Fig 1.
The 5’UTR of gs11 downregulates NSP5 expression.
A) Schematic representation of the genetic constructs used. B) Scheme of the different gs11 constructs with one or both UTRs deleted. C) 5’UTR sequences of wild-type gs11 and 5’ deletion mutant (gs11-Δ5’) and of the corresponding variants with mutations on the Kozak sequence (gs11-5’m and gs11-Δ5’m); start codon in bold. D-F) Anti-NSP5 WB of MA104 cells (D, E) or HeLa cells (F) transfected with the indicated constructs. G, H) Anti-EGFP WB of MA104 cells transfected with constructs containing the EGFP ORF with or without gs11 5’ and 3’ UTRs (G) and with a construct (EGFP-5’m3’ gs11) with mutated Kozak sequence (H). Tubulin used as loading control.
Fig 2.
A) Scheme of the constructs used. B-E) WB analysis with the indicated antibodies of MA104 cells transfected with: gs8 constructs with and without 5’ and 3’ UTRs (B); chimeric constructs with 5’UTR from gs8 and NSP5 ORF (C); gs2 constructs with or without 5’UTR (D); constructs with EGFP ORF downstream of the 5’UTR of either gs11 or gs2 (E). Tubulin used as loading control.
Fig 3.
Inhibition depends on 5’UTR primary sequence.
A) Predicted structures of gs11 5’UTR and mutants. B) Anti-NSP5 WB of MA104 cells transfected with the different mutant constructs shown in A; gs11-Δ5’ was included as a positive control. Tubulin used as loading control.
Fig 4.
A 6-nucleotide long motif on 5’UTR.
A) Alignment of 5’UTRs from gs11, gs8 and gs2. The 11-nucleotide long U-A rich motif is highlighted (bold). The different nucleotide in position 5 in gs2 is shown in red. B) Anti-VP2 WB of MA104 cells transfected with the indicated gs2 constructs. C) Right panel: anti-NSP5 WB of cells transfected with the mutant constructs shown on left panel. D) Anti-NSP5 WB of cells transfected with the indicated gs11 U-to-A mutants shown on left panel. E) Anti-NSP5 WB of cells transfected with constructs containing the 5’ terminal nucleotides of the U-A rich motif and with mutants of the pyrimidine tract. Tubulin used as loading control.
Fig 5.
IM 5’ terminal position is essential.
A) Schematic representation of constructs with the 11-nucleotide long U-A rich motif (containing IM) positioned within gs11 5’UTR (construct 5’-dIM), in EGFP ORF (construct IMORF) or in gs11 3’UTR (construct 3’IM). Within the 5’UTR the motif was placed in a non 5’-terminal position, downstream of a 15-nucleotide long not inhibitory sequence; as a control, a construct with the same sequence without the U-A rich motif was used (5’ctrl). In the construct with the motif within the ORF (IMORF), the amino acids encoded by the inserted motif and by the U5A control mutant [IM(A5)ORF] are shown. Within the 3’UTR, the motif or the U5A control mutant [3’IM(A5)] were positioned six nucleotides downstream of the stop-codon. B) Anti-EGFP WB of extracts from cells transfected with the indicated constructs. The EGFP construct with only gs11 3’UTR was used as a negative control while construct EGFP-5’3’gs11 was used as a positive control. C) Anti-NSP5 WB of cells transfected with a gs11 mutant construct with an additional 5’-terminal G (gs11-G3). Tubulin used as loading control.
Fig 6.
Compromised expression is due to T7-mediated transcription and translation.
Yield of transcripts and protein products of constructs containing EGFP ORF flanked by gs11 UTRs in wild-type and the U5A version. A) Comparison of mRNAs yield after in vitro transcription with T7 polymerase in transcription buffer. B) qPCR of the same constructs transfected in MA104 cells. C) WB of EGFP protein produced following in vitro coupled transcription/translation in a HeLa cell lysate-based kit. D) mRNA yields (by qPCR) from the same in vitro coupled transcription/translation HeLa cell lysate-based kit. E) Decay rate of transcripts in MA104 cells, quantified by qPCR. F) Anti-EGFP WB of samples obtained following in vitro translation (in the HeLa cell lysate-based kit) of mRNAs pre-synthesized as in A. G) Anti-EGFP WB of MA104 cells electroporated with mRNAs pre-synthesized as in A. Tubulin used as loading control.
Fig 7.
The UTRs of SA11 strain genome segments.
Anti-EGFP WB of cells transfected with constructs containing the EGFP ORF fused to the 5’UTR of the different gs indicated on the left. Tubulin used as loading control.
Fig 8.
Rotavirus partially reverts IM-mediated expression inhibition.
A) Anti-EGFP and anti-NSP5 WB of MA104 cells transfected with constructs containing the NSP5-EGFP (NE) ORF with or without gs11 5’ and 3’ UTRs, as indicated. B) Anti-EGFP and anti-NSP5 WB of MA104 cells infected with SA11 and the recombinant vaccinia virus vT7-NE expressing NSP5-EGFP from an mRNA with gs11 5’ and 3’ UTRs. α-actinin as loading control.