Enterovirus pathogenesis requires the host methyltransferase SETD3

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Abstract

Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification–mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections.

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Fig. 1: Genome-wide CRISPR–Cas9 screens identify SETD3 as a broad host factor for human EVs.
Fig. 2: SETD3 is required for EV replication in the cytoplasm.
Fig. 3: The SET and RSB domains of SETD3 are required for its role in viral replication but not its methyltransferase activity.
Fig. 4: SETD3 interacts with the enteroviral 2A protein to promote viral replication.
Fig. 5: SETD3 is important for EV infection of primary human cells and in vivo mouse models.

Data availability

Raw deep-sequencing data corresponding to the CRISPR KO screens reported in this paper are deposited in ArrayExpress (https://www.ebi.ac.uk/arrayexpress/) with accession number E-MTAB-8125.

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Acknowledgements

The authors thank the Carette Laboratory members for intellectual discussions and support. We acknowledge C. Marceau (Chan Zuckerberg Biohub) and A. Puschnik (Chan Zuckerberg Biohub) for invaluable advice and technical assistance. We thank K. Kirkegaard (Stanford University), M. Holtzman (Washington University School of Medicine in St. Louis), A. Palmenberg (University of Wisconsin-Madison) and K. Shokat (University of California San Francisco) for helpful discussions and valuable advice. We express our gratitude to the Stanford Shared FACS Facility and its former director M. Bigos, the Stanford Functional Genomics Facility and X. Ji, the Stanford University Mass Spectrometry Core and the Stanford Network Analysis of Proteins team under the guidance of P. Jackson, the Stanford Protein and Nucleic Acid Facility and the Stanford Mouse Facility for excellent services and professional technical assistance. We thank V. Masto for technical assistance with cloning, and M. Shales for assistance preparing figures related to the mass spectrometry data. We acknowledge the NIH Biodefense and Emerging Infections Research Repository (BEI Resources), NIAID, NIH for providing multiple viruses mentioned in the Methods. We thank J.-R. Wang (National Cheng Kung University, Taiwan) for providing the EV-A71 (MP4) infectious clone. This work was funded in part by the NSF GRFP (J.D.), Stanford Graduate Fellowship (J.D.), American Asthma Foundation 2014 Scholar Award (J.E.C.), NIH DP2 AI104557 (J.E.C.), NIH U19 AI109662 (J.E.C.), NIH R01 AI140186 (J.E.C.), Stanford Dean’s Fellowship (Y.S.O.), David and Lucile Packard Foundation (J.E.C.), NIH R01 GM079641 (O.G.), NIH R01 GM133051 (O.G.), NIH P01-AI091575 (R.A. and N.J.K.), NIH P50-GM082250 (N.J.K.), NIH P50-GM081879 (N.J.K.), NIH U19-AI135990 (N.J.K.), NIH R01 AI021362 (H.B.G.), NIH R56 AI021362 (H.B.G.), VA Merit review grant GRH0022 (H.B.G.), NIH K99 AI135031 (S.D.), Stanford Maternal and Child Health Research Institute (S.D.) and Thrasher Research Fund Early Career Award (S.D.).

Author information

J.D., Y.S.O. and A.W.W. were responsible for design and execution of the experiments, data analysis and manuscript preparation. C.E.P. analysed and generated the phylogenetic tree of SET domain family members, performed the CV-B3 2A alanine scan and analysed the three-dimensional structure of SETD3-binding mutants of 2A. E.F., J.R.J., O.L., G.J., T.Y., E.V., R.A. and N.J.K. designed, collected and analysed the CV-B3 virus–host interaction proteomics data. A.W.W. performed and analysed the actin methylation mass spectrometry experiments with help from J.E.E. J.Z. performed the statistical analyses for all datasets, and cloned and analysed the GFP-ΔVP1-2A constructs and CV-B3 infectious clones that harbour SETD3-binding mutants of 2A. R.H., G.J., J.X. and N.J.K. designed, collected and analysed the multiple EVs’ 2A AP-MS datasets. C.M.N., K.-F.W., J.D., K.J.K. and P.S. were responsible for the in vivo mice experiments and analysis. S.D. and H.B.G. were responsible for examining innate immune responses and expression levels of IFN-stimulated gene by transcriptome-wide RNA-Seq. J.E.C., O.G., N.J.K. and R.A. supervised the research, interpreted the data and prepared the manuscript.

Correspondence to Raul Andino or Nevan J. Krogan or Or Gozani or Jan E. Carette.

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Competing interests

O.G. is a co-founder of EpiCypher and Athelas Therapeutics.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–21 and Supplementary Table legends.

Reporting Summary

Supplementary Table 1

Datasets of RV-C15 and EV-D68 CRISPR screens.

Supplementary Table 2

RNA-Seq of WT versus SETD3KO cells.

Supplementary Table 3

AP-MS of SETD3 pull-downs.

Supplementary Table 4

AP-MS of CV-B3 viral proteins pull-downs.

Supplementary Table 5

AP-MS of multiple EV 2A pull-downs.

Supplementary Table 6

Precise P values for statistical analyses.

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