T7 mscript kit

DNA coding for the wild type CrPV-1A viral suppressor of RNA silencing was amplified from CrPV-1A-TOPO plasmid40 (link) using primers Cr1AT7F and Cr1ARev (Table S4). A mutant version missing 58 amino acids at the N-terminus was generated by PCR amplification of the same DNA with primers Cr1AT7∆N-F and Cr1ARev (Table S4). Final templates were cleaned by phenol extraction and sequenced. 5′ capped and 3′ polyadenylated in vitro transcripts were prepared using T7 mScript kit (CellScript) followed by phenol extraction as recommended by the manufacturer. Negative controls of non-infective RNA (NIR, Fig. 3B) were in vitro transcripts synthesized from an aphid lethal paralysis virus (ALPV) amplicon and IAPV cloned into the pJazz vector (Lucigen) (Supplementary Fig. S4 and Supplementary Information). The IAPV clone included an A25 sequence at the 3′ end resulting from a reverse primer that included a T25 sequence (Table S2).The resulting IAPV transcript was capped at the 5′ end and the 3′ A25 tail was extended with T7 mScript kit (CellScript).

Plasmids for IVT were designed using the full-length nucleotide sequences of RSV F and RSV G from RSV A2 (GenBank M74568.1). The coding region was followed by a 3′ untranslated region derived from the mouse alpha globin sequence. Sequences were codon optimized and inserted in a pMA-7 vector (Thermo Fisher Scientific, GeneArt) to be used as a template for mRNA synthesis. Plasmids were linearized with Not-I HF (New England Biolabs) overnight prior to IVT using a T7 mScript kit (Cellscript) following the manufacturer’s instructions. ATP, GTP, and CTP were used alongside m1Y-5′-triphosphate (TriLink). RNAs were capped using 2′-O-Methytransferase followed by enzymatic addition of a poly-A tail, both according to the mScript kit instructions. The capped and tailed mRNAs were then purified using an RNeasy kit (Qiagen), treated with Antarctic Phosphatase for 2 h (New England Biolaboratories), and purified again. Vero cells were transfected using a Neon electroporation system (Invitrogen) into a 24-well plate were transfected with 1 µg of synthetic mRNA encoding RSV F or RSV G, respectively, according to the manufacturer’s protocol. 20 h post-transfection, cells were stained with SBA-488 at 4 °C immediately before fixation and immunostaining without permeabilization.

Plasmids were linearized with Not-I HF (New England Biolabs) overnight and PCR purified using PCR clean-up kit (Qiagen), prior to in vitro transcription (IVT) using a T7 mScript kit (Cellscript) following the manufacturer’s instructions. Equimolar ratios of ATP, GTP, and CTP were used alongside N1-methylpseudouridine-5’-triphosphate (TriLink). RNAs were capped using 2’-O-Methytransferase followed by enzymatic addition of a poly-A tail, both according to the mScript kit instructions. The capped and tailed mRNAs were then purified using an RNeasy kit (Qiagen) and treated with Antarctic Phosphatase (New England Biolabs) for 1 h (New England Biolaboratories), and purified again. RNA concentration of the purified mRNA was measured the RNA was stored at −80 °C until further use.