. 2009 Feb 18;83(9):4153–4162. doi: 10.1128/JVI.02642-08

TABLE 1.

Phenotypic analyses of mutant NP proteins

NP^a	Virus rescue^b	Mean virus titer at 48 h postinfection (log₁₀ PFU/ml) ±SD^c		Group^d	Relative luciferase activity at 48 h posttransfection (%)^e
NP^a	Virus rescue^b	33°C	37°C	Group^d	33°C	37°C
Wild type	+	7.99 ± 0.01	8.85 ± 0.07	I	100.00 ± 6.88	100.00 ± 13.51
R19A	+	7.28 ± 0.02	9.00 ± 0.00	I	94.68 ± 4.14	88.26 ± 1.30
E24A	+	6.65 ± 0.05	8.67 ± 0.05	I	131.38 ± 14.44	95.84 ± 4.20
Y40A	+	7.98 ± 0.05	8.41 ± 0.01	I	91.83 ± 4.06	81.73 ± 5.45
L49A	+	8.12 ± 0.01	8.54 ± 0.07	I	151.00 ± 7.27	142.78 ± 8.76
R65A	+	6.29 ± 0.07	8.49 ± 0.02	I	138.46 ± 3.43	108.60 ± 17.82
R98A	+	8.30 ± 0.01	8.55 ± 0.06	I	108.01 ± 1.14	89.65 ± 3.67
R121A	+	6.37 ± 0.20	7.94 ± 0.02	I	144.83 ± 4.15	104.00 ± 2.76
G132A	+	7.62 ± 0.05	9.11 ± 0.02	I	145.46 ± 10.02	106.67 ± 4.86
S141A	+	8.09 ± 0.01	8.87 ± 0.05	I	133.72 ± 2.09	100.26 ± 3.02
Q149A	+	3.65 ± 0.01	7.51 ± 0.16	I	135.63 ± 6.60	102.19 ± 2.52
G169A	+	7.89 ± 0.03	8.43 ± 0.21	I	126.76 ± 2.21	94.92 ± 3.96
K184A	+	6.40 ± 0.08	7.68 ± 0.02	I	145.34 ± 11.35	96.27 ± 5.72
G185A	+	6.58 ± 0.16	7.46 ± 0.06	I	153.55 ± 6.32	95.09 ± 4.86
G187A	+	6.31 ± 0.23	8.43 ± 0.01	I	114.82 ± 5.33	99.31 ± 1.40
R214A	+	7.21 ± 0.01	8.01 ± 0.07	I	122.85 ± 4.95	87.02 ± 14.18
R216A	+	6.32 ± 0.06	6.92 ± 0.04	I	111.38 ± 4.64	85.36 ± 6.68
E220A	+	5.22 ± 0.06	8.57 ± 0.19	I	62.84 ± 3.39	103.46 ± 8.18
K229A	+	6.92 ± 0.01	8.03 ± 0.09	I	113.40 ± 0.68	97.47 ± 5.12
R236A	+	5.07 ± 0.05	7.61 ± 0.00	I	107.59 ± 2.69	90.13 ± 14.06
V242A	+	7.67 ± 0.13	8.15 ± 0.11	I	136.01 ± 4.81	101.33 ± 2.46
L256A	+	6.33 ± 0.10	6.97 ± 0.07	I	143.86 ± 3.42	87.44 ± 1.70
P277A	+	8.31 ± 0.01	8.79 ± 0.03	I	131.35 ± 3.02	98.03 ± 2.96
F291A	+	7.15 ± 0.05	8.41 ± 0.01	I	126.04 ± 3.53	89.44 ± 5.27
G300A	+	5.02 ± 0.06	7.75 ± 0.01	I	100.10 ± 3.52	86.88 ± 3.82
N319A	+	7.75 ± 0.11	8.36 ± 0.05	I	107.45 ± 2.23	81.28 ± 4.23
Q327A	+	6.74 ± 0.09	7.19 ± 0.27	I	125.66 ± 0.88	95.25 ± 7.13
C333A	+	8.06 ± 0.05	8.9 ± 0.01	I	105.19 ± 4.78	99.49 ± 6.63
K357A	+	7.78 ± 0.01	8.7 ± 0.08	I	105.22 ± 0.25	81.12 ± 1.17
E369A	+	7.27 ± 0.21	7.98 ± 0.02	I	114.80 ± 4.74	142.64 ± 1.72
W386A	+	5.44 ± 0.11	7.53 ± 0.05	I	88.39 ± 4.83	125.05 ± 8.59
G460A	+	6.68 ± 0.06	8.88 ± 0.04	I	118.59 ± 17.56	138.93 ± 10.31
P474A	+	6.04 ± 0.03	8.79 ± 0.08	I	115.52 ± 3.98	116.35 ± 3.24
G490A	+	7.63 ± 0.04	8.86 ± 0.13	I	113.78 ± 11.54	104.90 ± 6.71
E495A	+	7.21 ± 0.09	8.3 ± 0.02	I	73.02 ± 1.39	83.08 ± 7.90
R26A	+	1.54 ± 0.09	3.81 ± 0.03	II	79.44 ± 0.71	85.62 ± 1.65
*R74A	+	3.11 ± 0.09	4.42 ± 0.08	II	139.73 ± 9.61	109.25 ± 2.07
*R175A	+	2.91 ± 0.01	4.11 ± 0.05	II	170.32 ± 5.96	108.56 ± 6.54
E192A	+	4.16 ± 0.02	6.03 ± 0.01	II	78.03 ± 3.16	82.87 ± 1.66
R221A	+	4.56 ± 0.03	4.75 ± 0.13	II	111.94 ± 4.57	93.17 ± 4.51
M331A	+	1.00 ± 0.00	4.77 ± 0.1	II	99.45 ± 8.38	83.98 ± 7.56
R391A	+	2.89 ± 0.03	6.57 ± 0.02	II	70.66 ± 2.04	63.56 ± 0.46
S407A	+	1.00 ± 0.00	6.19 ± 0.03	II	5.40 ± 0.39	86.15 ± 0.28
V414A	+	1.69 ± 0.12	6.85 ± 0.06	II	47.29 ± 6.11	92.27 ± 1.14
D468A	+	3.54 ± 0.34	6.67 ± 0.10	II	97.46 ± 2.33	81.45 ± 5.87
D491A	+	5.09 ± 0.07	5.82 ± 0.09	II	52.09 ± 0.67	73.18 ± 4.46
N59A	+	ND	ND	III	159.52 ± 4.80	54.32 ± 1.72
E64A	+	ND	ND	III	193.40 ± 14.30	0.03 ± 0.00
E320A	+	ND	ND	III	145.78 ± 6.37	51.05 ± 1.47
*D72A	−	ND	ND	IV	121.17 ± 1.30	135.07 ± 1.05
G93A	−	ND	ND	IV	131.30 ± 10.53	55.13 ± 2.74
*K113A	−	ND	ND	IV	183.46 ± 12.21	106.16 ± 5.07
Y148A	−	ND	ND	IV	100.87 ± 3.15	82.89 ± 10.15
R150A	−	ND	ND	IV	12.46 ± 0.79	32.17 ± 1.44
R152A	−	ND	ND	IV	100.52 ± 1.78	77.61 ± 14.19
*R156A	−	ND	ND	IV	123.85 ± 6.03	95.24 ± 3.16
*R174A	−	ND	ND	IV	214.89 ± 17.15	100.56 ± 6.91
*R195A	−	ND	ND	IV	160.59 ± 14.23	105.41 ± 5.63
*R199A	−	ND	ND	IV	152.91 ± 10.97	102.41 ± 6.38
R208A	−	ND	ND	IV	0.19 ± 0.01	0.13 ± 0.01
R213A	−	ND	ND	IV	32.42 ± 0.36	38.29 ± 7.36
E254A	−	ND	ND	IV	0.26 ± 0.03	0.37 ± 0.13
A260R	−	ND	ND	IV	0.04 ± 0.00	0.02 ± 0.01
K273A	−	ND	ND	IV	0.19 ± 0.02	0.04 ± 0.01
*K325A	−	ND	ND	IV	157.38 ± 7.37	134.67 ± 12.73
A337R	−	ND	ND	IV	0.16 ± 0.00	0.03 ± 0.00
E339A	−	ND	ND	IV	0.16 ± 0.01	0.03 ± 0.01
R355A	−	ND	ND	IV	52.91 ± 0.47	45.07 ± 1.06
*R361A	−	ND	ND	IV	105.57 ± 0.49	104.60 ± 20.58
A387R	−	ND	ND	IV	0.15 ± 0.00	0.04 ± 0.00
Q405A	−	ND	ND	IV	0.16 ± 0.01	0.05 ± 0.01
F412A	−	ND	ND	IV	0.40 ± 0.32	0.03 ± 0.00
R416A	−	ND	ND	IV	0.21 ± 0.10	0.03 ± 0.00
F488A	−	ND	ND	IV	0.20 ± 0.01	8.05 ± 1.02
F489A	−	ND	ND	IV	0.16 ± 0.01	0.23 ± 0.22

We selected 74 amino acids that are conserved among influenza A, B, and C virus NPs for mutagenesis. NP mutants flagged with an asterisk were further tested for intracellular localization, and incorporation efficiency of a vRNA segment(s) into VLPs, in addition to replicative ability and polymerase activity.

Viruses were generated using an established plasmid-based reverse-genetics system. +, NP mutant virus recovery was verified by plaque assay, cytopathic effect, and NP gene sequencing; −, no replicating virus was recovered.

MDCK cells were infected with wild-type WSN or NP mutant virus at an MOI of 0.0001. At 48 h postinfection, the culture supernatants were harvested and subjected to plaque assays in MDCK cells. ND, plaques were not detected.

Based on virus rescue and viral-replication properties, NP mutants were categorized into four groups. Group I, at either 33°C or 37°C, viruses were attenuated by less than 2 log units; group II, mutant viruses were attenuated by more than 2 log units at both temperatures tested; group III, NP mutant viruses were rescued but did not form plaques at 48 h postinfection; group IV, NP mutant virus were not rescued.

The abilities of mutant NPs to support vRNA replication/transcription in an in vitro assay were examined in 293T cells as described in Materials and Methods. Since luciferase levels reflect the overall transcription and replication activity of the polymerase complex, we defined the polymerase activity levels as high, normal, and low for mutants that exhibited >120%, 80% to 120%, and <80%, respectively, of the wild-type activity.