Gaussia luciferase

Data analysis was performed as described (Liu et al., 2020 (link)). Briefly, raw reads were trimmed with Trimmomatic-0.39 (Bolger et al., 2014 (link)) and then aligned to the mouse genome (mm10) and transcriptome (GENCODE, version M19) together with spike-in genomes including unmodified control RNA (Cypridina Luciferase) and m⁶A methylated control RNA (Gaussia Luciferase) (New England Biolabs) using HISAT (version 2.1.0) (Kim et al., 2015 (link)) with “-k 5 --rna-strandness RF” parameters. Mapped reads were separated by strands using samtools (version 1.9) (Li et al., 2009 (link)) and m⁶A peaks were called using MACS2 (version 2) (Zhang et al., 2008 (link)) with parameter “-g 1.3e8 --tsize 150 --extsize 150 --nomodel --keep-dup 5” for each strand separately. Significant peaks with q < 0.01 identified by MACS2 were considered. Peaks identified in at least two biological replicates were intersected using bedtools (v.2.26.0) (Quinlan and Hall, 2010 (link)) and were used in the following analysis. The number of reads mapped to mouse genome divided by number of reads mapped to m⁶A modified spike-in represented whole m⁶A level.

Unless otherwise noted, protein extracts were prepared in Laemmli buffer. Immunoblot membranes were incubated with primary antibodies directed against DDX19 (NB100-760, Novus Biologicals), A/PR/8/34 virions59 (link), NS1 (kindly provided by Daniel Marc, INRA-Tours, France), M1 (GA2B, Pierce), HA (Genetex), NA (Genetex), GAPDH (Pierce), TPB (Cell Signaling), MEK1/2 (L38C12, Cell Signaling), Gaussia luciferase (New England Biolabs), and revealed with secondary antibodies (GE Healthcare) or peroxidase-conjugated Strep-Tactin (IBA), and with the ECL 2 substrate (Pierce). The chemiluminescence signals were acquired using G-Box and the GeneSnap software (SynGene).

The large extracellular domain of human CD81 (residues 113–201) and the E2 protein (residues 384–661) were fused with the gaussia luciferase (New England BioLabs) and the human IgG1 Fc moieties (Invivogen), respectively, to produce the CD81-Luc and E2-16Fc constructs. These constructs were transiently transfected separately into Huh7 cells which later secreted the resulting fusion proteins into the cell culture medium. The amount of protein in the cell culture supernatant was monitored either by Western blot analysis using a specific antibody to the human Fc tag or by measuring the activity of luciferase with the BioLUX® gaussia luciferase Flex Assay kit (New England BioLabs). The supernatant (100 µL/well) containing either E2-16Fc or its mutant forms was incubated in 96-well microtiter plates coated with protein A (Thermo Scientific) at room temperature for 1 h. After washing with PBS-T, CD81-Luc supernatant (2.5×10⁶ units of luciferase activity in 30 µL), with 1H8 (10 or 50 µg/reaction) or without 1H8, was added to the wells and incubated for 1 h. After rinsing the plates with PBS-T, a 50 µL mixture of the BioLUX kit substrate and buffer was added to the wells, and the bioluminescent signal intensity was immediately measured with an Infinite F500 microplate reader (TECAN, Männedorf, Switzerland).