Abstract
Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
Alignments and raw phylogenetic data related to this study can be found in the following GitHub repository: https://github.com/spyros-lytras/BTN3A3_IAV. Source data related to the animal experiments illustrated in Fig. 3h are available in Supplementary Table 9. Gel source data are available in Supplementary Fig. 1.
References
Lipsitch, M. et al. Viral factors in influenza pandemic risk assessment. eLife https://doi.org/10.7554/eLife.18491 (2016).
Afrache, H., Gouret, P., Ainouche, S., Pontarotti, P. & Olive, D. The butyrophilin (BTN) gene family: from milk fat to the regulation of the immune response. Immunogenetics 64, 781–794 (2012).
Ye, Q., Krug, R. M. & Tao, Y. J. The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature 444, 1078–1082 (2006).
Yoon, S.-W., Webby, R. J. & Webster, R. G. in Influenza Pathogenesis and Control Vol. I (eds W. Compans, R. A. & Oldstone, M. B. A.) 359–375 (Springer International Publishing, 2014).
Krammer, F. et al. Influenza. Nat. Rev. Dis. Primers 4, 3 (2018).
Harrington, W. N., Kackos, C. M. & Webby, R. J. The evolution and future of influenza pandemic preparedness. Exp. Mol. Med. 53, 737–749 (2021).
Short, K. R. et al. One health, multiple challenges: the inter-species transmission of influenza A virus. One Health 1, 1–13 (2015).
Liu, D. et al. Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses. Lancet 381, 1926–1932 (2013).
Wang, X. et al. Epidemiology of avian influenza A H7N9 virus in human beings across five epidemics in mainland China, 2013–17: an epidemiological study of laboratory-confirmed case series. Lancet Infect. Dis. 17, 822–832 (2017).
Liu, W. J. et al. Avian influenza A (H7N9) virus: from low pathogenic to highly pathogenic. Front. Med. 15, 507–527 (2021).
Long, J. S., Mistry, B., Haslam, S. M. & Barclay, W. S. Host and viral determinants of influenza A virus species specificity. Nat. Rev. Microbiol. 17, 67–81 (2019).
Rogers, G. N. & Paulson, J. C. Receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the H3 hemagglutinin based on species of origin. Virology 127, 361–373 (1983).
Di Lella, S., Herrmann, A. & Mair, C. M. Modulation of the pH stability of influenza virus hemagglutinin: a host cell adaptation strategy. Biophys. J. 110, 2293–2301 (2016).
Zaraket, H. et al. Increased acid stability of the hemagglutinin protein enhances H5N1 influenza virus growth in the upper respiratory tract but is insufficient for transmission in ferrets. J. Virol. 87, 9911–9922 (2013).
Long, J. S. et al. Species difference in ANP32A underlies influenza A virus polymerase host restriction. Nature 529, 101–104 (2016).
Blumenkrantz, D., Roberts, K. L., Shelton, H., Lycett, S. & Barclay, W. S. The short stalk length of highly pathogenic avian influenza H5N1 virus neuraminidase limits transmission of pandemic H1N1 virus in ferrets. J. Virol. 87, 10539–10551 (2013).
Park, S. et al. Adaptive mutations of neuraminidase stalk truncation and deglycosylation confer enhanced pathogenicity of influenza A viruses. Sci. Rep. 7, 10928 (2017).
Mänz, B. et al. Pandemic influenza A viruses escape from restriction by human MxA through adaptive mutations in the nucleoprotein. PLoS Pathog. 9, e1003279 (2013).
Riegger, D. et al. The nucleoprotein of newly emerged H7N9 influenza A virus harbors a unique motif conferring resistance to antiviral human MxA. J. Virol. 89, 2241–2252 (2015).
Shaw, A. E. et al. Fundamental properties of the mammalian innate immune system revealed by multispecies comparison of type I interferon responses. PLoS Biol. 15, e2004086 (2017).
Kane, M. et al. Identification of interferon-stimulated genes with antiretroviral activity. Cell Host Microbe. 20, 392–405 (2016).
Feeley, E. M. et al. IFITM3 inhibits influenza A virus infection by preventing cytosolic entry. PLoS Pathog. 7, e1002337 (2011).
Verhelst, J., Parthoens, E., Schepens, B., Fiers, W. & Saelens, X. Interferon-inducible protein Mx1 inhibits influenza virus by interfering with functional viral ribonucleoprotein complex assembly. J. Virol. 86, 13445–13455 (2012).
Wellington, D., Laurenson-Schafer, H., Abdel-Haq, A. & Dong, T. IFITM3: how genetics influence influenza infection demographically. Biomed. J. 42, 19–26 (2019).
Rhodes, D. A., Stammers, M., Malcherek, G., Beck, S. & Trowsdale, J. The cluster of BTN genes in the extended major histocompatibility complex. Genomics 71, 351–362 (2001).
Kumar, S., Stecher, G., Suleski, M. & Hedges, S. B. TimeTree: a resource for timelines, timetrees, and divergence times. Mol. Biol. Evol. 34, 1812–1819 (2017).
Afrache, H., Pontarotti, P., Abi-Rached, L. & Olive, D. Evolutionary and polymorphism analyses reveal the central role of BTN3A2 in the concerted evolution of the BTN3 gene family. Immunogenetics 69, 379–390 (2017).
Pinto, R. M., Lycett, S., Gaunt, E. & Digard, P. Accessory gene products of influenza A virus. Cold Spring Harb. Perspect. Med. https://doi.org/10.1101/cshperspect.a038380 (2021).
Naffakh, N., Tomoiu, A., Rameix-Welti, M. A. & van der Werf, S. Host restriction of avian influenza viruses at the level of the ribonucleoproteins. Annu. Rev. Microbiol. 62, 403–424 (2008).
Patrono, L. V. et al. Archival influenza virus genomes from Europe reveal genomic variability during the 1918 pandemic. Nat. Commun. 13, 2314 (2022).
van Lieshout, L. P. et al. A novel triple-mutant AAV6 capsid induces rapid and potent transgene expression in the muscle and respiratory tract of mice. Mol. Ther. Methods Clin. Dev. 9, 323–329 (2018).
Portela, A. & Digard, P. The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J. Gen. Virol. 83, 723–734 (2002).
Gabriel, G., Herwig, A. & Klenk, H. D. Interaction of polymerase subunit PB2 and NP with importin alpha1 is a determinant of host range of influenza A virus. PLoS Pathog. 4, e11 (2008).
Hu, Y., Sneyd, H., Dekant, R. & Wang, J. Influenza A virus nucleoprotein: a highly conserved multi-functional viral protein as a hot antiviral drug target. Curr. Top. Med. Chem. 17, 2271–2285 (2017).
Beaton, A. R. & Krug, R. M. Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid protein and the absence of a 5′ capped end. Proc. Natl Acad. Sci. USA 83, 6282–6286 (1986).
Chen, Y. et al. Rare variant MX1 alleles increase human susceptibility to zoonotic H7N9 influenza virus. Science 373, 918–922 (2021).
Pu, J. et al. Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus. Proc. Natl Acad. Sci. USA 112, 548–553 (2015).
He, J. et al. Genetic characterization of the first detected human case of avian influenza A (H5N6) in Anhui Province, East China. Sci. Rep. 8, 15282 (2018).
de Jong, J. C., Claas, E. C., Osterhaus, A. D., Webster, R. G. & Lim, W. L. A pandemic warning? Nature 389, 554 (1997).
Neumann, G., Chen, H., Gao, G. F., Shu, Y. & Kawaoka, Y. H5N1 influenza viruses: outbreaks and biological properties. Cell Res. 20, 51–61 (2010).
Su, S. et al. Epidemiology, evolution, and pathogenesis of H7N9 influenza viruses in five epidemic waves since 2013 in China. Trends Microbiol. 25, 713–728 (2017).
Harly, C. et al. Key implication of CD277/butyrophilin-3 (BTN3A) in cellular stress sensing by a major human γδ T-cell subset. Blood 120, 2269–2279 (2012).
Arnett, H. A. & Viney, J. L. Immune modulation by butyrophilins. Nat. Rev. Immunol. 14, 559–569 (2014).
Gu, S., Borowska, M. T., Boughter, C. T. & Adams, E. J. Butyrophilin3A proteins and Vγ9Vδ2 T cell activation. Semin. Cell Dev. Biol. 84, 65–74 (2018).
Galão, R. P. et al. TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction. PLoS Pathog. 18, e1010530 (2022).
Kuroda, M. et al. Identification of interferon-stimulated genes that attenuate Ebola virus infection. Nat. Commun. 11, 2953 (2020).
Staeheli, P., Grob, R., Meier, E., Sutcliffe, J. G. & Haller, O. Influenza virus-susceptible mice carry Mx genes with a large deletion or a nonsense mutation. Mol. Cell. Biol. 8, 4518–4523 (1988).
Guénet, J. L. & Bonhomme, F. Wild mice: an ever-increasing contribution to a popular mammalian model. Trends Genet. 19, 24–31 (2003).
Zhang, B. et al. The nucleoprotein of influenza A virus inhibits the innate immune response by inducing mitophagy. Autophagy https://doi.org/10.1080/15548627.2022.2162798 (2023).
Philippon, D. A. M., Wu, P., Cowling, B. J. & Lau, E. H. Y. Avian influenza human infections at the human-animal interface. J. Infect. Dis. 222, 528–537 (2020).
Adlhoch, C. et al. Avian influenza overview December 2021 - March 2022. EFSA J. 20, e07289 (2022).
Agüero, M. et al. Highly pathogenic avian influenza A(H5N1) virus infection in farmed minks, Spain, October 2022. Euro. Surveill. https://doi.org/10.2807/1560-7917.Es.2023.28.3.2300001 (2023).
Bussey, K. A., Bousse, T. L., Desmet, E. A., Kim, B. & Takimoto, T. PB2 residue 271 plays a key role in enhanced polymerase activity of influenza A viruses in mammalian host cells. J. Virol. 84, 4395–4406 (2010).
Burke, S. A. & Trock, S. C. Use of influenza risk assessment tool for prepandemic preparedness. Emerg. Infect. Dis. 24, 471–477 (2018).
Ramirez, R. D. et al. Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res. 64, 9027–9034 (2004).
Wit, E. D. et al. Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments. Virus Res. 103, 155–161 (2004).
Schoggins, J. W. et al. A diverse range of gene products are effectors of the type I interferon antiviral response. Nature 472, 481–485 (2011).
Rihn, S. J. et al. TRIM69 inhibits vesicular stomatitis Indiana virus. J. Virol. https://doi.org/10.1128/JVI.00951-19 (2019).
Rihn, S. J. et al. A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research. PLoS Biol. 19, e3001091 (2021).
Kawakami, E. et al. Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA. J. Virol. Methods 173, 1–6 (2011).
Bakshi, S., Taylor, J., Strickson, S., McCartney, T. & Cohen, P. Identification of TBK1 complexes required for the phosphorylation of IRF3 and the production of interferon β. Biochem. J. 474, 1163–1174 (2017).
Zufferey, R., Donello, J. E., Trono, D. & Hope, T. J. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J. Virol. 73, 2886–2892 (1999).
Rghei, A. D. et al. Production of adeno-associated virus vectors in cell stacks for preclinical studies in large animal models. J. Vis. Exp. https://doi.org/10.3791/62727 (2021).
MacLeod, M. K. et al. Vaccine adjuvants aluminum and monophosphoryl lipid A provide distinct signals to generate protective cytotoxic memory CD8 T cells. Proc. Natl Acad. Sci. USA 108, 7914–7919 (2011).
Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinf. 10, 421 (2009).
Cunningham, F. et al. Ensembl 2022. Nucleic Acids Res. 50, D988–d995 (2022).
Mistry, J., Finn, R. D., Eddy, S. R., Bateman, A. & Punta, M. Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. Nucleic Acids Res. 41, e121 (2013).
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
Suyama, M., Torrents, D. & Bork, P. PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Res. 34, W609–W612 (2006).
Nguyen, L. T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).
Steinegger, M. & Söding, J. MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nat. Biotechnol. 35, 1026–1028 (2017).
Sagulenko, P., Puller, V. & Neher, R. A. TreeTime: maximum-likelihood phylodynamic analysis. Virus Evol. 4, vex042 (2018).
Yu, G. Using ggtree to visualize data on tree-like structures. Curr. Protoc. Bioinformatics 69, e96 (2020).
Huerta-Cepas, J., Serra, F. & Bork, P. ETE 3: reconstruction, analysis, and visualization of phylogenomic data. Mol. Biol. Evol. 33, 1635–1638 (2016).
Beare, A. S. & Hall, T. S. Recombinant influenza-A viruses as live vaccines for man. Report to the Medical Research Council’s Committee on Influenza and other Respiratory Virus Vaccines. Lancet 2, 1271–1273 (1971).
Beare, A. S., Schild, G. C. & Craig, J. W. Trials in man with live recombinants made from A/PR/8/34 (H0 N1) and wild H3 N2 influenza viruses. Lancet 2, 729–732 (1975).
Acknowledgements
We are thankful to the authors, originating and submitting laboratories of the sequences from GISAID’s EpiFlu Database on which some of this research is based (Supplementary Tables 7 and 8). We thank P. Murcia (MRC-University of Glasgow Centre for Virus Research) for providing clinical virus isolates and fruitful discussions, and M. Peiris (The University of Hong Kong) who kindly provided A/ruddy shelduck/Mongolia/963V/2009 (H3N8). We are grateful to S. Bhat, M. Iqbal (The Pirbright Institute), L. Tiley, R. Fouchier (Erasmus MC) and D. Perez (The University of Georgia) for providing the reverse genetics systems of the A/Anhui/1/2013 (H7N9) and A/mallard/Netherlands/10-Cam/1999 (H1N1), A/Puerto Rico/8/1934 (H1N1) and A/California/04-061-MA/2009 (H1N1) viruses, respectively. We acknowledge J. Mccauley (The Crick Institute) for sharing the MDCK-SIAT cells. This work was supported by the UK Medical Research Council (grant no. MC_UU_12016/10) awarded to M.P. and S.J.W. and the following grants: Wellcome Trust grant no. 206369/Z/17/Z (to M.P.); Biotechnology and Biological Sciences Research Council (BBSRC) grant no. BB/P013740/1 awarded to F.G. and P.D.; grant no. BBSRC BB/S00114X/1 awarded to F.G., P.D. and S.J.W.; EU Horizon2020: DELTA-FLU (grant no. 727922) awarded to P.D. and I.M.; Natural Sciences and Engineering Research Council of Canada Discovery grant (no. RGPIN-2018-04737) awarded to S.K.W.; Daphne Jackson Fellowship funded by Medical Research Scotland awarded to S.S.; MRC Career Development Award and Transition Support Award (grant nos. MR/N008618/1 and MR/V035789/1) to E.H.; Wellcome Trust grant no. 210703/Z/18/Z awarded to M.K.L.M. and Medical Research Council grant no. MC_UU_12014/5 awarded to C.B., Q.G. and J.H. are funded by Medical Research Council grant no. MC_UU_12014/12.
Author information
Authors and Affiliations
Contributions
Conceptualization was done by R.M.P., S.J.W. and M.P. The methodology was developed by R.M.P., S.B., S.L., M.K.Z., S.S., J.C.W., V.H., K.E.H., L.O., S.K.W. and M.K.L.M. Software was provided by S.L., J.H. and Q.G. Validation was done by R.M.P., S.B., S.L., M.K.Z., J.C.W., V.H. and K.E.H. Formal analysis was carried out by R.M.P., S.B., S.L., M.K.Z., S.S., V.H., M. Varjak, N.C.-R., M.C.R., M. Varela and J.H. The investigation was carried out by R.M.P., S.B., S.L., M.K.Z., S.S., J.C.W., V.H., M. Varjak, N.C.-R., M.C.R., M. Varela, L.O., A.W., C.L., Y.P., E.V., M.L.T., W.F., K.E.H., A.T., O.D. and C.D. Resources were provided by R.M.P., A.T., C.B., E.H., P.D., I.M., S.K.W., S.J.W. and M.P. Data were curated by R.M.P., S.L., M. Varjak, N.C.-R., M.C.R., M. Varela, A.W. and E.V. The original draft preparation and writing were done by R.M.P., S.B., S.L., M.K.Z., J.C.W., V.H., M.L.T., S.K.W. and M.P. Review and editing of the draft were done by R.M.P., S.B., S.L., M.K.Z., S.S., J.C.W., V.H., M.V.k., N.C.-R., M.C.R., M. Varela, A.W., C.L., J.H., E.V., M.L.T., W.F., K.E.H., Q.G., L.O., A.T., O.D., F.G., E.H., P.D., I.M., S.K.W., M.K.L.M., S.J.W. and M.P. Visualization was done by R.M.P., S.B., S.L. and M.P. Supervision was done by R.M.P., J.H., C.B., S.K.W., M.K.L.M., S.J.W. and M.P. Project administration was done by S.J.W. and M.P. Funding was acquired by S.S., C.B., F.G., P.D., I.M., S.K.W., M.K.L.M., S.J.W. and M.P.
Corresponding author
Ethics declarations
Competing interests
P.D. is a member of the Science Advisory Council’s Exotic and Emerging Animal Diseases subgroup (SAC-ED) for the UK Government’s Department for Environment, Food & Rural Affairs (Defra) and was part of SAC-ED’s independent expert Scientific Advisory Group in highly pathogenic avian influenza. S.K.W. is an inventor on issued patents in Canada and the United States for the AAV6.2FF capsid, which are owned by the University of Guelph, and licensed to Avamab Pharma Inc., Inspire Biotherapeutics, and Cellastra Inc. M.P. is a member of the Standing Committee on Pandemic Preparedness of the Scottish Government. The remaining authors declare no competing interests.
Peer review
Peer review information
Nature thanks Andrew Mehle, Peter Staeheli, Michael Worobey and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Activity and expression of BTN3A1 and BTN3A3 in cell lines and human tissues.
a, siRNA knock-down of BTN3A1 in MRC5T and hBEC3-KT. Cells were transfected with scrambled (Neg ctrl) or BTN3A1-targeting siRNAs, and protein levels in the resulting cell lysates were assessed by western blotting. α-Tubulin was used as loading control. Arrows indicate the band corresponding to BTN3A1. For gel source data, see Supplementary Fig. 1. b, Graphs showing the replication kinetics of PR8 and Mallard in siRNA-treated MRC5T and hBEC3-KT cells. Cells were infected with a MOI of 0.001, supernatants were collected at the indicated times post infection and viruses titrated by plaque assay. Data are mean +/− SEM of 3 independent experiments (each using 2 technical replicates). Statistical significance between groups was measured by a 2-way ANOVA. Comparisons were made between area under the curve of the different BTN3A1 siRNA treatment conditions and the average of the two negative controls. No statistically significant differences were found. c, Organ-dependent bulk tissue gene expression. Lung samples are highlighted in blue. d, Lung single cell tissue expression. Data in c and d were obtained from the GTEx Portal (www.gtexportal.org). e, Western blotting of cell lysates obtained from A549, MRC5T, hBEC3-KT and Calu-3 treated with either IFN-γ or universal type-I IFN. Treatment with IFN was for 16h in A549, MRC5T, hBEC3-KT and for 24h in Calu-3 cells. pSTAT1 and RSAD2/Viperin were used as IFN induction controls and α-Tubulin as loading control. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 2 Evolution of BTNs.
a, Maximum likelihood phylogeny of concatenated protein domain coding sequences of the Haplorrhini BTN3 genes (K2P+G4 substitution model). Nodes with bootstrap support below 60 have been collapsed. Branches confirmed to have or not have anti-avian IAV activity (described in b) are highlighted in yellow and blue, respectively. Branches not tested were kept black. Relation to more distant tested homologues and orthologous/paralogous gene families are shown as a schematic in grey. IgV homogenization events, major gene duplications and gene subfamilies are annotated on the phylogeny. Presence of each of the four protein domains (IgV, IgC, PRY and SPRY) is annotated on the right of each tree tip and coloured by pairwise amino acid similarity to the respective domain of the human BTN3A3. Species names and taxonomic classification is annotated on the right. The median divergence time between Catarrhini and Platyrrhini was retrieved from TimeTree (http://timetree.org/). b, Schematic representation of domain organisation and sequence similarity of the indicated proteins.
Extended Data Fig. 3 Evolution of antiviral activity of BTNs.
a, A549 cells were transiently transduced with SCRPSY lentiviruses expressing the indicated BTN proteins and challenged with PR8- or Mallard-GFP. Eight hours post infection, the percentage of GFP-positive cells was measured by flow cytometry. Data are mean +/− SEM of 2 independent experiments which both gave similar results. b, Western blotting of cell lysates obtained from A549 cells transiently transduced with SCPRSY lentiviruses expressing different BTN proteins. GAPDH was used as loading control. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 4 Phylogeny of BTN3 domains and antiviral activity of BTN orthologues and paralogues.
a-d, maximum likelihood Haplorrhini BTN3 gene coding sequence phylogenies of separate domains: IgV (a), IgC (b), PRY (c), SPRY (d) under a K2P+G4 substitution model. Trees are rooted at the C. syrichta branch and node confidence values (10,000 bootstrap replicates) are annotated on each node. Tip shapes are coloured by whether each gene exhibits anti-AIV activity (consistent with Fig. 3a). Phylogenies were visualised using FigTree. e–f, A549 cells were transiently transduced with SCRPSY lentiviruses expressing the indicated BTN proteins and challenged with PR8- or Mallard-GFP. Eight hours post-infection, percentage of RFP-positive cells and its subpopulation of GFP-positive cells was measured by flow cytometry. Data are mean +/− SEM of 2 independent experiments which both gave similar results. Detection of these proteins was not possible using commercially available antibodies, due to their genetic divergence compared to human BTN3A1-3. Therefore, tagging of these BTN genes was attempted by introducing a C-terminal FLAG. Despite their protein expression being successfully detected using an anti-FLAG antibody, the addition of FLAG to human BTN3A3 and other BTN genes resulted in the abolishment of their antiviral activity. Cloning of the genes indicated above was conducted in the same way as those constructs shown in Extended Data Fig. 3.
Extended Data Fig. 5 Determinants of BTN3A3 sensitivity.
a, PR8:Mallard reassortants and Udorn wild type (WT) were used to perform plaque assays in MDCK cells expressing BTN3A3 and empty vector control cells. Note that reassortant PR8 7:1 Mallard seg 1, Mallard 7:1 PR8 seg 2 and Mallard 7:1 PR8 seg 5 failed to rescue. Reassortant PR8 Mallard 3PNP is formed by PR8 (segments 4, 6, 7 and 8) and segments 1, 2, 3 and 5 from Mallard. Conversely, reassortant Mallard PR8 3PNP contain segments 4, 6, 7 and 8 from Mallard and segments 1, 2, 3 and 5 from PR8. Data are mean +/− SEM of 2 technical replicates from 2 independent experiments. Statistical differences between cells expressing BTN3A3 and control cells were calculated using multiple t-tests and corrected for multiple comparisons using the Holm-Šídák method. NS-Non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. b, Identity of amino acid residues in positions 100 (left) and 313 (right) of all NP proteins available in our dataset collected from GenBank. c, Identity of combinations between amino acid residues 100 and 313 (left) and 52 and 313 (right). d, Infectious virus titres obtained in A549-Empty and A549-BTN3A3 cells infected with either Mallard or Cal04 residues 100 and 313 mutants. Cells were infected with an MOI of 0.001 for 48h and viruses were titrated by plaque assay. Data are mean +/− SEM of 2 technical replicates from 3 independent experiments. Statistical analysis was carried out as in (a). e, Viral replication assays in avian cells were carried out in chicken fibroblasts (DF1 cells). Cells were infected an MOI of 0.001 for 48 h. Infectious virus titres were determined by plaque assay. Data are mean +/− standard error of the mean (SEM) of 3 independent experiments (each using two technical replicates).
Extended Data Fig. 6 Molecular dating of the F313V NP substitution on the classical swine H1N1 lineage.
a, Tip-dated maximum likelihood phylogeny of all classical H1N1 lineage NP sequences annotated by position 313 residue (left) and isolation host (mirrored tree, right). b, Zoomed in snippet of the part of the ML phylogeny shown in A where the F313V change has occurred. Tip shapes are coloured by 313 residue, estimated dates for key nodes are annotated, and strain names are shown on the right of the tips. c, Zoomed in snippet of the part of the BEAST maximum clade credibility phylogeny where the F313V change has occurred. Tip shapes are coloured by 313 residue, median node age and 95% highest posterior density confidence intervals are annotated for key nodes, posterior probability values are shown for each node, and strain names are shown on the right of the tips. The branch where F313V is believed to have taken place on is annotated in colour (pink and green). Phylogenies were visualised using FigTree.
Extended Data Fig. 7 BTN3A3 activity and its relation to Mx1 and vRNP complexes.
a, Western blotting of cell lysates obtained from A549 cells transfected with the indicated amounts of control or Mx1-targeting pooled siRNAs. Cells were transfected for 48h followed by a 16h type-I IFN treatment. pSTAT1 and α-Tubulin were used as IFN-treatment and loading controls, respectively. For gel source data, see Supplementary Fig. 1. b, Upon siRNA treatment, A549 Empty and BTN3A3 cells were infected with the indicated viruses at an MOI of 0.001. Supernatants were harvested at 48 hpi and infectious viral titres were measured by plaque assay. Data are mean +/− SEM of 2 technical replicates from 3 independent experiments. c, Quantification of cytoplasmic and nuclear levels of vRNP complex proteins at early stages post infection in the presence or absence of BTN3A3. Quantification of 3 independent western blots, one set of which is shown in Fig. 4a. A549-Empty and BTN3A3 overexpressing cells were synchronously infected with PR8 or Mallard at MOI 3. Nuclear/cytoplasm fractionation was performed at 45, 90 mins and 6h post infection. Quantification of vRNP-complex proteins was performed by fluorescence measurements. Cytoplasmic and nuclear viral proteins were normalized to GAPDH and H3, respectively. All values were further normalised to values of A549-Empty cells. Data are mean +/− SEM of 3 independent experiments (each using 2 technical replicates). Statistical significance between groups was measured by a 2-way ANOVA. Comparisons were made between A549-Empty and A549-BTN3A3. NS = non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Extended Data Fig. 8 Minireplicon assays with avian/mammalian NP reassortant RNP complexes.
a, 293T-Empty and 293T-BTN3A3 cells were transfected with pcDNA plasmids encoding for PB2, PB1, PA and NP of the indicated viruses alongside firefly luciferase-coding vRNA- or cRNA-like reporter plasmids. A transfection control plasmid expressing Renilla firefly was also added. Forty-eight hours post-transection cells were lysed and firefly and Renilla luciferase activities were measured. Values were normalised to PR8 or Cal04 WT replicons with respective NPs transfected in 293T-Empty cells. Data are mean +/− SEM of 3 independent experiments (each using 2 technical replicates). Statistical differences between Empty and HsBTN3A3 overexpressing cells were calculated using multiple t-tests and corrected for multiple comparisons using the Holm-Šídák method. NS = non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. b. Expression levels of PB2, PB1, PA and NP transfected in 293T-Empty (E) or 293T-BTN3A3 (B3) cells were assessed by western blot. α-Tubulin was used as loading control. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 9 Interactions between NP and BTN3A3.
a, Western blotting of hBEC3-KT cells infected with IAV Mallard WT or Mallard NP F313Y for 6 h. Expression of NP, PA, PB1, PB2 and GAPDH (loading control) from total cell lysates is shown. b, Total cell lysates of infected cells were used to perform NP immunoprecipitation followed by the detection of the remaining RNP complex-forming proteins. c, Supernatants of the NP immunoprecipitates was used to perform an additional immunoprecipitation using an anti-BTN3A3 antibody followed by the detection of PB2, PB1 and NP. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 10 Correlation between NP signal and nuclear BTN3A3.
a, Representative images of confocal microscopy of hBEC3-KT cells infected with Cal04WT or Cal04 NP V313F at MOI 3. Six hours post infection, cells were immunostained with NP (red) and BTN3A3 (green). DAPI staining (blue) was used as a nuclear marker. Scale bar = 35µm. b, Images from >3500 cells from four independent experiments performed as in (a) were used to quantify total NP and nuclear BTN3A3 for Cal04 WT and Cal04 NP V313F. c, Values from b were stratified based on nuclear BTN3A3 intensity (<0.2 or ≥0.2). Data represents relative abundance of total infected cells present in each of the two nuclear BTN3A3 intensities ranges, taking values obtained with Cal04 WT as 100%.
Extended Data Fig. 11 Distinct requirements of NP residues for BTN3A3 and Mx1 evasion, and GWAS analysis of H7N9 patients.
a, A549 cells overexpressing human Mx1 or BTN3A3 were infected with the mentioned viruses at a MOI of 0.001. Supernatants were harvested at 48 hpi and infectious viral titres were measured by plaque assay. Data are mean +/− SEM of 3 independent experiments (each using 2 technical replicates). Statistical significance between groups was measured by a 2-way ANOVA. NS- non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (b-c) GWAS analysis of H7N9-infected patients. Manhattan plots of MX1 (b) and BTN3A3 (c) genomic regions. Gene-level p-values were acquired using RACER in R from data obtained from Chen, et al., Science 373, 918-922, doi:10.1126/science.abg5953 (2021).
Extended Data Fig. 12 BTN3A3 restriction by highly pathogenic H5N1.
a, Schematic representation of numbers and characteristics of avian IAV NP sequences identified in human spillover events. Total number of sequences was divided into BTN3A3-resistant or sensitive genotypes based on amino acid residues 313 and 52. NP sequences were matched with their respective HA sequences and highly pathogenic avian influenza (HPAI) viruses were separated from the low pathogenic (LPAI) based on the presence of a polybasic cleavage site (only present in HPAI isolates). b, Replication of 7:1 reassortants of PR8 encoding segment 5 from Mallard or H5N1 HPAI viruses in A549-Empty or A549-BTN3A3 expressing cells. Cells were infected at an MOI of 0.001 for 48h and titres were measured by plaque assay. Data are mean +/− SEM of 3 independent experiments (each using 2 technical replicates) with the exception of 52Q which was instead a single technical replicate from 3 independent experiments. Statistical differences between values obtained in empty and BTN3A3 expressing cells were calculated using multiple t-tests and corrected for multiple comparisons using the Holm-Šídák method. NS = non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Supplementary information
Supplementary Discussion
Molecular dating of the F313V NP change.
Supplementary Fig. 1
Original source images for data obtained by electrophoretic separation.
Supplementary Fig. 2
Example flow cytometry gating of an IAV-GFP arrayed ISG expression screen.
Supplementary Table 1
ISG screen results. Percentage of transduced cells and relative virus infectivity are shown for screens illustrated in Figs. 1b and 2e. Mock-transfected and/or mock-infected controls are also included.
Supplementary Table 2
Dataset of avian IAV isolated from human patients downloaded from the GISAID EpiFlu database. Nature of residues NP 52 and 313, and PB2 271, 590, 591, 627 and 701 for each isolate are shown.
Supplementary Table 3
Genetic variation of human BTN3A3. Haplotype allelic frequencies of all detected variants of BTN3A3 shown by region. Data were acquired from Ensembl transcript ENST00000244519.7. FLAG definitions: S, Introduction of a STOP codon; D, Deleterious shift or polyphen.
Supplementary Table 4
Dataset of avian IAV isolated from birds downloaded from GISAID EpiFlu database. Nature of residues NP 52 and 313, and PB2 271, 590, 591, 627 and 701 for each isolate are shown.
Supplementary Table 5
NCBI accessions of all BTN3 homologues presented in this study.
Supplementary Table 6
Classical H1N1 swine clade NP sequence NCBI accessions with subclade and BEAST dataset annotations.
Supplementary Table 7
GISAID EpiFlu acknowledgements for the avian IAV human isolates used in this study.
Supplementary Table 8
GISAID EpiFlu acknowledgements for the avian IAV bird isolates used in this study.
Supplementary Table 9
Source data for animal experiments.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Pinto, R.M., Bakshi, S., Lytras, S. et al. BTN3A3 evasion promotes the zoonotic potential of influenza A viruses. Nature 619, 338–347 (2023). https://doi.org/10.1038/s41586-023-06261-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-023-06261-8
This article is cited by
-
Evolution of enhanced innate immune suppression by SARS-CoV-2 Omicron subvariants
Nature Microbiology (2024)
-
A human protein that holds bird flu viruses at bay
Nature (2023)
-
Avian influenza takes flight in humans by evading restriction
Nature Reviews Microbiology (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
