Summary auto-generated
This 1996 study by Toyoda and colleagues investigates how the three subunits of influenza virus RNA polymerase (PB1, PB2, and PA) assemble into a functional complex. Using transfection of COS-7 cells with various combinations of wild-type and deletion mutant polymerase subunit genes, the researchers employed co-immunoprecipitation to identify subunit-subunit contact sites. They created nested sets of N- and C-terminal deletion mutants for each subunit and expressed them alone or in pairs to map binding regions. The results demonstrate that PB1 functions as a core scaffolding subunit through which both PB2 and PA are incorporated. Binary complexes form between PB1-PB2 and PB1-PA, but PB2 and PA do not directly interact when expressed without PB1. The C-terminal 158 amino acids of PB1 bind to the N-terminal 249 amino acids of PB2, while the N-terminal 140 amino acids of PB1 bind to the C-terminal two-thirds of PA. These findings provide the first detailed mapping of protein-protein interaction sites within the influenza polymerase complex and establish PB1 as the central organizational component of this essential RNA synthesis enzyme.
Key findings
- PB1 functions as the core scaffolding subunit of influenza RNA polymerase, with both PB2 and PA binding directly to PB1 but not to each other
- The PB2-binding domain on PB1 is located in the C-terminal 158 amino acids, while the PA-binding domain is in the N-terminal 140 amino acids
- The PB1-binding site on PB2 maps to the N-terminal 249 amino acids; the PB1-binding site on PA maps to the C-terminal two-thirds
- Binary PB1-PB2 and PB1-PA complexes form in transfected cells, but PB2-PA binding does not occur without PB1 present
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Abstract
Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome. By transfection of various combinations of cDNA encoding wild-type and serial deletion mutants of each P protein subunit and co- immunoprecipitation with subunit-specific antibodies, the subunit- subunit contact sites on all three of the P proteins were determined. Results indicate that binary complexes are formed between PB1-PB2 and PB1-PA but not between PB2-PA. Therefore, we concluded that PB1 is the core subunit for assembly of the virus RNA polymerase. The C-terminal 158 amino acids of PB1 bound to the N-terminal 249 amino acids of PB2, while the N-terminal 140 amino acids of PB1 bound to the C-terminal two- thirds of PA. PB2-PA binding was not detected when they were expressed in the absence of the PB1 subunit.