Influenza Polymerase Activity Correlates with the Strength of Interaction between Nucleoprotein and PB2 through the Host-Specific Residue K/E627

The ribonucleoprotein (RNP) complex is the essential transcription-replication machinery of the influenza virus. It is composed of the trimeric polymerase (PA, PB1 and PB2), nucleoprotein (NP) and RNA. Elucidating the molecular mechanisms of RNP assembly is central to our understanding of the control of viral transcription and replication and the dependence of these processes on the host cell. In this report, we show, by RNP reconstitution assays and co-immunoprecipitation, that the interaction between NP and polymerase is crucial for the function of the RNP. The functional association of NP and polymerase involves the C-terminal ‘627’ domain of PB2 and it requires NP arginine-150 and either lysine-627 or arginine-630 of PB2. Using surface plasmon resonance, we demonstrate that the interaction between NP and PB2 takes place without the involvement of RNA. At 33, 37 and 41°C in mammalian cells, more positive charges at aa. 627 and 630 of PB2 lead to stronger NP-polymerase interaction, which directly correlates with the higher RNP activity. In conclusion, our study provides new information on the NP-PB2 interaction and shows that the strength of NP-polymerase interaction and the resulting RNP activity are promoted by the positive charges at aa. 627 and 630 of PB2

The oligomeric states of wild-type BNP, BNP-Δ38 and BNP-Δ66 examined by election microscopy.

<p>Electron microscopy pictures of NP and its variants. <i>a)</i> wild-type BNP (<i>a1</i>), BNP-Δ38 (<i>a2)</i> and BNP-Δ66 (<i>a3</i>) in 100 mM sodium phosphate, 100 mM NaCl, pH6.8. b) Wild-type BNP (<i>b1</i>), BNP-Δ38 (<i>b2)</i> and BNP-Δ66 (<i>b3</i>) in PBS when RNA was added at 0h. c) Wild-type BNP (<i>c1</i>), BNP-Δ38 (<i>c2)</i> and BNP-Δ66 (<i>c3</i>) in PBS 16hr after RNA was added. The red arrow in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137802#pone.0137802.g003" target="_blank">Fig 3B</a>-c1 indicates the RNA-BNP complexes that displays a double layered structure.</p

The viral RNP activity of wild-type BNP and variants.

<p>Viral RNP activity of wild-type and BNP N-terminal deletion variants. For each mutants triplicate wells were set and recorded. Experiments are repeated twice. P values are calculated by T-test algorithm. *, <i>P</i> < 0.01; <i>**</i>, <i>P</i> < 0.001.</p

Static light scattering analysis of purified wild-type NP and variants.

<p>Horizontal lines on top of the curves illustrate the calculated molecular weights.</p

The construction and growth curves of influenza B virus with BNP variants.

<p>(A) Construction of influenza B NP mutants by systematically deleting the N-terminal coding region of NP. (B) Growth curves of influenza B wild-type and NP N-terminal deletion mutants after virus rescue. The growth curves are averages from three independent replicates.</p

Wild-type BNP and BNP-Δ66 have comparable RNA binding activities.

<p>Indicated amount of purified BNP and BNP-Δ66 were incubated with 10 μM of 24 nt 2-O-methylated RNA and analyzed by agarose gel electrophoresis. Band intensities were calculated by ImageJ (National Institutes of Health)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137802#pone.0137802.ref019" target="_blank">19</a>], using the RNA without the protein as a control (100%).</p

Functional Analysis of the Influenza Virus H5N1 Nucleoprotein Tail Loop Reveals Amino Acids That Are Crucial for Oligomerization and Ribonucleoprotein Activities ▿

Homo-oligomerization of the nucleoprotein (NP) of influenza A virus is crucial for providing a major structural framework for the assembly of viral ribonucleoprotein (RNP) particles. The nucleoprotein is also essential for transcription and replication during the virus life cycle. In the H5N1 NP structure, the tail loop region is important for NP to form oligomers. Here, by an RNP reconstitution assay, we identified eight NP mutants that had different degrees of defects in forming functional RNPs, with the RNP activities of four mutants being totally abolished (E339A, V408S P410S, R416A, and L418S P419S mutants) and the RNP activities of the other four mutants being more than 50% decreased (R267A, I406S, R422A, and E449A mutants). Further characterization by static light scattering showed that the totally defective protein variants existed as monomers in vitro, deviating from the trimeric/oligomeric form of wild-type NP. The I406S, R422A, and E449A variants existed as a mixture of unstable oligomers, thus resulting in a reduction of RNP activity. Although the R267A variant existed as a monomer in vitro, it resumed an oligomeric form upon the addition of RNA and retained a certain degree of RNP activity. Our data suggest that there are three factors that govern the NP oligomerization event: (i) interaction between the tail loop and the insertion groove, (ii) maintenance of the tail loop conformation, and (iii) stabilization of the NP homo-oligomer. The work presented here provides information for the design of NP inhibitors for combating influenza virus infection

Biophysical characterization of the NP-PB2 ‘627-domain’ interactions.

<p>(A) Pull down assay to analyze NP-PB2 interaction. Purified PB2 ‘627-domain’ was covalently immobilized onto an NHS column. Purified NP was then applied and eluted after extensive washing. (B) SPR analysis of NP-PB2 interaction. Wild-type PB2 ’627-domain’ was immobilized on a CM5 chip. NP in increased concentration was applied to the chip surface. The response differences of experimental and control flow cells are reported (individual diamonds). Solid lines are the fitted curves. (C) Wild-type NP alone (2 µM), and NP with different molar ratios of RNA were passed through the chip. (D, E and F) Wild-type and R150A mutant NP were analyzed with immobilized (D) WT, (E) [E627K,R630G] and (F) R630G H5 PB2 ‘627-domain’ by SPR.</p

The R150A NP mutant shows different activities in H5 and WSN(H1) polymerase backgrounds.

<p>(A) SPR of different concentrations of NP R150A mutant against immobilized RNA. (B) Co-immunoprecipitation of flag-tagged NP mutants with their myc-tagged counterparts. ‘+’ refers to the presence of the anti-myc antibodies while ‘−’ indicates their absence. (C) The wild-type and R150A mutant NP were subjected to RNP reconstitution assay in an H5 background and viral RNA (NA) levels were quantified by primer extension. A representative result of three independent experiments is shown. RNA levels of the NP R150A mutant were compared to those of wild-type NP, which was set to 100%. 5S rRNA was used to normalize the m-, c- and v-RNA levels. The quantitation represents the mean percentage ± standard deviations from three experiments. (D) RNP reconstitution assay of wild-type and R150A mutant NP in an WSN(H1) background (*, <i>P</i><0.05; **, <i>P</i><0.005).</p

K627 and R630 are crucial for the RNP activity and NP-PB2 interaction.

<p>(A) Sequence alignment of H5 and WSN(H1) PB2 C-terminal region (aa. 551–759). The arrows denote the differences. (B and C) RNP reconstitution assay of PB2 point mutants with (B) wild-type or (C) R150A mutant NP. The mean RNP activities from three independent experiments of the PB2 mutants were compared to those of wild-type H5-PB2 (*, <i>P</i><0.05). (D) Co-immunoprecipitation of wild-type and R150A mutant NP with Myc-tagged polymerase variants in 293T cells at 37°C.</p