3D structure of the influenza virus polymerase complex: localization of subunit domains

Abstract

The 3D structure of the influenza virus polymerase complex was determined by electron microscopy and image processing of recombinant ribonucleoproteins (RNPs). The RNPs were generated by in vivo amplification using cDNAs of the three polymerase subunits, the nucleoprotein, and a model virus-associated RNA containing 248 nt. The polymerase structure obtained is very compact, with no apparent boundaries among subunits. The position of specific regions of the PB1, PB2, and PA subunits was determined by 3D reconstruction of either RNP-mAb complexes or tagged RNPs. This structural model is available for the polymerase of a negative-stranded RNA virus and provides a general delineation of the complex and its interaction with the template-associated nucleoprotein monomers in the RNP.

Fig. 1.

Purification of recombinant mini-RNPs. RNPs were reconstituted and amplified in vivo by using His-tagged PB2 (His). As a control, a reconstitution was performed in the absence of RNA (CTRL). RNP preparations after glycerol gradient centrifugation (Input) and after elution from Ni²⁺-NTA agarose (RNPs) were analyzed by silver staining. The positions of the RNP components are indicated to the right and that of vRNA is indicated by a star. The molecular mass markers (in kDa) are indicated to the left. MW, molecular weight.

Fig. 2.

3D reconstruction of the influenza virus polymerase complex. The images of the polymerase complex from isolated RNPs were used for 3D reconstruction. (A–D) The isosurface representations at a threshold that corresponds to the mass of the polymerase (Upper) and a lower threshold representation (pink) under a transparent isosurface (Lower). They represent the side views (A and C), front view (B), and rear view (D) of the model. The arrows point to a possible groove in the complex. (E and F) Side views of the 3D model of a recombinant RNP (36) showing the relative topology of the new model of the polymerase with regard to the NP ring. (Scale bars, 50 Å.)

Fig. 3.

2D average images of recombinant polymerase–mAb complexes. Images obtained from negative-stained preparations of RNP–PB2–25 and RNP–PA-2 complexes were centered and aligned. The average image obtained is presented in A. Sorting by a self-organizing feature map led to data collections lacking or containing bound IgG. The average images obtained for the PB2–25- and PA-2-containing collections are presented in B and C, respectively. The arrows indicate the additional masses observed. (Scale bar, 50 Å.)

Fig. 4.

3D reconstruction of polymerase–anti-PB2 and polymerase–anti-PA monoclonal complexes. The images at the bottom are the result of a 90° rotation of those at the top. The images whose 2D averages are presented in B and C were used for 3D reconstruction. (A) The isosurface representation of the WT polymerase is shown. (B and C) The corresponding models for polymerase–anti-PB2 and polymerase–anti-PA complexes, respectively, are shown. The arrows point to the additional masses representing the domains of interaction of the mAb IgGs.

Fig. 5.

3D reconstruction of PB1-tagged influenza virus polymerase. (A) Purification of RNP–TAP–IgG complexes. RNPs were amplified by using TAP-tagged PB1 and His-tagged PB2, purified, and incubated with an excess of purified, unrelated rabbit IgGs. The RNP–TAP and RNP–TAP–IgG complexes were purified by affinity chromatography on Ni²⁺-NTA agarose. The successive Western blotting of the affinity-purified material is shown; unrelated rabbit serum was used to reveal the TAP tag (anti-TAP), anti-PB2 rabbit serum (anti-PB2), and anti-rabbit IgG biotin (anti-IgG). Molecular mass markers (in kDa) are indicated to the left. (B and C) The images obtained for RNP–TAP–IgG complexes were used for 3D reconstruction. Relevant views of the tagged polymerase (C) are presented in comparison with WT polymerase (B). The arrows point to additional masses contributed by the TAP tag and the bound IgG. The lower images are the result of a 90° rotation of the upper images.

Fig. 6.

3D model for the influenza virus polymerase. The colored areas indicate the location of specific subunit domains: red, N-terminal region of PB2; blue, C-terminal region of PA; green, C-terminal region of PB1. (A) The model obtained by 3D reconstruction from electron micrograph images. (B) The position of the polymerase subunits in the context of the structure of the RNP.