1 10-1 10-2 10-3 1 10-1 10-2 10-3

WT PB1a

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 + - + -+ -

0 0 1 1 2 2

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.

+ ++ +

+ -+ -

4 4 6 6h pi

PB1a, PB2, PA 1 2 3 30 0.5

NP 1 1 1 00 1

1 2 3 4 5 6

mRNA

cRNA

vRNA

PB1a, PB2, PA 1 1 1 10 0

NP 1 2 4 00 4

1 2 3 4 5 6

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

1 2 3

1 10-1 10-2 10-3 1 10-1 10-2 10-3

WT PB1-Y559A

WT

-pol

ymer

ase

PB

1-Y

559A

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 (α32P-GTP-labelled) is

shown.

PB2, PA, NP 1 11 1

PB1ar 2 40 1

PB1a 0 01 0

1 2 3 4

1 0

0 0

0 0

5 6

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.