Wheat germ extract

The CFA packaging assay was carried out as described previously (Lourenco & Roy, 2011 (link)). Briefly, VP1, VP4, VP6, VP3 and VP7 were in vitro translated sequentially using wheatgerm extract (Promega) and mixed with the 10 BTV uncapped ssRNAs to form viral cores. The whole mixture was then fractionated through a continuous sucrose gradient and tested for RNA content. In relevant fractions, unpackaged RNAs were eliminated by RNase One (Promega) digestion. Packaged ssRNAs were detected by reverse transcription PCR using primers for S4 of BTV-9 as described above (for Fig. 5) or S4 of BTV-9 5′ and 3′ end primers: SBF1-T7-S4-BTV9, 5′-AAAACCTGCAGGTAATACGACTCACTATAGTTAAAACATGCCTGAGCCAC-3′; S4-BTV9-terminus, 5′-GTAAGTTTTACATGCCCCCC-3′ (for Fig. 7). Primers for S8 of BTV-1 were: for reverse transcription reaction of S8, B1S8R 5′-GTAAGTGTAAAATCCCCC-3′; for detection of S8, B1S8F 5′-GTTAAAAAATCCTTGAGTCATGGAG-3′; B1S8 627R, 5′-CAGCTTCTCCAATCTGCTGG-3′.

Reference fragments of B. subtilis MgtE encompassing the first 300, 315, 330, 340, 355 and 370 residues of MgtE were generated by PCR amplification of the respective coding regions from B. subtilis 168 genomic DNA, and in vitro‐transcribed and in vitro‐translated as described previously (Lemberg & Martoglio, 2003; Strisovsky et al, 2009). Briefly, PCR was performed using set of mgtE‐specific primers containing SP6 RNA polymerase promoter and ribosome‐binding site in the forward primer, and stop codon in the reverse primer. Messenger RNAs were purified by LiCl precipitation and translated using Wheat Germ Extract (Promega). The resulting crude translation mixtures containing the MgtE reference fragments were separated using SDS–PAGE and visualised by polyclonal anti‐MgtE(2–275) antibody and immunoblotting.

Plasmids containing full-length CYVaV (pUC19-CYVaV) or PEMV2 (pUC19-PEMV2) were linearized with Hind III or SmaI, respectively, and served as templates for RNA transcription using T7 RNA polymerase. Quantification of the synthesized RNA was performed using a DeNovix DS-II FX spectrophotometer. For in vitro translation, 10 μL translation mixtures contained 5 μL wheat germ extract (Promega), 0.5 pmol RNA template, 0.8 μL 1 mM amino acids mix (without methione), 100 mM potassium acetate and 0.5 μL (5 μCi) ³⁵S-methionine. The translation mixture was incubated at 25 °C for 45 min and then resolved on a 10% SDS-PAGE gel. The gel was dried and exposed to Fuji phosphorimager screen for 3 h. The screen was subsequently scanned by an Amersham Typhoon fluorescent image analyzer. The intensity of radioactive bands was quantified using ImageQuant TL 8.1 (GE Lifesciences). All experiments were repeated at least three times.

A total of 0.5 to 2 μg of purified RNA were translated using wheat germ extract (Promega) at 25°C for 90 min in a final reaction volume of 12.5 μl according to the manufacturer’s instructions. Protein output was analyzed via Western blotting as described above.

In vitro translation was carried out using increasing concentrations of mRNA transcripts with self-made untreated RRL, amino acid mixture containing all the amino acids except methionine (1 mM of each), RNasin (Promega ), 75 mM KCl, 0.5 mM MgCl₂, 3.8 mCi [³⁵S] methionine, and autoclaved milli-Q water. Reaction mixture was incubated at 30°C for 1 hr. Aliquots of translation mixture were analysed by SDS-PAGE (10%) (Laemmli, 1970 (link)) and translation products were visualized by phosphor imaging. In vitro translation assays with wheat germ extract (Promega) were performed according to manufacturer’s instructions. In vitro translation assays with HeLa cell extract and Drosophila S2 cell extract were performed as previously described (Thoma et al., 2004 (link); Wakiyama et al., 2006 (link)).