110 -1 10 -2 10 -3 110 -1 10 -2 10 -3 WTPB1a Acetylated products chloramphenicol SUPPLEMENTARY FIG. 1. RNA polymerase containing the PB1- D445A/D446A mutant subunit (PB1a) is functionally inactive. 293T cells were transfected with pPOLI-vCAT and pcDNA expression vectors for PB1 (wild-type [WT] or mutant [PB1a] as indicated), PB2, PA and NP proteins. Transfections and CAT expression assays, using 10 fold serially diluted transfected cell extracts as indicated, were carried out as described previously (8).
PB2, PA, NP ++++++ PB1a +-+-+- 001122 mRNA cRNA vRNA SUPPLEMENTARY FIG. 2. Cycloheximide-mediated inhibition of influenza A/WSN/33 viral cRNA synthesis can be rescued by expression of PB1, PB2, PA and NP. Time course of viral infection in the presence of 100 μg/ml cycloheximide following prior (12-14 h) transfection of expression plasmids expressing viral proteins as indicated. Viral RNA species were analysed by primer extension with NS gene-specific primers, generating cDNA with an expected size of 157 nucleotides from vRNA, 226 nucleotides from cRNA, and 235 - 243 nucleotides from mRNA depending on the length of the capped primer. mRNA is a broad heterogeneous band 9-17 nucleotides longer than cRNA due to its 5 cap- snatched sequence. The bands appearing above the cRNA band position are background bands of unknown origin. PB1a = PB1-D445A/D446A; h pi = hours post-infection. ++++ +-+- 4466h pi
PB1a, PB2, PA123300.5 NP111001 123456 mRNA cRNA vRNA PB1a, PB2, PA111100 NP124004 123456 mRNA cRNA vRNA A B SUPPLEMENTARY FIG. 3. cRNA signals are not affected by the polymerase concentration (A) but are enhanced by increasing the NP concentration (B). Varying quantities (as shown in μg) of pcDNA expression vectors for PB1-D445A/D446A (abbreviated as PB1a), PB2, PA and NP were transfected into 293T cells prior to influenza A/WSN/33 viral infection in the presence of 100 μg/ml cycloheximide. Viral RNA species were analysed by primer extension with NA gene-specific primers.
14 nt product 123 110 -1 10 -2 10 -3 110 -1 10 -2 10 -3 WTPB1-Y559A WT -polymerase PB1-Y559A A B Acetylated products chloramphenicol SUPPLEMENTARY FIG. 4. RNA polymerase containing the PB1-Y559A mutant subunit is functionally inactive. (A) 293T cells were transfected with pPOLI-vCAT and pcDNA expression vectors for PB1 (wild-type [WT] or mutant [PB1-Y559A] as indicated), PB2, PA and NP proteins. Transfections and CAT expression assays, using 10 fold serially diluted transfected cell extracts as indicated, were carried out as described previously (8). (B) ApG-primed transcription reactions were carried out in vitro on a 14 nucleotide (nt) 3-end vRNA template in the presence of a 15 nucleotide 5-end vRNA as described previously (7). RNA polymerase was partially purified from 293T cells transiently transfected with pcDNA expression plasmids for PB1 (wild-type [WT] or mutant [PB1-Y559A] as indicated), PB2 and PA-His6 (8). Lane 2 is a control lacking PB1, PB2 and PA-His6. The 14 nt long major transcription run-off product (α 32 P-GTP-labelled) is shown.
PB2, PA, NP1111 PB1ar2401 PB1a0010 1234 10 00 00 56 SUPPLEMENTARY FIG. 5. PB1 mutants PB1-D445A/D446A (abbreviated PB1a) and PB1-D445A/D446A/Y559A (abbreviated PB1ar) are expressed at detectable and similar levels in transfected cells, and in proportion to the quantity of transfected plasmid. 293T cells were transfected with varying quantities of pcDNA expression vectors for the polymerase subunits and NP as indicated in μg. Cells were harvested at 12 hours post-transfection and the proteins analysed by western immunoblotting with a polyclonal PB1-specific antibody (Santa Cruz Biotechnology). An arrow shows the specific product.
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