Amino acid substitutions affecting aspartic acid 605 and valine 606 decrease the interaction strength between the influenza virus RNA polymerase PB2 '627' domain and the viral nucleoprotein

Abstract

The influenza virus RNA genome is transcribed and replicated in the context of the viral ribonucleoprotein (vRNP) complex by the viral RNA polymerase. The nucleoprotein (NP) is the structural component of the vRNP providing a scaffold for the viral RNA. In the vRNP as well as during transcription and replication the viral polymerase interacts with NP but it is unclear which parts of the polymerase and NP mediate these interactions. Previously the C-terminal '627' domain (amino acids 538-693) of PB2 was shown to interact with NP. Here we report that a fragment encompassing amino acids 146-185 of NP is sufficient to mediate this interaction. Using NMR chemical shift perturbation assays we show that amino acid region 601 to 607 of the PB2 '627' domain interacts with this fragment of NP. Substitutions of these PB2 amino acids resulted in diminished RNP activity and surface plasmon resonance assays showed that amino acids D605 was essential for the interaction with NP and V606 may also play a partial role in the interaction. Collectively these results reveal a possible interaction surface between NP and the PB2 subunit of the RNA polymerase complex.

Fig 1. Interaction between NP fragment 146–185 and PB2 involves PB2 amino acids 601 to 607.

(A) SPR sensorgrams of interaction between NP fragment 146–185 and PB2 ‘627’ domain. ‘627’ domain was immobilized on a CM5 sensor chip and NP 146–185 of 0, 312.5, 625, 1250, 2500, 10000 nM was injected. (B) ¹⁵N-labelled PB2 ‘627’ domain was perturbed by either full-length NP (blue, colour code: #0000FF) or NP fragment 146–185 (red, colour code: #FF0000), and the chemical shift of each amino acid was recorded. For some amino acids on the PB2 ‘627’ domain the perturbation data were unavailable and the values were assigned as zero. (C) Amino acids 601 to 607 (highlighted in red, colour code: #FF0000) of the PB2 ‘627’ domain are located on an alpha-helix adjacent to the characteristic phi-loop of the domain (PDB ID: 2VY7).

Fig 2. Substitutions of amino acids 601 to 607 of the PB2 ‘627’ domain disrupt RNP activity.

Plasmids expressing wild-type or mutant PB2, PA, PB1, NP and a vRNA encoding luciferase were co-transfected along with a plasmid expressing GFP as control. The overall polymerase activity is represented by a ratio of luminescence signal to GFP signal. The activity of polymerase with wild-type PB2 was set to 1. Level of PB2 expression of each mutant was detected by anti-PB2 polyclonal antibodies in Western blotting with karyopherin ß3 as loading control. The bar represents the mean ratio ± standard deviations from three independent experiments. *The p-values are smaller or equal to 0.05 in two-tailed Student’s t-test.

Fig 3. Substitutions of amino acids 605 and 606 in PB2 affect RNP activity in an NP-dependent manner.

(A) Primer extension analysis of vRNA and mRNA form vRNP reconstitution assays using a full-length segment 6 vRNA template. PB2 was omitted as negative control and the levels of 5S rRNA acted as internal control. Level of PB2 expression of each mutant was detected by anti-PB2 polyclonal antibodies in Western blotting with karyopherin ß3 as loading control. Quantification was performed by phosphoimage analysis of data from three independent experiments. The mRNA and vRNA levels of WT were set to 1. (B) Similar to (A) but without NP and using a truncated 47 nt-long vRNA-like template. *The p-values are smaller or equal to 0.05 in two-tailed Student’s t-test.

Fig 4. Amino acids 605 and 606 of the PB2 ‘627’ domain are involved in interaction with NP.

SPR was performed with wild-type PB2 (A), PB2 variant D605A (B) or PB2 variant V606A (C) using increasing concentrations of NP mutant R416A produced by 2-fold serial dilutions. The NP concentration series covers a range from 7 nM to 8000 nM. Curves produced by 500, 250, 125, 62.5, 31.25, 15.63 and 7.82 nM NP are shown in the sensorgrams.

Fig 5. PB2 variants D605A and V606A do not affect the secondary structures of the PB2 ‘627’ domain.

Circular dichroism spectroscopy was performed using purified PB2 ‘627’ domain. Spectra were recorded from 260 to 200 nm and were averaged over three measurements. The spectrum obtained for wild-type PB2 is compared with that of PB2 mutant D605A (A) and V606A (B).