TABLE 2.
Summary of phenotypes induced by mutations in the PA hinge
| Rescue virus | Phenotypea | Virus titer (PFU/ml) atb: |
Replication kinetics phenotypec | Polymerase activity atd: |
Revertant selection and isolation | Nuclear import of PB1 ate: |
Pathogenicityf | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 33°C | 37°C | 39.5°C | 33°C | 39.5°C | 33°C | 39.5°C | |||||
| PA wt | + | 1.7 ′ 108 | 4.8 ′ 108 | 1.3 ′ 108 | wt | +++ | +++ | ++ | +++ | 3.5 | |
| PA wt FS | + | 9.5 ′ 107 | 4.8 ′ 107 | wt | |||||||
| E198P | − | − | |||||||||
| E198A | + | + | |||||||||
| I201P | − | − | |||||||||
| I201A | ++ | + | |||||||||
| T210P | +/ts | 3.8 ′ 108 | 1.6 ′ 108 | 5 ′ 107 sp | ts++ | ++ | − | + | |||
| T210P FS | +/ts | 2.4 ′ 107 | 4.0 ′ 105 | ts+++ | |||||||
| T210A | + | +++ | + | ||||||||
| K213P | +/ts | 6 ′ 108 | 5 ′ 108 | 107 sp | ts+ | +++ | − | ||||
| K213A | + | +++ | +++ | ||||||||
| L214P | − | 0 | 0 | 0 | − | − | +/− | − | |||
| L214A | + | − | − | ||||||||
| D216P | +/ts | 4.4 ′ 108 | 5 ′ 107 | <103 | ts+++ | + | − | + | ++ | +/− | 5.5 |
| D216P FS | +/ts | 1.0 ′ 108 | 2.4 ′ 105 | ts+++ | |||||||
| D216A | + | +++ | + | ||||||||
| Q217P | + | ||||||||||
| Q217A | + | ||||||||||
| S218P | − | − | − | ||||||||
| S218A | +++ | ++ | |||||||||
| L219P | +/ts | 4 ′ 108 sp | 4 ′ 108 sp | 1.2 ′ 104 sp | ts+++ | − | − | + | ++ | +/− | >6 |
| L219A | +/ts | ||||||||||
| P220A | − | − | − | ||||||||
| P221A | +/ts | ||||||||||
| N222P | +/ts | ||||||||||
| N222A | + | ||||||||||
| F223P | +/ts | 6 ′ 108 | 8 ′ 107 | <103 | ts+++ | + | − | + | ++ | − | |
| F223A | + | +++ | + | ||||||||
| S224P | + | ||||||||||
| S225P | +/ts | ||||||||||
| L226P | +/ts | 6 ′ 108 | 1.6 ′ 108 | 1.2 ′ 105 | ts+ | + | − | ||||
| L226A | + | +++ | − | ||||||||
| E227P | + | 5 ′ 108 | 4 ′ 108 | 1.2 ′ 108 sp | wt | ++ | ++ | ||||
| E227A | + | +++ | ++ | ||||||||
| Y232P | − | − | |||||||||
| Y232A | +++ | + | |||||||||
| E237P | − | ||||||||||
| E237A | + | ||||||||||
| M249P | − | − | |||||||||
| M249A | + | − | |||||||||
| T210P + D216P | +/ts | 5.0 ′ 107 | 2.0 ′ 105 | ts++ | |||||||
+, the virus demonstrated efficient rescue; -, efficient rescue did not occur; ts, the virus had the temperature-sensitive phenotype.
Titers followed by the notation “sp” signify low titers.
Replication kinetics symbols: ts+++, no virus amplification at 39.5°C; ts++, a latency phase was observed at 39.5°C; ts+, a delay in virus replication compared to that of the wild-type virus was observed.
+++, 50 to 100% of the wild-type polymerase activity; ++, 30 to 50% of the wild-type activity; +, 5 to 30% of the wild-type activity; -, 0 to 5% of the wild-type activity.
Symbols for nuclear import of PB1: +++, >80% of the cells were exclusively labelled in the nucleus; ++, between 50% and 80% of the cells were exclusively labelled in the nucleus; +/−, between 30% and 0% of the cells were exclusively labelled in the nucleus; −, no labelled nucleus.
Pathogenicity was quantified based on the LD50 (expressed in log PFU).