Functional and structural studies of the Influenza A virus polymerase subunit PB1 and its complex with the PA subunit
Master of Arts
The influenza virus RNA polymerase is a heterotrimeric complex of PA, PB1, and PB2. PB1 functions in RNA elongation, 5' and 3' viral RNA binding, and has an endonuclease activity. For detailed biochemical and structural studies, PB1 was expressed in the baculovirus expression system (BVES). However, the majority of recombinant PB1 was insoluble. Nine PB1 truncation mutants expressed in E. coli or insect cells were also largely insoluble. The coexpression of PA and PB1 in the BVES improved PB1 solubility, and recombinant PA:PB1 appeared stable. Purified PA:PB1 possessed RNA elongation, 5' and 3' vRNA binding activities, and appeared compact under EM. Using negative staining images, a ∼30 Å resolution reconstruction of PA:PB1 was obtained. Promising crystallization conditions were also identified. By showing that heterodimeric PA:PB1 is biologically active and that highly purified samples can be obtained in preparative amounts, this study represents a significant step towards the determination of the polymerase atomic structure, which will have important applications in anti-influenza drug development.
Microbiology; Biochemistry; Biophysics; Pure sciences; Biological sciences