Influenza virus polymerase subunits co-evolve to ensure proper levels of dimerization of the heterotrimer

Abstract

The influenza A virus RNA-dependent RNA polymerase complex consists in three subunits, PB2, PB1 and PA, that perform transcription and replication of the viral genome through very distinct mechanisms. Biochemical and structural studies have revealed that the polymerase can adopt multiple conformations and form oligomers. However so far it remained unclear whether the available oligomeric crystal structures represent a functional state of the polymerase. Here we gained new insights into this question, by investigating the incompatibility between non-cognate subunits of influenza polymerase brought together through genetic reassortment. We observed that a 7:1 reassortant virus whose PB2 segment derives from the A/WSN/33 (WSN) virus in an otherwise A/PR/8/34 (PR8) backbone is attenuated, despite a 97% identity between the PR8-PB2 and WSN-PB2 proteins. Independent serial passages led to the selection of phenotypic revertants bearing distinct second-site mutations on PA, PB1 and/or PB2. The constellation of mutations present on one revertant virus was studied extensively using reverse genetics and cell-based reconstitution of the viral polymerase. The PA-E349K mutation appeared to play a major role in correcting the initial defect in replication (cRNA -> vRNA) of the PR8xWSN-PB2 reassortant. Strikingly the PA-E349K mutation, and also the PB2-G74R and PB1-K577G mutations present on other revertants, are located at a dimerization interface of the polymerase. All three restore wild-type-like polymerase activity in a minigenome assay while decreasing the level of polymerase dimerization. Overall, our data show that the polymerase subunits co-evolve to ensure not only optimal inter-subunit interactions within the heterotrimer, but also proper levels of dimerization of the heterotrimer which appears to be essential for efficient viral RNA replication. Our findings point to influenza polymerase dimerization as a feature that is controlled by a complex interplay of genetic determinants, can restrict genetic reassortment, and could become a target for antiviral drug development.

Fig 1. Phenotypic and genotypic characterization of the wild-type, attenuated and revertant viruses.

(A) Plaque phenotype and titers of the wild-type PR8 virus, attenuated PR8xWSN-PB2 reassortant virus (att-PxW) and revertant virus (rev-PxW) isolated upon 5 serial passages of att-PxW. Following reverse genetics (PR8, att-PxW) or plaque purification (rev-PxW), the viruses were amplified once and titrated on MDCK cells. Crystal violet staining of infected cell monolayers is shown. (B) Growth kinetics under multi-cycle conditions. A549 cells were infected at a m.o.i. of 0.001 with the indicated viruses. At the indicated times post-infection, viral titers were determined by plaque assay on MDCK cells. The results are shown as the mean ± SD of three independent experiments. (C-E) Levels of NP v-, c- and mRNAs under single cycle conditions. A549 cells were infected at a m.o.i. of 5 and the levels of NP vRNAs (C), cRNAs (D) and mRNAs (E) were determined at the indicated times post-infection by strand-specific RT-qPCR. The copy numbers are shown as the mean ± SD of four independent experiments in duplicate. Significance was assessed using Student’s paired t-test after log transformation (**p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001, rev-PxW compared to att-PxW). (F) Whole genome sequence. The three amplified viral stocks were subjected to vRNA extraction, RT-PCR of the eight genomic segments and next-generation sequencing. The amino acid changes observed in the rev-PxW virus compared to the initial att-PxW virus are indicated.

Fig 2. Contribution of FluPol amino acid changes to the titer and plaque phenotype of the rev-PxW virus.

(A) Titers and plaque phenotype of recombinant viruses bearing one or several reversion mutations in the att-PxW genetic background, as follows: PB2-D701N (d), PB1-M195T (e), PA-L28R (f), PA-E349K (g), PA-L28R/E349K (h) PB2-D701N+PA-L28R/E349K (i) and PB2-D701N+PB1-M195T+PA-L28R/E349K (j). Recombinant PR8 (a) and att-PxW (b) viruses were rescued in parallel. Following one round of amplification on MDCK cells, the titers and plaque phenotypes were compared to that of the rev-PxW virus (c). Grey squares represent a WSN-PB2 background. Residues that are mutated compared to the att-PxW virus are framed. Green frames correspond to the green color code used in the following figures. (B-C) Growth kinetics under multi-cycle conditions. A549 cells were infected at a m.o.i. of 0.001 with the indicated viruses. At the indicated times post-infection, viral titers were determined by plaque assay on MDCK cells. The results are shown as the mean ± SD of three independent experiments. The data shown for PR8, att-PxW and rev-PxW viruses in (B) are the same as in Fig 1B. The significance was tested with a Student’s paired t-test after log transformation (*p≤0.05; **p≤0.01; *** p ≤ 0.001, PA-28/349 compared to att-PxW in (B), PB2+PB1+PA28/349 compared to att-PxW in (C).

Fig 3. Contribution of FluPol amino acid changes to viral replication capacity.

(A-C) Levels of NP v-, c- and mRNAs under single cycle conditions. A549 cells were infected at a m.o.i. of 5 and the levels of NP vRNAs (A), cRNAs (B) and mRNAs (C) were determined at 6 hpi by strand-specific RT-qPCR. The copy numbers are shown as the mean ± SD of three independent experiments in duplicate. Significance was assessed using Student’s paired t-test after log transformation (*p ≤ 0.05; **p ≤ 0.01; *** p ≤ 0.001). (D-E) The levels of NP v-, c-, and m-RNAs determined at 6 hpi were used to calculate the vRNA/cRNA (D) and mRNA/vRNA (E) copy number ratios. (F) Accumulation of primary NP transcripts in the presence of cycloheximide (CHX). A549 cells were infected as in (A-C) in the presence of 100 μg/mL of CHX i.e. under conditions of primary transcription. The levels of NP v- and m-RNAs determined at 6 hpi were used to calculate the mRNA/vRNA ratio. The results are shown as the mean ± SD of three (D-E) or two (F) independent experiments in duplicates. Significance was assessed using Student’s unpaired t-test (*p ≤ 0.05; **p ≤ 0.01; ns: non significant).

Fig 4. Polymerase activities measured in a minigenome assay.

(A) Polymerase activities of mutant FluPols. HEK-293T cells were co-transfected with plasmids expressing the att-PxW polymerase, using wild-type (dotted bar) or mutant WSN-PB2 and/or PR8-PA plasmids (grey and green bars) as indicated, together with the PR8-NP, pPolI-Firefly and pTK-Renilla plasmids. Plasmids expressing the polymerase and NP from the PR8 (black bar) and WSN (white bar) viruses were used as controls. Firefly luciferase activities were measured at 24 hours post-transfection and normalized relative to Renilla luciferase activities. The results are expressed as percentages (PR8: 100%) and are shown as mean ± SD of three independent experiments in triplicates. Significance was assessed using one sample t-test (for pairwise comparison with the 100% PR8 reference) or Student’s unpaired t-test for pairwise comparison among other samples (*p ≤ 0.05; **p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). (B) Transcription/replication activity in the presence of trans-complementing transcription-deficient or replication-deficient PB2 proteins. HEK-293T cells were co-transfected with plasmids expressing the PR8 (a) or att-PxW (b-e) polymerase, together with the PR8-NP, pPolI-Firefly and pTK-Renilla plasmids. Where indicated, plasmids encoding a transcription-deficient (PB2-E361A, R+/T-) or replication-deficient (PB2-R142A, R-/T+) PB2 protein were co-transfected to trans-complement the att-PxW polymerase (b-e). As a control, they were transfected instead of the PR8-PB2 plasmid in (a). Firefly luciferase activities were measured at 48 hours post-transfection and normalized relative to Renilla luciferase activities. The results are expressed as percentages (PR8: 100%) and are shown as mean ± SD of three independent experiments in triplicates. Significance was assessed using a one sample t-test in (a) and Student’s unpaired t-test in (b-e) (**p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns: non significant).

Fig 5. FluPol mutations observed on revertant viruses derived from five independent serial passages of the att-PxW virus.

(A) Schematic representation of five independent serial passaging of plaque-purified att-PxW viruses. As soon as large plaque forming viruses were observed, at passages 5 to 8 (P5 to P8) as indicated, they were isolated by plaque purification and subjected to whole genome next-generation sequencing. Only reversion mutations on the FluPol genes that were present on >98% of the reads are indicated (other mutations are shown in S2 Table). (B) Mapping of amino-acids of interest onto the three-dimensional structure for a cRNA-bound FluPol (influenza B/Memphis/13/03 virus, PDB: 5EPI). The PB2, PB1 and PA subunits are represented in purple, blue and green, respectively. The amino-acids that differ between the PR8 and WSN viruses are colored in bright orange and labelled as PR8/WSN residues. The amino acids mutated in the revertant viruses isolated upon serial passaging of the att-PxW virus are colored in red. Grey labeling indicates residues that can be clearly visualised in one of the previous views of the cRNA-bound FluPol structure in the figure. (C) Growth kinetics under multi-cycle conditions. A549 cells were infected at a m.o.i. of 0.001 with the indicated viruses. At the indicated times post-infection, viral titers were determined by plaque assay on MDCK cells. The results are shown as the mean ± SD of three independent experiments, except for the 72 h time point of Rev 1 that was only measured twice.

Fig 6. Dimerization of mutant FluPol heterotrimers.

(A) FluPol dimerization assessed by co-immunoprecipitation. HEK-293T cells were co-transfected with plasmids encoding the PR8 polymerase (both PB1-3xFlag and PB1-Gluc2, together with the wild-type or mutant PR8-PA and PR8-PB2 as indicated, lanes 1-5), or the att-PxW polymerase (both PR8-PB1-3xFlag and PR8-PB1-Gluc2 together with PR8-PA and WSN-PB2, lane 6). Controls in the absence of PB1-Gluc2, PA or PB2 were also performed (lanes 7-9). FluPol complexes were purified at 48 hours post-transfection using anti-Flag antibody-magnetic beads, in the presence of either 0.5 (upper panel) or 0.4% (lower panel) Igepal CA-630 and analysed by SDS-PAGE and silver staining. Molecular weight markers are indicated. Quantification of the PB1-Gluc2 and PB1-3xFlag signals was performed by gel densitometry on a BioRad ChemiDoc Imaging apparatus, using the BioRad software. The PB1-Gluc2/PB1-3xFlag ratios are presented, with the wild-type PR8 condition used as a reference (PR8-PB1-Gluc2/PR8-PB1-3xFlag ratio: 1). MW: Molecular Weight Marker (kDa). (B) Schematic representation of the split luciferase complementation-based assay for FluPol dimerization and validation of the assay for FluPol dimerization. The split luciferase Gluc1 and Gluc2 domains only interact to reconstitute an active luciferase enzyme when two fully reconstituted FluPol heterotrimers are associated. HEK-293T cells were co-transfected with the same plasmid combinations as in (A) except for PR8-PB1-3xFlag which was replaced by PR8-PB1-Gluc1. After 24 hours, the Gaussia princeps luciferase activities were measured. (C) FluPol dimerization for the wild-type PR8 and att-PxW FluPols, as well as for att-PxW FluPols bearing one or several of the reversion mutations observed in revertant viruses Rev 1 (green and light grey bars), Rev2 (medium grey bar), and Rev3-4-5 (dark grey bar), assessed as in (B). (D) Polymerase activity of the same series of FluPols as in (C), plus the WSN FluPol (white bar) assessed using a minigenome assay as described previously in Fig 4A. (E) FluPol dimerization in the context of a cRNP. FluPol dimerization was measured for the Apo FluPol and compared to dimerization in the presence of NA-cRNA or NA-cRNA + NP, for the att-PxW, PA-349, PB2-74 and PB1-577 FluPol complexes. (B-E) The results are expressed as percentages (PR8: 100%) and are shown as mean ± SD of three (B, D and E) or four (C) independent experiments in triplicates. Significance was assessed using one sample t test (for pairwise comparison with the 100% PR8 reference) and Student’s unpaired t-test for pairwise comparison among other samples (*p ≤ 0.05; **p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns: non significant). In (E), the significance of pairwise comparison with att-PxW in each experimental condition is indicated.