1. Microbiology and Infectious Disease
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Species specific differences in use of ANP32 proteins by influenza A virus

  1. Jason S Long
  2. Alewo Idoko-Akoh
  3. Bhakti Mistry
  4. Daniel Goldhill
  5. Ecco Staller
  6. Jocelyn Schreyer
  7. Craig Ross
  8. Steve Goodbourn
  9. Holly Shelton
  10. Michael A Skinner
  11. Helen Sang
  12. Michael J McGrew
  13. Wendy Barclay  Is a corresponding author
  1. Imperial College London, United Kingdom
  2. University of Edinburgh, United Kingdom
  3. St. George's, University of London, United Kingdom
  4. The Pirbright Institute, United Kingdom
Research Article
Cite this article as: eLife 2019;8:e45066 doi: 10.7554/eLife.45066
8 figures, 1 table, 1 data set and 1 additional file

Figures

Figure 1 with 2 supplements
Phylogenetic and sequence analysis reveals avian ANP32B to be a paralog of mammalian ANP32B.

The best maximum-likelihood tree was calculated from a set of ANP32 proteins with mapmodulin from Drosophila melanogaster as an outgroup using RAxML with 100 bootstraps. This figure is a cladogram showing the relationships between mammalian ANP32s, avian ANP32s and ANP32s from Xenopus tropicalis. Selected bootstrap values show the relationship between different ANP32 protein clades. Avian ANP32B clade is shown in green. The full tree is shown in Figure 1—figure supplement 1.

https://doi.org/10.7554/eLife.45066.003
Figure 1—figure supplement 1
Phylogenetic and sequence analysis reveals avian ANP32B to be a paralog of mammalian ANP32B.

Full tree used to make the cladogram in Figure 1. This tree was made using RAxML with 100 bootstraps and mapmodulin as an outgroup.

https://doi.org/10.7554/eLife.45066.004
Figure 1—figure supplement 2
Synteny of ANP32 genes.

Chromosome locations of ANP32 family members A, B, C and E from coelacanth (Latimeria chalumnae), Xenopus (Xenopus tropicalis), chicken (Gallus gallus), zebra finch (Taeniopygia guttata), opossum (Monodelphis domestica), mouse (Mus musculus) and Human (Homo sapiens).

https://doi.org/10.7554/eLife.45066.005
Figure 2 with 1 supplement
Chicken ANP32B is not functional for IAV polymerase.

Cells were transfected with avian H5N1 50–92 polymerase (PB2 627E or 627K) together with NP, firefly minigenome reporter, Renilla expression control, either Empty vector (control) or ANP32 expression plasmid and incubated at 37°C for 24 hr. (a) Minigenome assay in human eHAP1 cells with co-expressed Empty vector, FLAG-tagged chANP32A or chANP32B. (b) Minigenome assay in double knockout (dKO) eHAP1 cells. (c) Western blot analysis of dKO eHAP1 cell minigenome assay confirming expression of PB2 and FLAG-tagged chANP32A and B. (d) Minigenome assay in WT DF-1 cells with either co-expressed Empty vector or chANP32B. (e) Minigenome assay in DF-1 ANP32B knockout (bKO) cells with either co-expressed Empty vector or chANP32B. Data shown are firefly activity normalised to Renilla, plotted as mean ± SEM (n = 3 biological replicates). Two-way ANOVA with Dunnet’s multiple comparisons to Empty vector. ns = not significant, ****p<0.0001.

https://doi.org/10.7554/eLife.45066.006
Figure 2—figure supplement 1
Sequence analysis of ANP32 in genome edited DF-1 chicken cells.

(a) DNA sequence analysis of ANP32B from genomic DNA of DF-1 WT and ΔB clones, showing the target sequence of the gRNA pair used in the CRISPR/Cas9 reaction. Allele A had a 16 bp deletion and allele B a 40 bp deletion, resulting in premature stop codons in the ANP32B sequence. (b) qRT-PCR analysis of mRNA isolated from WT and bKO DF-1 cells. Data are Δct of RPL30, ANP32A, B or E to RS17. Data are Δct of RS17, ANP32A, B or E to RPL30. Annotated alignments generated using Geneious R6 software.

https://doi.org/10.7554/eLife.45066.007
Figure 3 with 2 supplements
Chicken PGC derived fibroblast cells lacking ANP32A or the 33 amino acid insertion do not support avian IAV polymerase activity.

(a) Schematic of CRISPR/Cas9 RNA guide targets used to generate aKO (exon1) and Δ33 (exon 5) PGC cell lines. (b) Western blot analysis of ANP32A and β-actin expression in WT, KO and Δ33 PGC-derived fibroblast cells. (c) Minigenome assay in WT, Δ33 or aKO PGC derived fibroblast cells with either PB2627E (black) or 627K (grey) polymerase derived from avian H5N1 50–92 virus. (d) Minigenome assay in WT, Δ33 or aKO cells with avian H5N1 50–92 PB2 627E polymerase co-transfected with Empty vector (black) or FLAG-tagged chANP32A (grey). (e) Western blot analysis of PB2, FLAG and Histone 3. Data shown are firefly activity normalised to Renilla, plotted as mean ± SEM (n = 3 biological replicates). Two-way ANOVA with Dunnet’s multiple comparisons to WT. ns = not significant, **p<0.01, ***p<0.001, ****p<0.0001.

https://doi.org/10.7554/eLife.45066.008
Figure 3—figure supplement 1
Sequence analysis of ANP32 in genome edited PGC chicken cells.

Alignment of DNA sequence from WT, Δ33 and KO PGCs, showing the target sequence of the gRNAs used in the CRISPR/Cas9 reaction. (a) Comparison between WT and KO PGCs showed an 8 bp deletion in exon 1 of ANP32A in KO PGC cells, resulting in a truncated ANP32A protein. (b) Intron and exon five sequence comparison of WT and Δ33 cells revealed a 400 bp deletion resulting in the loss of exon 5. (c) qRT-PCR analysis of mRNA isolated from WT, Δ33 or aKO PGC derived fibroblast cells. Data are Δct of RS17, ANP32A, B or E to RPL30. Annotated alignments generated using Geneious R6 software.

https://doi.org/10.7554/eLife.45066.009
Figure 3—figure supplement 2
In vitro reprogramming of chicken PGCs into adherent fibroblast-like cells.

(a) Diagram describing the method to differentiate PGCs from day three chicken embryos. (b) Fluorescent images of live cells demonstrating the differentiation of GFP+ PGCs into fibroblast-like cells.

https://doi.org/10.7554/eLife.45066.010
Figure 4 with 2 supplements
Lack of functional support for IAV polymerase by chicken ANP32B maps to differences in LRR5 domain.

(a) Schematic of chicken ANP32A protein highlighting the different domains and LRR sequences (LRR 1–5). (b) Human 293 T cells were transfected with avian H5N1 50–92 polymerase (PB2 627E) together with NP, pHOM1-firefly minigenome reporter, Renilla expression control, either Empty vector or FLAG-tagged ANP32 expression plasmid and incubated at 37°C for 24 hr. Western blot analysis shown below (FLAG and Vinculin). (c) Minigenome assay in 293 T cells (PB2 627E) with FLAG-tagged WT or mutant chANP32A expression plasmids with associated western blot (FLAG and PCNA). (d) huANP32A crystal structure (PDB 4 × 05) with residues K116, N127, N129, D130 and K137 highlighted using UCSF Chimaera (Pettersen et al., 2004). (e) Minigenome assay of avian H5N1 50–92 polymerase with either PB2 627E or 627K in PGC-derived fibroblast aKO cells, together with co-expressed Empty vector, chANP32A or chANP32AN129I. Data shown are firefly activity normalised to Renilla, plotted as mean ± SEM (n = 3 biological replicates). One-way ANOVA with Tukey’s comparison to chANP32A (b and c) or two-way ANOVA with Dunnet’s multiple comparisons to chANP32A (e). ns = not significant, *p<0.05, **p<0.01, ****p<0.0001.

https://doi.org/10.7554/eLife.45066.011
Figure 4—figure supplement 1
Nuclear localisation of exogenously expressed ANP32 proteins in 293 T cells.

Immunofluorescent images of 293 T cells expressing ANP32 constructs fixed and stained with DAPI to highlight the nucleus. (a) FLAG-tagged ANP32A constructs were imaged by probing with mouse α-FLAG antibody and detected by α-mouse AlexaFluor-568: Empty vector(1), huANP32B(2), huANP32B33(3), chANP32B(4), chANP32B33(5), huANP32B33LRR(6), huANP32B33CENT(7), huANP32B33LCAR(8), huANP32B33N+LRR1(9), huANP32B33LRR2+3 (10), huANP32B33LRR4+5 (11), chANP32A(12), chANP32Ascr149-175(13), chANP32Ascr176-208(14). (b) ANP32A with mCherry fused to the C-terminus: Empty Vector(1), chANP32A(2), chANP32B(3), chANP32AK116H(4), chANP32AN127M(5), chANP32AN129I(6), chANP32AD130N(7), chANP32AK137T(8). All images were prepared using ImageJ (Rueden et al., 2017) and Microsoft PowerPoint.

https://doi.org/10.7554/eLife.45066.012
Figure 4—figure supplement 2
Avian ANP32B proteins share the I129 and N130 residues in LRR5.

Alignment comparing sequence of LRR5 from chicken ANP32A and ANP32B sequences from Homo sapiens and 22 avian species (residues 115 to 141). Protein sequences downloaded from NCBI and aligned using Geneious R6 software.

https://doi.org/10.7554/eLife.45066.013
Sequence of amino acids 149–175 of the central domain of chANP32A are required to support activity of both avian and human-adapted IAV polymerase.

(a) Schematic of chANP32A showing the sequence of amino acids in the central domain (149–175 or 33 amino acid insertion (176-208) and the randomly scrambled sequence in red. (b) Minigenome assay of avian H5N1 50–92 polymerase with either PB2 627E or 627K in PGC-derived fibroblast aKO cells with co-expressed Empty plasmid or FLAG-tagged WT chANP32A, chANP32Ascr149-175 or chANP32Ascr176-208 expression plasmids. (c) Western blot analysis of PB2 (627E), lamin B1 and FLAG. Data shown are firefly activity normalised to Renilla, plotted as mean ± SEM (n = 3 biological replicates). Two-way ANOVA with Dunnet’s multiple comparisons to chANP32A. ns = not significant, ****p<0.0001.

https://doi.org/10.7554/eLife.45066.014
Figure 6 with 1 supplement
A single amino acid change (N129I) derived from chANP32B disrupts chANP32A support of influenza polymerase activity by abrogating binding to IAV polymerase.

(a) Diagram of the split Gaussia luciferase system, demonstrating how ANP32 fused to luciferase fragment luc2 may bind to polymerase containing PB1 fused to luciferase fragment luc1 and complement full luciferase, which then reacts with substrate to generate a measurable bioluminescent signal. (b) Human 293 T cells were transfected with PB1 fused to luc1 (PB1luc1), PB2 (627E or K), PA and either chANP32A, chANP32B or chANP32B33 fused to luc2 (control wells were transfected with all components but with unfused PB1 and luc1 or chANP32 and luc2). (c) As (b) but with either chANP32Aluc2 or chANP32AN129Iluc2. (d) 293 T cells transfected with either chANP32Aluc2 or chANP32AN129Iluc2 and histone four fused to luc1 (or with unfused controls) or with H4luc1 and histone three fused to luc2. All data are Normalised Luciferase Ratio (n = 3 biological replicates) (Figure 6—figure supplement 1). One-way ANOVA (d) or two-way ANOVA with Dunnet’s multiple comparisons to chANP32A (b and c). ns = not significant, ****p<0.0001.

https://doi.org/10.7554/eLife.45066.015
Figure 6—figure supplement 1
Western blot analysis of split luciferase constructs.

(a) Normalised luciferase Ratio was calculated by the equation described in this diagram, whereby the bioluminescence measured by interacting partners A and B fused to luc1 and luc2 are divided by the sum of the bioluminescence of the unfused controls. (b) Western blot analysis of luc-fused constructs (α-Vinculin, PB1, Gaussia luciferase and histone 3).

https://doi.org/10.7554/eLife.45066.016
Viral replication is abrogated in chicken PGC fibroblast cells lacking ANP32A.

WT (black lines) or aKO (red lines) PGC-derived fibroblast cells were infected with recombinant viruses (containing PR8 HA, NA and M genes and internal genes from either H5N1 50–92 or H7N9 Anhui), at an MOI of either 0.0001 (a,b) or 1.0 (c,d), incubated at 37°C in the presence of trypsin, cell supernatants harvested at described time-points and PFU ml−1 measured by plaque assay on MDCK cells. (a) H5N1 50–92 (MOI 0.0001). b. H7N9 Anhui (MOI 0.0001). c. H5N1 50-−92 (MOI 1.0). d. H7N9 Anhui (MOI 1.0). vRNAs from supernatants 24 hr post infection MOI 1.0 (c and d) were extracted, PCR amplified and sequenced by Sanger sequencing. Limit of detection by plaque assay shown by dotted line (10PFU ml−1). n = 3 biological replicates. Multiple t-tests with Holm-Sidak comparison. ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

https://doi.org/10.7554/eLife.45066.017
Author response image 1

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent (G. gallus)GFP+/Hyline crossRoslin InstituteFertile heterozygous eggs for PGC derivations (Pettersen et al., 2004)
Primary cells (G. gallus)Primordial Germ Cells (PGCs)G. gallus GFP+/Hyline cross, Roslin Institute (This study)
Cell line (G. gallus)DF-1 fibroblastsAmerican Type Culture CollectionCRL-12203; RRID:CVCL_0570
Cell line (C. sapiens)MDCKAmerican Type Culture CollectionCCL- 34; RRID:CVCL_0422
Cell line (H. sapiens)293TAmerican Type Culture CollectionCRL-3216; RRID:CVCL_0063
Cell line (H. sapiens)eHAP1Horizon DiscoveryC669
Antibodyrabbit polyclonal α-ANP32ASigma-AldrichAV40203; RRID:AB_1844874Dilution 1:500-1:1000
Antibodymouse monoclonal α-β-actinSigma-AldrichA2228; RRID:AB_476697Dilution 1:1000
Antibodymouse monoclonal α-FLAGSigma-AldrichF1804; RRID:AB_262044Dilution 1:1000 (WB), 1:300 (IF)
Antibodymouse monoclonal α-Lamin B1MerckMAB5492; RRID:AB_2085944Dilution 1:1000
Antibodymouse monoclonal α-PCNASanta Cruzsc-25280, RRID:AB_628109Dilution 1:1000
Antibodyrabbit polyclonal α-Histone 3AbcamAB1791; RRID:AB_302613Dilution 1:2000
Antibodyrabbit monoclonal α-vinculinAbcamAB129002; RRID:AB_11144129Dilution 1:1000
Antibodyrabbit polyclonal α-Gaussia LucNEBE80235Dilution 1:2000
Antibodyrabbit polyclonal α-PB1InvitrogenPA5-34914; RRID:AB_2552264Dilution 1:2000
Antibodyrabbit polyclonal α-PB2GeneTexGTX125926; RRID:AB_11162999Dilution 1:2000
Antibodygoat polyclonal anti-rabbit HRPCST7074Dilution 1:2000
AntibodyHorse polyclonal anti-mouse HRPCST7076Dilution 1:2000
AntibodySheep polyclonal α-rabbit HRPMerckAP510PDilution 1:20000
Antibodygoat polyclonal α-mouse HRPAbD SerotecSTAR117P; RRID:AB_323839Dilution 1:10000
Antibodygoat polyconal α-mouse AlexaFluor-568InvitrogenA11031; RRID:AB_144696Dilution 1:1000
Recombinant DNA reagentpGEM-T Easy vectorPromegaA1360
Recombinant DNA reagentpSpCas9(BB)−2A-Puro PX459 V2.0 vectorGift from Dr. Feng ZhangRRID:Addgene_62988
Recombinant DNA reagentpSpCas9n(BB)−2A-GFP PX461 vecotraddgenePlasmid 48140; RRID:Addgene_48140
Recombinant DNA reagentpCAGGS vectorBelgium Co-ordinated Collections of Microorganisms (BCCM), University of Ghent, Belgium
Recombinant DNA reagentH5N1 A/turkey/England/50-92/1991 polI plasmidsAPHA, Weybridge, UK
Recombinant DNA reagentH7N9 Anhui/1/2013 pHW2000 plasmidsPirbright Institute, UK
Recombinant DNA reagentH1N1 A/PR/8/34 (PR8) polI or pHW2000 plasmids(Neumann et al., 1999)
Commercial assay or kitRNeasy mini kitQiagen74106
Commercial assay or kitQuick Start Bradford Protein Assay KitBiorad5000202
Commercial assay or kitDual-luciferase Reporter assay systemPromegaE1910
Commercial assay or kitRenilla luciferase kitPromegaE2810
Commercial
assay or kit
QIAamp Viral RNA Mini KitQiagen52906
Chemical compound, drugLipofectamine 3000InvitrogenL3000008
Chemical
compound, drug
Lipofectamine 2000Invitrogen11668019
Software, algorithmImage JImageJ (http://imagej.nih.gov/ij/)
Software, algorithmGraphPad PrismGraphPad Prism (https://graphpad.com)version 6
Software, algorithmGeneiousGeneious https://www.geneious.comR6

Data availability

The data used to generate Figure 1 and Figure 1- figure supplement 1 was downloaded from Ensembl (http://www.ensembl.org/Multi/GeneTree/Image?gt=ENSGT00950000182907). Source data for Figures 2, 3, 4, 5, 6 & 7 are available on Dyrad https://dx.doi.org/10.5061/dryad.338t920.

The following data sets were generated
  1. 1
    Dryad Digital Repository
    1. JS Long
    2. A Idoko-Alewo
    3. B Mistry
    4. DH Goldhill
    5. E Staller
    6. J Schreyer
    7. C Ross
    8. S Goodbourn
    9. H Shelton
    10. MA Skinner
    11. HM Sang
    (2019)
    Data from: Species specific differences in use of ANP32 proteins by influenza A virus.
    https://doi.org/10.5061/dryad.j1fd7

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