TABLE 2.
Expression and maturation of NA protein in virus-infected cells and percentage of NA protein incorporated in virion envelope
| Virus | Amt of NA protein expresseda (avg ± SD)d | Maturation of NA (% of endo H resistance)b (avg ± SD)d | NA incorporation in virion envelopec (avg ± SD)d |
|---|---|---|---|
| WT | 100 | 60 ± 4 | 100 |
| NA3A2 | 27 ± 3 | 58 ± 3 | 12.7 ± 2.1 |
| NA2A5 | 105 ± 4 | 57 ± 3 | 50.5 ± 3.2 |
| NA3A7 | 60 ± 3 | 57 ± 4 | 27.3 ± 2.6 |
| NA4A10 | 71 ± 4 | 59 ± 3 | 44.2 ± 3.6 |
| NA5A14 | 23 ± 3 | 49 ± 3 | 3.6 ± 0.7 |
| NA4A19 | 53 ± 3 | 28 ± 3 | 7.1 ± 1.2 |
| NA4A23 | 103 ± 4 | 24 ± 3 | 6.4 ± 0.9 |
| NA5A27 | 50 ± 5 | 65 ± 5 | 5.6 ± 0.8 |
| NA5A31 | 44 ± 4 | 55 ± 6 | 5.4 ± 0.7 |
| NA(1T2N)NA | 41 ± 4 | 15 ± 3 | 2.5 ± 0.6 |
| NATRNA | 82 ± 3 | 64 ± 4 | 10.3 ± 1.1 |
Virus-infected MDCK cells were metabolically labeled (at 4 h p.i.) for 20 min with 300 μCi of 35S-protein labeling mix/ml, labeled cells were lysed and immunoprecipitated with anti-NA and anti-M1 antibodies, and proteins were eluted, treated with PNGase F, analyzed by SDS-PAGE, autoradiographed, and quantified as described in Materials and Methods. The M1 band was used for normalization, and the results are shown with the WT result as 100%.
Virus-infected MDCK cells were metabolically labeled for 20 min as described above and chased for 90 min. Labeled cells were lysed and immunoprecipitated with anti-NA and anti-M1 antibodies, and proteins were eluted, divided into two parts, treated with either endo H or PNGase F, analyzed by SDS-PAGE, autoradiographed, and quantified. Percentages of endo H resistance proteins were calculated with the following formula: (PNGase F sensitive − Endo H sensitive) × 100/PNGase F sensitive.
Virus-infected MDCK cells were metabolically labeled (at 4 h p.i.) for 12 h with 200 μCi of 35S-protein labeling mix/ml with 2:8 media (see Materials and Methods). At 16 h p.i., medium was harvested and clarified by low-speed centrifugation, and virions were pelleted by ultracentrifugation through a 25% sucrose cushion. Labeled viruses were lysed in RIPA buffer and immunoprecipitated with anti-NA and anti-M1 antibodies, and proteins were eluted, treated with PNGase F, analyzed by SDS-PAGE, autoradiographed, and quantified (Fig. 6). Amounts of NA proteins were normalized with M1 proteins for each mutant virus and presented as percentages of that in WT virus.
Results represent averages of those from three to five independent experiments.