Abstract
Orthoflaviviruses are RNA viruses that cause serious diseases in humans, with currently no antivirals available. Targeting host factors is emerging as an attractive antiviral approach. However, as a first step, there is a need to understand which host proteins are hijacked and for what purpose. Here, using a combination of fluorescence microscopy, knock-down, crosslinking immunoprecipitation sequencing, mass spectrometry, and in vitro and biophysical assays, we identify nucleoporin-153 (NUP153) as a proviral factor during orthoflavivirus infection. We show that NUP153 is recruited to the virus amplification site on the endoplasmic reticulum to impact the structural to nonstructural viral protein ratios. We find that NUP153 interacts with both the viral proteins NS3 and NS5, and a highly conserved G-rich motif on the viral RNA. These interactions specifically promote the production of viral structural proteins, leading to an efficient virion assembly, virus release and spread to new cells. We propose that NUP153 acts as a key regulator in viral protein ratios, a mechanism that appears conserved among orthoflaviviruses.
Data availability
Source data are provided with this paper. The affinity purification mass spectrometry data generated in this study have been deposited in the PRIDE database under accession code PXD052523. The mass spectrometry data from WNV-infected cells generated in this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD067546. The CLIP-seq data generated in this study have been deposited in the Sequence Read Archive database under accession code PRJNA1137903. Source data are provided with this paper.
References
Mandl, C. W., Ecker, M., Holzmann, H., Kunz, C. & Heinz, F. X. Infectious cDNA clones of tick-borne encephalitis virus European subtype prototypic strain Neudoerfl and high virulence strain Hypr. J. Gen. Virol. 78, 1049–1057 (1997).
Panayiotou, C. et al. Viperin restricts Zika virus and tick-borne encephalitis virus replication by targeting NS3 for proteasomal degradation. J. Virol. 92, https://doi.org/10.1128/JVI.02054-17 (2018).
Staples, J. E. & Monath, T. P. Yellow fever: 100 years of discovery. JAMA 300, 960–962 (2008).
Mansfield, K. L. et al. Tick-borne encephalitis virus—a review of an emerging zoonosis. J. Gen. Virol. 90, 1781–1794 (2009).
Rodrigues, R., Danskog, K., Överby, A. K. & Arnberg, N. Characterizing the cellular attachment receptor for Langat virus. PLoS ONE 14, e0217359 (2019).
Miorin, L. et al. Three-dimensional architecture of tick-borne encephalitis virus replication sites and trafficking of the replicated RNA. J. Virol. 87, 6469–6481 (2013).
Lindqvist, R., Upadhyay, A. & Överby, A. K. Tick-borne flaviviruses and the type I interferon response. Viruses 10, https://doi.org/10.3390/v10070340 (2018).
Doye, V. & Hurt, E. From nucleoporins to nuclear pore complexes. Curr. Opin. Cell Biol. 9, 401–411 (1997).
Schwartz, T. U. The structure inventory of the nuclear pore complex. J. Mol. Biol. 428, 1986–2000 (2016).
Beck, M. & Hurt, E. The nuclear pore complex: understanding its function through structural insight. Nat. Rev. Mol. Cell Biol. 18, 73–89 (2017).
Ullman, K. S., Shah, S., Powers, M. A. & Forbes, D. J. The nucleoporin nup153 plays a critical role in multiple types of nuclear export. Mol. Biol. Cell 10, 649–664 (1999).
Walther, T. C. et al. The nucleoporin Nup153 is required for nuclear pore basket formation, nuclear pore complex anchoring and import of a subset of nuclear proteins. EMBO J. 20, 5703–5714 (2001).
Neufeldt, C. J. et al. Hepatitis C virus-induced cytoplasmic organelles use the nuclear transport machinery to establish an environment conducive to virus replication. PLoS Pathog. 9, e1003744 (2013).
Nofrini, V., Di Giacomo, D. & Mecucci, C. Nucleoporin genes in human diseases. Eur. J. Hum. Genet. 24, 1388–1395 (2016).
Davis, L. I. & Blobel, G. Nuclear pore complex contains a family of glycoproteins that includes p62: glycosylation through a previously unidentified cellular pathway. Proc. Natl. Acad. Sci. USA 84, 7552–7556 (1987).
Moreno, H., Moller, R., Fedeli, C., Gerold, G. & Kunz, S. Comparison of the innate immune responses to pathogenic and nonpathogenic clade B new world arenaviruses. J. Virol. 93, https://doi.org/10.1128/JVI.00148-19 (2019).
Ci, Y. & Shi, L. Compartmentalized replication organelle of flavivirus at the ER and the factors involved. Cell. Mol. Life Sci. 78, 4939–4954 (2021).
Aksenova, V. et al. Nucleoporin TPR is an integral component of the TREX-2 mRNA export pathway. Nat. Commun. 11, 4577 (2020).
Liao, P. et al. A positive feedback loop between EBP2 and c-Myc regulates rDNA transcription, cell proliferation, and tumorigenesis. Cell Death Dis. 5, e1032 (2014).
Su, L., Hershberger, R. J. & Weissman, I. L. LYAR, a novel nucleolar protein with zinc finger DNA-binding motifs, is involved in cell growth regulation. Genes Dev. 7, 735–748 (1993).
Kondoh, H., Yuasa, T. & Yanagida, M. Mis3 with a conserved RNA binding motif is essential for ribosome biogenesis and implicated in the start of cell growth and S phase checkpoint. Genes Cells 5, 525–541 (2000).
Chen, D. et al. RPS12-specific shRNA inhibits the proliferation, migration of BGC823 gastric cancer cells with S100A4 as a downstream effector. Int. J. Oncol. 42, 1763–1769 (2013).
Yuan, F., Li, G. & Tong, T. Nucleolar and coiled-body phosphoprotein 1 (NOLC1) regulates the nucleolar retention of TRF2. Cell Death Discov. 3, 17043 (2017).
Calo, E. et al. RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. Nature 518, 249–253 (2015).
Liang, W. L. et al. miR-892a regulated PPP2R2A expression and promoted cell proliferation of human colorectal cancer cells. Biomed. Pharmacother. 72, 119–124 (2015).
Song, D., Peng, K., Palmer, B. E. & Lee, F. S. The ribosomal chaperone NACA recruits PHD2 to cotranslationally modify HIF-alpha. EMBO J. 41, e112059 (2022).
Selinger, M. et al. Tick-borne encephalitis virus inhibits rRNA synthesis and host protein production in human cells of neural origin. PLoS Negl. Trop. Dis. 13, e0007745 (2019).
Finkel, Y. et al. SARS-CoV-2 uses a multipronged strategy to impede host protein synthesis. Nature 594, 240–245 (2021).
Pulkkinen, L. I. A. et al. Simultaneous membrane and RNA binding by tick-borne encephalitis virus capsid protein. PLoS Pathog. 19, e1011125 (2023).
Diosa-Toro, M., Prasanth, K. R., Bradrick, S. S. & Garcia Blanco, M. A. Role of RNA-binding proteins during the late stages of flavivirus replication cycle. Virol. J. 17, 60 (2020).
Griffis, E. R., Craige, B., Dimaano, C., Ullman, K. S. & Powers, M. A. Distinct functional domains within nucleoporins Nup153 and Nup98 mediate transcription-dependent mobility. Mol. Biol. Cell 15, 1991–2002 (2004).
Nakielny, S., Shaikh, S., Burke, B. & Dreyfuss, G. Nup153 is an M9-containing mobile nucleoporin with a novel Ran-binding domain. EMBO J. 18, 1982–1995 (1999).
Duheron, V., Chatel, G., Sauder, U., Oliveri, V. & Fahrenkrog, B. Structural characterization of altered nucleoporin Nup153 expression in human cells by thin-section electron microscopy. Nucleus 5, 601–612 (2014).
Bastos, R., Lin, A., Enarson, M. & Burke, B. Targeting and function in mRNA export of nuclear pore complex protein Nup153. J. Cell Biol. 134, 1141–1156 (1996).
Di Nunzio, F. et al. Nup153 and Nup98 bind the HIV-1 core and contribute to the early steps of HIV-1 replication. Virology 440, 8–18 (2013).
Ball, J. R. et al. Sequence preference in RNA recognition by the nucleoporin Nup153. J. Biol. Chem. 282, 8734–8740 (2007).
Kikin, O., D’Antonio, L. & Bagga, P. S. QGRS mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences. Nucleic Acids Res. 34, W676–W682 (2006).
Fleming, A. M., Ding, Y., Alenko, A. & Burrows, C. J. Zika virus genomic RNA possesses conserved G-quadruplexes characteristic of the Flaviviridae family. ACS Infect. Dis. 2, 674–681 (2016).
Holoubek, J. et al. Guanine quadruplexes in the RNA genome of the tick-borne encephalitis virus: their role as a new antiviral target and in virus biology. Nucleic Acids Res. 50, 4574–4600 (2022).
Guo, J. U. & Bartel, D. P. RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria. Science 353, https://doi.org/10.1126/science.aaf5371 (2016).
Feng, H. & Kwok, C. K. Spectroscopic analysis reveals the effect of hairpin loop formation on G-quadruplex structures. RSC Chem. Biol. 3, 431–435 (2022).
Weldon, C., Eperon, I. C. & Dominguez, C. Do we know whether potential G-quadruplexes actually form in long functional RNA molecules? Biochem. Soc. Trans. 44, 1761–1768 (2016).
Fajardo, T. et al. The flavivirus polymerase NS5 regulates translation of viral genomic RNA. Nucleic Acids Res. 48, 5081–5093 (2020).
Lubick, K. J. et al. Flavivirus antagonism of type I interferon signaling reveals prolidase as a regulator of IFNAR1 surface expression. Cell Host Microbe 18, 61–74 (2015).
Mazzon, M., Jones, M., Davidson, A., Chain, B. & Jacobs, M. Dengue virus NS5 inhibits interferon-alpha signaling by blocking signal transducer and activator of transcription 2 phosphorylation. J. Infect. Dis. 200, 1261–1270 (2009).
Gracias, S. et al. Tick-borne flavivirus NS5 antagonizes interferon signaling by inhibiting the catalytic activity of TYK2. EMBO Rep. 24, e57424 (2023).
Fujiwara, T., Oda, K., Yokota, S., Takatsuki, A. & Ikehara, Y. Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum. J. Biol. Chem. 263, 18545–18552 (1988).
De Jesús-González, L. A. et al. The nuclear pore complex: a target for NS3 protease of dengue and Zika viruses. Viruses 12, https://doi.org/10.3390/v12060583 (2020).
Neufeldt, C. J. et al. The hepatitis C virus-induced membranous web and associated nuclear transport machinery limit access of pattern recognition receptors to viral replication sites. PLoS Pathog. 12, e1005428 (2016).
Overby, A. K., Popov, V. L., Niedrig, M. & Weber, F. Tick-borne encephalitis virus delays interferon induction and hides its double-stranded RNA in intracellular membrane vesicles. J. Virol. 84, 8470–8483 (2010).
Welsch, S. et al. Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe 5, 365–375 (2009).
Peters, M. B. A. et al. NUP98 regulates orthoflavivirus replication through interaction with vRNA and can be targeted for antiviral purposes. Nucleic Acids Res. 54, https://doi.org/10.1093/nar/gkag027 (2026).
Majee, P. et al. Inhibition of Zika virus replication by G-quadruplex-binding ligands. Mol. Ther. Nucleic Acids 23, 691–701 (2021).
Bourdon, S. et al. QUADRatlas: the RNA G-quadruplex and RG4-binding proteins database. Nucleic Acids Res. 51, D240–D247 (2023).
Nagata, T. et al. Structure and interactions with RNA of the N-terminal UUAG-specific RNA-binding domain of hnRNP D0. J. Mol. Biol. 287, 221–237 (1999).
Belachew, B., Gao, J., Byrd, A. K. & Raney, K. D. Hepatitis C virus nonstructural protein NS3 unfolds viral G-quadruplex RNA structures. J. Biol. Chem. 298, 102486 (2022).
Butovskaya, E., Solda, P., Scalabrin, M., Nadai, M. & Richter, S. N. HIV-1 nucleocapsid protein unfolds stable RNA G-quadruplexes in the viral genome and is inhibited by G-quadruplex ligands. ACS Infect. Dis. 5, 2127–2135 (2019).
Stern-Ginossar, N., Thompson, S. R., Mathews, M. B. & Mohr, I. Translational control in virus-infected cells. Cold Spring Harb. Perspect. Biol. 11, https://doi.org/10.1101/cshperspect.a033001 (2019).
Moomau, C., Musalgaonkar, S., Khan, Y. A., Jones, J. E. & Dinman, J. D. Structural and functional characterization of programmed ribosomal frameshift signals in West Nile virus strains reveals high structural plasticity among cis-acting RNA elements. J. Biol. Chem. 291, 15788–15795 (2016).
Plumet, S., Duprex, W. P. & Gerlier, D. Dynamics of viral RNA synthesis during measles virus infection. J. Virol. 79, 6900–6908 (2005).
Aviner, R., Li, K. H., Frydman, J. & Andino, R. Cotranslational prolyl hydroxylation is essential for flavivirus biogenesis. Nature 596, 558–564 (2021).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Stirling, D. R. et al. CellProfiler 4: improvements in speed, utility and usability. BMC Bioinforma. 22, 433 (2021).
Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).
Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Cox, J. et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteom. 13, 2513–2526 (2014).
Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).
Kurhade, C. et al. Type I interferon response in olfactory bulb, the site of tick-borne flavivirus accumulation, is primarily regulated by IPS-1. J. Neuroinflamm. 13, 22 (2016).
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Galaxy, C. The galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2022 update. Nucleic Acids Res. 50, W345–W351 (2022).
Hahne, F. & Ivanek, R. Visualizing genomic data using gviz and bioconductor. Methods Mol. Biol. 1418, 335–351 (2016).
Larsson, A. AliView: a fast and lightweight alignment viewer and editor for large datasets. Bioinformatics 30, 3276–3278 (2014).
Ge, S. X., Jung, D. & Yao, R. ShinyGO: a graphical gene-set enrichment tool for animals and plants. Bioinformatics 36, 2628–2629 (2020).
Szklarczyk, D. et al. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 51, D638–D646 (2023).
Acknowledgements
We thank G. Dobler (Bundeswehr Institute of Microbiology, Munich, Germany) for providing stocks of LGTV TP21, TBEV Neudörfl, and ZIKV MR766 strains, and VeroB4 cells; S. Vene (Public Health Agency of Sweden) for providing stocks of JEV (Nakayama strain), WNV (WNV_0304h_ISR00), YFV (Asibi), and DENV (serotype-2; PNG/New Guinea C). DLD-1 and NUP153(NG)AID cells were provided by M. Dasso (Division of Molecular and Cellular Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA). We thank S. Lind (Chemistry-BMC, Uppsala University, Sweden), Bettina Sarg (Protein Core Facility, Institute of Medical Biochemistry, University of Innsbruck, Austria) and the Protein Core Facility of the Medical University of Innsbruck for the help with mass spectrometry data analysis. We thank C. Patthey (Department of Radiation Sciences, Oncology, Umeå University, Umeå, Sweden) for help with the CLIP-seq experiments. Funding: Laboratory for Molecular Infection Medicine Sweden (MIMS) VR2021-06602 to A.K.Ö.; Swedish Research Council, grants 2018-05851, 2020-06224 and 2024-00390 to A.K.Ö., 2018-05851 and 2024-00390 to R. Lu., 2020-03380 to Y.I., 2021-02468 to N.S.; the Swedish Foundation for Strategic Research, grant SB16-0039 to Y.I.; Swedish Cancer Society 22 2380 Pj to N.S.; and Knut and Alice Wallenberg foundations (KAW2021-0173) to N.S., (KAW2024-0039) to A.K.Ö., R. Lu. and German Research Foundation (INST 193/90-1 FUGG; project-ID: 497694394) to G.G. We also acknowledge Umeå Center for Microbial Research (UCMR); the Biochemical Imaging Center at Umeå University (BICU), the National Microscopy Infrastructure for Microscopy Support (NMI; VR-RFI 2019-00217); and BioMolecular Characterization Umeå (BMCU) for CD spectroscopy. We also thank Elevate Science for their editorial service. Illustrations (Figs. 4A, 5A, and 8L) were created in BioRender. Överby, A (2026) https://BioRender.com/67626ap, https://BioRender.com/r30wsap, https://BioRender.com/rw0asnh, https://BioRender.com/3sto0kr.
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M.B.A.P., A.K.Ö. conceived the study. All authors designed the experiments. M.B.A.P. acquired and analyzed data from confocal microscopy experiments, Co-IP, CLIP assays, purified proteins, RNA-EMSA, in vitro assays, knock-down experiments, and infections, and performed bioinformatic analysis of CLIP-seq and mass spectrometry data. R. Li., processed samples for mass spectrometry and performed infection experiments. E.K. processed samples, performed and analyzed the data for the mass spectrometry analysis. W.L.Y. scientific discussions and writing. P.S. performed the CD spectroscopy experiments. I.N. processed samples for NUP153 (NG)AID WNV-infected mass spectrometry experiment. M.B.A.P. prepared the figures. G.G., N.S., Y.I., R. Lu., and A.K.Ö. supervised the experiments. M.B.A.P., A.K.Ö. wrote the manuscript. All authors revised the manuscript.
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Peters, M.B.A., Lindqvist, R., Kassa, E. et al. Proviral NUP153 binding to viral proteins and RNA regulates structural–nonstructural protein ratios in orthoflavivirus infection. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71449-1
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DOI: https://doi.org/10.1038/s41467-026-71449-1
