TABLE 1.

Functional assessment of PA-binding mutants of PB1^a

Strain or mutant	% PA binding (two-hybrid)b	P5 polymerase activity (CAT) (%)b	PB1 mutant virus yield (PFU/ml)	Plaque purified and sequenced
WT	100	100	4.0 × 107	+
D2V	53 ± 7	0	0	−
V3D	21 ± 8	0	0	−
N4D	22 ± 5	24 ± 1	4.0 × 104	+
P5Lc		NDd	1.0 × 103	+
T6D	75 ± 5	56 ± 4	5.1 × 106	+
L7De	0	21 ± 2	2.0 × 103e	+e
L8D	0	0		−
F9D	0	3 ± 1	0	−
L10De	0	15 ± 5	2.0 × 103e	+e
K11D	35 ± 7	48 ± 2	3.0 × 103	+
V12D	40 ± 5	54 ± 7	4.5 × 103	+
P13D	100 ± 2	78 ± 2	1.9 × 105	+
A14D	100 ± 2	0	0	−

^aSingle amino acid substitutions were introduced within the first 14 N-terminal residues of PB1, and the effect of these mutations was tested in two functional assays: the influenza virus RG system (38), and the rescue of mutant influenza virus (23). The polymerase activity of each mutant is expressed as a percentage relative to the WT PB1 protein in the P5 RG system. Rescued PB1 mutant viruses were plaque purified and stocks were prepared. Sequence analysis was performed with three independent plaques from each mutant (except L7D and L10D, from which 10 plaques were used in sequence analysis). Plaque-purified mutant viruses were propagated on MDCK cells for 2 days (P5L mutant virus was grown for 3 days), and their yields (in PFU per milliliter) were analyzed by plaque assay (two independent experiments). 

^bMeans ± standard deviations. 

^cMutant P5L polymerase activity was demonstrated using a green fluorescent protein RG (not shown). 

^dND, not determined. 

^eMutants L7D and L10D were propagated on MDCK cells for 6 days before analysis of infectious virus yield by plaque assay.