Determinants of Zika virus host tropism uncovered by deep mutational scanning

Abstract

Arboviruses cycle between, and replicate in, both invertebrate and vertebrate hosts, which for Zika virus (ZIKV) involves Aedes mosquitoes and primates1. The viral determinants required for replication in such obligate hosts are under strong purifying selection during natural virus evolution, making it challenging to resolve which determinants are optimal for viral fitness in each host. Herein we describe a deep mutational scanning (DMS) strategy2,3,4,5 whereby a viral cDNA library was constructed containing all codon substitutions in the C-terminal 204 amino acids of ZIKV envelope protein (E). The cDNA library was transfected into C6/36 (Aedes) and Vero (primate) cells, with subsequent deep sequencing and computational analyses of recovered viruses showing that substitutions K316Q and S461G, or Q350L and T397S, conferred substantial replicative advantages in mosquito and primate cells, respectively. A 316Q/461G virus was constructed and shown to be replication-defective in mammalian cells due to severely compromised virus particle formation and secretion. The 316Q/461G virus was also highly attenuated in human brain organoids, and illustrated utility as a vaccine in mice. This approach can thus imitate evolutionary selection in a matter of days and identify amino acids key to the regulation of virus replication in specific host environments.

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Fig. 1: DMS of C-terminal region of ZIKV E protein.
Fig. 2: In vitro characterization of ZIKV mutants.
Fig. 3: Immunofluorescence and TEM of infected C6/36 or Vero cells.
Fig. 4: Molecular analysis and modelling of the S461G and K316 mutations.
Fig. 5: Infection of iPSC-derived human brain organoids.
Fig. 6: 316G/461Q mutant as an attenuated ZIKV vaccine candidate.

Data availability

Deep sequencing data are deposited on the Sequence Read Archive under PRJNA449413. Data files are also provided as Supplementary Data 1 and 2. The software suite for DMS data analysis can be found on https://github.com/jbloomlab/dms_tools2. The actual code used to run the suite in this study is at https://github.com/jbloomlab/ZIKV_DMS_w_Khromykh

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Acknowledgements

This work was supported by the National Health and Medical Research Council (NHMRC) of Australia (grant No. APP1144950). A.K. and A.S. are Research Fellows with the NHMRC. E.N. was supported in part by the Daiichi Sankyo Foundation of Life Science, Japan. We thank R. Sullivan from the Queensland Brain Institute Microscopy and Histology Facility for help with preparation and imaging of the brain organoid slides. Organoid confocal microscopy work was performed in part at the Queensland node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano- and microfabrication facilities for Australia’s researchers.

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Contributions

Y.S. and A.K. conceptualized the study. A.K., A.S. and Y.S. performed funding acquisition. Y.S., A.A, N.P., R.G., J.C., E.N., S.O., D.W., N.M., M.F., B.T., A.S., F.T., F.N. and P.P. performed the experiments. Y.S., A.A., R.G., J.H., E.W., S.O., J.H., A.K., J.B. and J.M. analysed the data. J.B. developed the software. T.C. established the computing resources. J.P., R.H., N.P., R.G., J.M., E.W., P.Y. and J.W. provided critical reagents and models. Y.S. and A.K. wrote the original draft. Y.S., A.K., A.S., D.W., J.B., E.W., R.H. and A.A. reviewed and edited the article.

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Correspondence to Yin Xiang Setoh or Alexander A. Khromykh.

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Supplementary information

Supplementary Information

Supplementary Notes, Supplementary Tables 1–5 and Supplementary Figures 1–9.

Reporting Summary

Supplementary Table 1

DMS mutagenesis primers.

Supplementary Data 1

Data analysis Jupyter Notebook.

Supplementary Data 2

Raw data files.

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Setoh, Y.X., Amarilla, A.A., Peng, N.Y.G. et al. Determinants of Zika virus host tropism uncovered by deep mutational scanning. Nat Microbiol 4, 876–887 (2019). https://doi.org/10.1038/s41564-019-0399-4

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