Configuration of viral ribonucleoprotein complexes within the influenza A virion

Abstract

The influenza A virus possesses an eight-segmented, negative-sense, single-stranded RNA genome (vRNA). Each vRNA segment binds to multiple copies of viral nucleoproteins and a small number of heterotrimeric polymerase complexes to form a rod-like ribonucleoprotein complex (RNP), which is essential for the transcription and replication of the vRNAs. However, how the RNPs are organized within the progeny virion is not fully understood. Here, by focusing on polymerase complexes, we analyzed the fine structure of purified RNPs and their configuration within virions by using various electron microscopies (EM). We confirmed that the individual RNPs possess a single polymerase complex at one end of the rod-like structure and that, as determined using immune EM, some RNPs are incorporated into budding virions with their polymerase-binding ends at the budding tip, whereas others align with their polymerase-binding ends at the bottom of the virion. These data further our understanding of influenza virus virion morphogenesis.

Fig 1

Isolation and purification of RNPs. (A) Gel electrophoresis of the proteins of each fraction after ultracentrifugation through 30% to 70% glycerol gradients. Fractions 8 and 9, containing NP and polymerase proteins, were used for the RNP observations. M, molecular marker. (B) A representative electron micrograph of the RNPs at low magnification. Scale bar, 200 nm.

Fig 2

Negative-staining immuno-EM of purified RNPs with an anti-NP (A), anti-PA (B), anti-PB1 (C), or anti-PB2 (D) antibody or anti-FLAG antibody (E) conjugated with 5-nm gold particles. We analyzed 21 RNPs labeled with an anti-PA antibody, 16 RNPs labeled with an anti-PB1 antibody, 23 RNPs labeled with an anti-PB2 antibody, and 35 RNPs labeled with an anti-NP antibody. All of the RNPs labeled with antipolymerase antibodies had only a single gold bead, whereas those labeled with the anti-NP antibody had one to four gold beads which were distributed throughout the RNP. Scale bar, 50 nm.

Fig 3

Reconstructed 0.18- to 0.26-nm-thick digital slice views of purified RNPs by STEM tomography. Large panels on the left show representative contrast-turned slice views of the RNPs; small panels on the right show serial sections of RNPs at 0.18- to 0.26-nm intervals. (A) At one end of the RNP, NP molecules form a loop structure (arrowheads). A molecule with a holey or grooved structure (arrow on the left panels) of about 10 nm in diameter, which is morphologically different from NP molecules, is visible at the opposite end of the loop structure. (B) An RNP containing a molecule that is not clearly distinguishable from NP (arrow on the left panel). (C) An RNP that probably does not contain a polymerase. Scale bars, 50 nm.

Fig 4

Immunostaining of thin-sectioned virions. The sections were sequentially incubated with an anti-NP (A), anti-PA (B), anti-PB1 (C), anti-PB2 (D), or anti-FLAG (E) monoclonal antibody or without a primary antibody (F) and with a secondary antibody conjugated to 10-nm gold particles. The numbers of virions examined were as follows: 211 for NP, 198 for PA, 178 for PB1, and 218 for PB2. (G) In some virions, the gold signals were simultaneously found at both the top and bottom of the same budding virion. (H) Distances between the budding tip of the virion and the gold-particle signals were measured. (I) Histograms indicating the distribution of NP, PA, PB1, and PB2 within the virions. Scale bars, 100 nm.

Fig 5

Molecular distribution models on RNPs. (A) Schematic diagrams of models showing even distribution on the RNPs (NP model), localization only at the budding tip (polymerase model 1; top only), localization only at the bottom (polymerase model 2; bottom only), or localization at both positions (polymerase model 3; half-and-half). (B) Probability distribution curves for each vRNA segment for NP and polymerase models 1 to 3. The x axis represents the number of signals; the y axis represents the distance between the virion-budding tip and the gold signals. (C) Combined distribution curves for each model. For the respective models, three curves were constructed by using different standard deviations of 20 (red), 15 (green), or 10 nm (blue).

Fig 6

Schematic diagrams of RNPs within a virion. (A) The diagram showing a polymerase complex binding to the promoter region, which is composed of the complementary sequences of the 5′ and 3′ ends of the vRNA (6, 7, 10, 26). (B) A possible model, based on the results of this study, in which differently oriented RNPs are vertically packaged in a virion. (C and D) These models depict all eight RNPs as being aligned in the same direction within the virion, with the polymerase complex being at the bottom (C) or at the budding tip (D).