In vitro assembly of PB2 with a PB1-PA dimer supports a new model of assembly of influenza A virus polymerase subunits into a functional trimeric complex

Abstract

Influenza virus RNA-dependent RNA polymerase is a heterotrimeric complex of PB1, PB2, and PA. We show that the individually expressed PB2 subunit can be assembled with the coexpressed PB1-PA dimer in vitro into a transcriptionally active complex. Furthermore, we demonstrate that a model viral RNA promoter can bind to the PB1-PA dimer prior to assembly with PB2. Our results are consistent with a recently proposed model for the sequential assembly of viral RNA polymerase complex in which the PB1-PA dimeric complex and the PB2 monomer are transported into the nucleus separately and then assembled in the nucleus.

FIG. 1.

(A) Purification of TAP-tagged RNA polymerase subunits from 293T cells transfected with expression plasmids for various combinations of the three polymerase subunits, PB1, PB2, and PA, analyzed by silver staining of an 8% SDS-PAGE gel. The plasmids transfected into 293T cells in different combinations to generate monomers and dimers are indicated above the lanes. Sizes of protein standards in kilodaltons are shown on the left; positions of PB1, PAtap, PB1tap, PB2, and PB2tap are shown either on the right or on the left. The star indicates the position of PB2tap in lane 6. The circles indicate the positions of Hsp90 identified by both matrix-assisted laser desorption ionization-time of flight and liquid chromatography-tandem mass spectrometry. In lanes 4 to 7, the tagged subunit is in higher yield than the untagged subunit(s) because of copurification of the tagged monomer. (B) PB1-PA dimers can assemble with PB2 in vitro to produce functional polymerase (solid-phase assembly). Lanes 1 to 8: as assayed by in vitro ApG-primed transcription with all possible combinations of monomers, dimers, and 3P. Lanes 9 to 15: as assayed by in vitro globin mRNA-primed transcription with the all possible combinations of monomers, dimers, and 3P. The transcription products (TP) are indicated on the right with a line. The products were analyzed by 20% PAGE in 7 M urea followed by autoradiography. (C) The amount of assembled PB2 with the PB1-PAtap dimer, compared with coexpressed 3P complex, was analyzed by Western blotting using both an anti-PB2 antibody (rabbit polyclonal antibody from Inglis [7]) and an anti-PB1 antibody (rabbit polyclonal antibody from T. Jung). PAtap alone was used as a negative control for nonspecific binding of PB2 to IgG Sepharose beads (lane 1). The positions of PB1 and PB2 are shown on the right and size markers on the left in kilodaltons.

FIG. 2.

Analysis of promoter binding capacity by UV cross-linking using the ³²P-labeled 5′ end of the vRNA promoter in the presence of the unlabeled 3′ end. Size standards are shown on the left in kilodaltons. Positions of cross-linked products are shown on the right, identified by immunoprecipitation (see reference for methodology). The products were analyzed by 8% SDS-PAGE and autoradiography. Lanes 1 to 7 are a short run. Lanes 8 and 9 are longer runs to resolve the positive signals corresponding to lanes 4 and 7, respectively.

FIG. 3.

Functional analysis of complexes assembled from immobilized PB1-PA and the vRNA promoter and PB2 added in different orders. (A) Schematic of assembly of RNA polymerase formed by binding of PB1-PA, vRNA promoter, and PB2 in different orders. (B) Analysis of assembled complexes by transcription primed by ApG or globin mRNA. The order of assembly is shown above the lanes. The reaction volumes of lanes 6 and 7 are five times the volume of lane 8. For lanes 6 and 7, the RNA was phenol-chloroform extracted and ethanol precipitated with 20 μg glycogen carrier. No globin mRNA was added in lane 9 (negative control). A longer exposure of lanes 5 to 9 is shown below to emphasize the activity in lanes 6 and 7 of the in vitro-assembled complexes. The transcription products (TP) and size are shown on the right.

FIG. 4.

Model for assembly of the influenza virus RNA polymerase complex. In vivo evidence from reference is showed by dotted arrows. Bold arrows emphasize the in vitro evidence for polymerase assembly described in this paper. The promoter is shown binding to both the PB1-PA dimer and the trimeric complex (see the text for full details).