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Identification of antiviral roles for the exon–junction complex and nonsense-mediated decay in flaviviral infection

Nature Microbiology volume 4, pages 985–995 (2019)Cite this article

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Abstract

West Nile virus (WNV) is an emerging mosquito-borne flavivirus, related to dengue virus and Zika virus. To gain insight into host pathways involved in WNV infection, we performed a systematic affinity-tag purification mass spectrometry (APMS) study to identify 259 WNV-interacting human proteins. RNA interference screening revealed 26 genes that both interact with WNV proteins and influence WNV infection. We found that WNV, dengue and Zika virus capsids interact with a conserved subset of proteins that impact infection. These include the exon–junction complex (EJC) recycling factor PYM1, which is antiviral against all three viruses. The EJC has roles in nonsense-mediated decay (NMD), and we found that both the EJC and NMD are antiviral and the EJC protein RBM8A directly binds WNV RNA. To counteract this, flavivirus infection inhibits NMD and the capsid–PYM1 interaction interferes with EJC protein function and localization. Depletion of PYM1 attenuates RBM8A binding to viral RNA, suggesting that WNV sequesters PYM1 to protect viral RNA from decay. Together, these data suggest a complex interplay between the virus and host in regulating NMD and the EJC.

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Fig. 1: Generation of the WNV–host protein interaction map.
Fig. 2: The WNV–human PPI network.
Fig. 3: Flaviviral capsid proteins interact with overlapping host factors.
Fig. 4: RNAi screening reveals important roles for host proteins that interact with WNV proteins.
Fig. 5: PYM1 is antiviral against flaviviruses and interacts with flavivirus capsid proteins.
Fig. 6: NMD is inhibited by flaviviruses through targeting of the EJC and the EJC protein RBM8A binds to viral RNA.

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Data availability

Mass spectrometry data in this study are deposited in the PRIDE database (https://www.ebi.ac.uk/pride/archive/, Project Accession no. PXD011728). A complete list of interaction scores are provided in Supplementary Tables 1, 2 and 5. Gene ontology enrichment analyses are provided in Supplementary Tables 3, 6 and 7. Interactors found in previous flavivirus proteomic or genetic studies are detailed in Supplementary Table 4. RNAi screening data are provided in Supplementary Table 8. Additional supporting data are available from the corresponding authors upon request.

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Acknowledgements

We thank members of the Cherry, Krogan and Ramage laboratories for discussion, advice and reagents. We thank the University of Pennsylvania High-Throughput Screening Core for providing reagents and technical expertise. We thank J. To for virus preparations, D. Tatomer for RNA analyses and M. Shales for graphical support. This work was supported by The American Liver Foundation Liver Scholar Award and the Creative and Novel Ideas in HIV Research Award to H.R.; NIH grants nos. R01AI074951 and RO1AI122749 and the Burroughs Wellcome Investigators in the Pathogenesis of Infectious Disease Award to S.C.; and NIH grants nos. U19 AI118610, U19 AI135990 and P50 GM082250 to N.J.K.

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Authors and Affiliations

  1. Department of Microbiology, University of Pennsylvania, Philadelphia, PA, USA

    Minghua Li, Billy Truong, Grace Kim, Nathan Weinbren, Mark Dittmar, Sara Cherry & Holly Ramage

  2. Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA

    Jeffrey R. Johnson, Priya S. Shah, John Von Dollen, Billy W. Newton, Gwendolyn M. Jang & Nevan J. Krogan

  3. Quantitative Biosciences Institute, University of California San Francisco, San Francisco, CA, USA

    Jeffrey R. Johnson, Priya S. Shah, John Von Dollen, Billy W. Newton, Gwendolyn M. Jang & Nevan J. Krogan

  4. The J. David Gladstone Institutes, San Francisco, CA, USA

    Jeffrey R. Johnson, Priya S. Shah, John Von Dollen, Billy W. Newton, Gwendolyn M. Jang & Nevan J. Krogan

  5. Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA, USA

    Priya S. Shah

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Contributions

H.R. and G.M.J. performed affinity purification of flaviviral proteins. B.W.N. and J.R.J. performed mass spectrometry. J.V.D., P.S.S. and J.R.J. performed proteomic scoring and bioinformatics analyses. H.R. and M.L. performed immunostaining and microscopy. H.R., M.L., B.T. and G.K. performed RNAi, infections, RT–qPCR and western blotting. M.L. performed co-immunoprecipitation assays, cell fractionation and RNA immunoprecipitations. N.W. and M.D. performed TCID50 assays. H.R., S.C. and N.J.K. supervised research. H.R. and S.C. wrote the manuscript with input from N.J.K. and M.L.

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Correspondence to Nevan J. Krogan, Sara Cherry or Holly Ramage.

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Supplementary information

Supplementary Information

Supplementary Figures 1–10 and legends for Supplementary Tables.

Reporting Summary

Supplementary Table 1

WNV-interacting proteins identified by AP/MS.

Supplementary Table 2

High confidence WNV-host protein–protein interactions.

Supplementary Table 3

GO Enrichment for WNV Interactome.

Supplementary Table 4

Overlap of WNV interactome with previous flavivirus AP-MS and genetic screens.

Supplementary Table 5

Overlap of WNV capsid interactors with DENV and ZIKV capsids.

Supplementary Table 6

GO Enrichment for WNV capsid interactors.

Supplementary Table 7

Enrichment of WNV-interactor localization and GO terms for individual WNV bait proteins.

Supplementary Table 8

Complete RNAi screening results (robust z-scores).

Supplementary Table 9

siRNAs used in this study.

Supplementary Table 10

qPCR primers used in this study.

Supplementary Table 11

Complete table of precise P-values for all statistical analyses.

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Li, M., Johnson, J.R., Truong, B. et al. Identification of antiviral roles for the exon–junction complex and nonsense-mediated decay in flaviviral infection. Nat Microbiol 4, 985–995 (2019). https://doi.org/10.1038/s41564-019-0375-z

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