasmid of ZIKV Puerto Rico (PRVABC59) strain cDNA was provided by Dr. Ren Sun from the University of California, Los Angeles, USA, and the virus particles were prepared by reverse genetic system. SFTSV was kindly provided by Professor Xuejie Yu (School of Health Sciences, Wuhan University, China).

Reagents and antiboties

The linear form of the tick salivary peptide HIDfsin2 was synthesized by Nanjing Yuanpeptide Biotech Co., Ltd. (Nanjing, China). ST2345 was synthesized by GL Biochem Co., Ltd. (Shanghai, China). IRAK1/4 inhibitor I (HY-13329) and SB203580 (HY-10256) were purchased from MedChemExpress (MCE) and dissolved in 100% dimethyl sulfoxide (DMSO). Lipopolysaccharide (LPS) was purchased from Beyotime (S1732). Cell Counting Kit (CCK-8) was purchased from Yeasen (40203ES60). The P-p38 MAPK antibody (4511), p38 MAPK antibody (9212) and P-MKK3/6 antibody (12280) were ordered from the Cell Signaling Technology (CST). The MKK3/6 (A19830) antibody and HA (AE008) antibody were purchased from ABclonal Technology. The GAPDH antibody (60004-1-lg) and β-tubulin (10094-1-AP) were the products of Proteintech (Rosemont, USA). The flag antibody (F1084) was purchased from Sigma. SFTSV NP rabbit polyclonal antibody was customized by Atagenix Biological Technology Co., Ltd. (Wuhan, China).

Cell viability assay

The cytotoxicity of the compounds to A549 and Huh7 cells was evaluated using cell CCK8. 5 × 104 cell suspension was seeded to the 96-well plates for 24 h, and then different concentrations of HIDfsin2 were added to the 96-well plates. After incubation for 72 h, the medium was discarded and 100 μL fresh DMEM medium containing 10 μL CCK8 solution was added to each well for 1.5 h. The absorbance of each well was measured at 450 nm using a BioTek microplate reader (BioTek, Winooski, VT, USA).

Quantitative real-time PCR (qRT-PCR)

According to the manufacturer’s instructions, total RNA was extracted from cells using RNAiso Plus reagent (Takara, Japan). Then, 1 μg of total RNA was reversed to cDNA by HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The target gene amplification system contained 10 μL of 2 × ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China), 0.4 μL of upstream and downstream primers and 1 μL of cDNA template, and 8.2 μL ddH2O up to 20 μL. The qRT-PCR primers used for SFTSV were 5′-ATGTCAGAGTGGTCCAGGA-3′(upstream) and 5′-TCTCCACCTGTCTCCTTCAG-3′ (downstream). The qRT-PCR primers used for ZIKV were 5′-TTGTGGAAGGTATGTCAGGTG-3′ (upstream) and 5′-ATCTTACCTCCGCCATGTTG-3′ (downstream). The qRT-PCR primers used for GAPDH were 5′-TGATGACATCAAGAAGGTGGTGAAG-3′ (upstream) and 5′-TCCTTGGAGG CCATGTGGGCCAT-3′ (downstream). The copy number of genes was determined by a 7500 real-time PCR system (Applied Biosystems, USA). The experimental results were analyzed by the comparative method (ΔΔCT). Experiments were performed in triplicate and data were presented as mean ± standard deviations (SD).

Western blotting

1% sodium dodecyl sulfate (SDS) with protease and phosphatase inhibitors (Topscience, Shanghai, China) was used to lyse cells. The concentration of total protein was quantified with BCA protein quantification kit (Vazyme, Nanjing, China). Samples containing 20 μg of total protein were separated by 10% or 12% SDS-PAGE. Proteins were transferred to nitrocellulose (NC) membrane, and the membrane was then blocked with 5% skim milk for 2 h at room temperature. Then primary antibodies were added and the membrane were incubated overnight at 4 °C. Subsequently, the membrane was incubated with 3% skim milk containing HRP-conjugated secondary antibodies at room temperature for 2 h. Finally, the results were analyzed by Clarity™ Western ECL Substrates (#1705060, Bio-Rad) with FuJi medical X-ray film.

CRISPR/Cas9

p38α was knocked down in A549 cells using CRISPR/Cas9 technology. sgRNA was designed by the online website of Zhang Feng’s laboratory (https://zlab.bio/guide-design-resources), and then the synthesized sgRNA was ligated to the pGL3-U6-sgRNA-PGK-puromycin plasmid. Subsequently, the constructed pGL3-U6-sgRNA-PGK-puromycin-p38α and Cas9 plasmids were co-transfected into A549 cells. After 24 h, fresh DMEM medium was replaced and the final concentrations of 1 μg/μL puromycin and 15 μg/μL blasticidin were added for screening, and the screening lasted for 5–7 days. After that, the cells were collected and the knocked-down effect of p38α was detected by Western blotting.

Oxidation and purification of peptide

The synthesized linear HIDfsin2 peptide was dissolved in 0.1 M Tris–HCl (pH = 7.8) and cyclized for 48 h at 25 °C in a shaker (80–100 rpm). Then the reduced and oxidized forms of HIDfsin2 were separated and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a C18 column (Elite HPLC, Dalian, China, 10 × 250 mm, 5 µm, 300 A). The elution conditions were as follows: the flow rate was 4 mL/min, the mobile phase was 90% acetonitrile + 0.1% trifluoroacetic acid, eluted with 5 ~ 95% linear gradient within 60 min, and the detection was performed at 230 nm. The target peptide was collected and lyophilized for preservation. The molecular weight of the target peptide was detected by matrix-assisted laser desorption lonization–time of flight mass spectrometer (MALDI–TOF).

Plasmids and cloning

The plasmid with a full-length p38α cDNA was gifted by Prof. Jiahuai Han (Xiamen University, China). Then, p38α cDNA was cloned into pCS2 + plasmid expressing 3 × Flag. The primer sequences for p38αAA and p38αDD were as follows: 5′-ACAGATGATGAAATGGCAGGCGCCGTGGCCAC TAGGTGG-3′ (upstream) and 5′-CCACCTAGTGGCCACGGCGCCTGCCATTTCATCATCTGT-3′ (downstream) for p38αAA; 5′-ACAGATGATGAAATGGACGGCGACGTGGCCACTAGGTGG-3′ (upstream) and 5′-CCACCTAGTGGCCACGTCGCCGTCCATTTCATCATCTGT-3′ (downstream) for p38αDD.

Statistical analysis

All data and graphics were accomplished and analyzed by GraphPad Prism 8, Adobe Photoshop CS6, and UniProt website. Data represent at least three repeated independent experiments, and all experimental results are presented as means ± SD. Statistical significance was analyzed using a two-tailed unpaired Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; and ns, no significance).