Envelope protein ubiquitination drives entry and pathogenesis of Zika virus

Abstract

Zika virus (ZIKV) belongs to the family Flaviviridae, and is related to other viruses that cause human diseases. Unlike other flaviviruses, ZIKV infection can cause congenital neurological disorders and replicates efficiently in reproductive tissues1,2,3. Here we show that the envelope protein (E) of ZIKV is polyubiquitinated by the E3 ubiquitin ligase TRIM7 through Lys63 (K63)-linked polyubiquitination. Accordingly, ZIKV replicates less efficiently in the brain and reproductive tissues of Trim7−/− mice. Ubiquitinated E is present on infectious virions of ZIKV when they are released from specific cell types, and enhances virus attachment and entry into cells. Specifically, K63-linked polyubiquitin chains directly interact with the TIM1 (also known as HAVCR1) receptor of host cells, which enhances virus entry in cells as well as in brain tissue in vivo. Recombinant ZIKV mutants that lack ubiquitination are attenuated in human cells and in wild-type mice, but not in live mosquitoes. Monoclonal antibodies against K63-linked polyubiquitin specifically neutralize ZIKV and reduce viraemia in mice. Our results demonstrate that the ubiquitination of ZIKV E is an important determinant of virus entry, tropism and pathogenesis.

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Fig. 1: ZIKV-E ubiquitination on K38 and K281 promotes virus replication in cells and in vivo.
Fig. 2: TRIM7 ubiquitinates ZIKV E and promotes virus replication.
Fig. 3: Ubiquitination of ZIKV E promotes virus attachment and fusion of the virus and the endosome membrane.
Fig. 4: Specific anti-K63-linked polyubiquitin antibody neutralizes ZIKV in vitro and in vivo.
Fig. 5: Ubiquitination of ZIKV E promotes binding to the TIM1 receptor.

Data availability

All data are available in the Article and its Supplementary Information. Mutant viruses may be available upon request to the corresponding authors, after respective material transfer agreements are completed. Source data are provided with this paper.

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Acknowledgements

We thank M. A. Garcia-Blanco, S. Bradrick and R. Soto for their generosity in sharing reagents and advice; Y. Liang for his technical advice on flow cytometry; M. Fan for providing the lentivirus to establish stable cell lines that express HA–Ub (pLenti puro HA-Ub), through Addgene; A. Gamarnik, R. Stephens and V. Menachery for suggestions and helpful discussions; T. Wang for providing some control mice; and L. Yeager for editing. The laboratory of R.R. was supported in part by the John Sealy Memorial Endowment Fund for Biomedical Research (UTMB), a research career development award (K12HD052023: BIRCWH program, from NIH ORWH/NICHD), and NIH/NIAID grants R21 AI132479-01, R21 AI126012-01A1 and R01 AI134907-01. S.V.T. was supported by NIH/NIAID T32-AI060549, and A.H. by NIH/NIAID T32 AI007526. P.-Y.S. was supported by NIH grants AI142759, AI134907, AI145617 and UL1TR001439, and awards from the Sealy & Smith Foundation, Kleberg Foundation, John S. Dunn Foundation, Amon G. Carter Foundation, Gilson Longenbaugh Foundation and Summerfield Robert Foundation. The laboratory of S.M.B. was supported in part by the Division of Intramural Research of the NIH/NIAID. J.R.J. and N.J.K. were supported by NIH/NIAID grant U19 AI118610.

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Authors

Contributions

M.I.G. performed all aspects of this study. M.I.G., H.X., L.A.-A., S.R.A., S.L.R., A.H., S.v.T., C.S. and X.X. performed experiments and analysed data. H.X. generated ZIKV recombinant viruses. S.R.A. and S.L.R. performed in vivo experiments. P.-Y.S. and R.R. organized, conceptualized the study and provided funding. G.L.S., S.J.R., K.L.M., K.M.-W. and S.M.B. generated the Trim7−/− mice. W.M. provided the AB6 Tim-1−/− mice. M.W. and M.C.M. performed cryo-electron microscopy. H.R., J.R.J. and N.J.K. performed mass spectrometry studies. M.I.G., L.A.-A, P.-Y.S. and R.R. prepared the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Pei-Yong Shi or Ricardo Rajsbaum.

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The authors declare no competing interests.

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Peer review information Nature thanks Jan Carette and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Ubiquitination of flavivirus E protein.

a, Proteasome inhibition blocks ZIKV replication. JEG-3 cells were pretreated with DMSO or MG132 (2 h) followed by ZIKV infection (MOI 2, 24 h, visualized by immunofluorescence with anti-E 4G2). b, Ubiquitinated peptides from flavivirus-infected cells identified by mass spectrometry (peptides highlighted in yellow, diglycine residues indicating ubiquitination in red and conserved residues in green). Sequences for strains ZIKV FSS13025, GenBank: KU955593.1; DENV-2 Y98P, JF327392.1; West Nile virus (WNV) NY99, DQ211652; and yellow fever virus (YFV), ANC33490.1. JEG-3 cells were used for ZIKV infections, Huh7 cells for DENV infections and A549 cells for WNV infections (repeated in U2OS cells with identical results, two independent experiments). Representative mass spectra for ubiquitinated peptides found for WNV are shown. b and y ions are indicated in blue and red, respectively. c, Whole-cell extracts from DENV- or ZIKV-infected (MOI 2, 20 h) Huh7 cells transfected with HA–Ub, followed by DMSO or MG132 treatment (6 h) were used for HA immunoprecipitation. Immunoblots are shown. NT, non-treated d, Whole-cell extract from cells transfected with vectors expressing E and wild-type ubiquitin or all K-to-R mutants except for K63 or K48 (only), or Ub(K48R) and Ub(K63R), followed by immunoprecipitation. e, JEG-3 cells pretreated with MG132 or DMSO. Cells were incubated with ZIKV at 4 °C for 30 min, followed by a wash with or without glycine to test virus adsorption. Additional samples were then switched to 37 °C to allow virus internalization. Viral RNA detection by quantitative reverse-transcription PCR (qRT–PCR). Representative of two independent experiments, n = 3 technical replicates, mean ± s.e.m., unpaired t-test, two-sided, *P < 0.05; NS, not significant. All experiments are representatives from two independent experiments, with similar results. Source data

Extended Data Fig. 2 Differences in Zika virus cell tropism are associated with ubiquitination of E.

a, JEG-3 cells stably expressing HA–Ub were infected with wild-type ZIKV, or recombinant infectious E(K38R)- and E(K281R)-mutant ZIKV. Whole-cell extracts were used for immunoprecipitation with HA beads (same experiment as shown in Fig. 1b, but without normalizing the input for the immunoprecipitation). Reduced replication of E(K38R)-mutant ZIKV can be seen represented by the levels of E in the whole-cell extract. Representative of two independent experiments. be, Different cell types were infected with either wild-type ZIKV, E(K38R)- or E(K281R)-mutant ZIKV (MOI 0.5). Cells were lysed for RNA extraction and virus quantification by qRT–PCR (c), and supernatants were collected for plaque assays at different time-points, for HTR-8 (b), 15P-1 (d) and HuH7 (e) cells. f, Back titration for the virus used on these experiments, and for Fig. 1e, f. Representatives from two independent experiments. n = 3 technical replicates, mean ± s.e.m., multiple t-test, Holm–Sidak correction, *P < 0.05, ***P < 0.001. Source data

Extended Data Fig. 3 Ubiquitination of E in tissues from infected mice.

a, b, Tissues from testis (a) and brain (b) from mock-infected or wild-type-ZIKV-infected A129 mice were collected at day 8 after infection. Tissues were homogenized and 200 μg of total input protein was used for immunoprecipitation of E using 4G2 antibody or an IgG control. Ubiquitination of wild-type E was detected with anti-ubiquitin antibody by immunoblot. Immunoprecipitations shown are from mixed tissue lysates from three different mice. c, d, A129 mice (male and females) were mock-treated (5 mice) or infected with wild-type ZIKV, E(K38R)- or E(K281R)-mutant ZIKV (1 × 104 PFU, 9 mice per group, combined from 2 independent experiments). Weight loss and survival is shown in Fig. 1e, f. c, Serum titres (viraemia), were determined at day 2 after infection by plaque assay, after blood collection from 6 mice for wild-type and E(K281R)-mutant ZIKV, and 7 mice for E(K38R)-mutant ZIKV. d, Virus titres (at day 8 after infection) in brain (14 mice for wild-type ZIKV, and 9 mice for E(K38R)- and E(K281R)-mutant ZIKV), testis (6 mice per group) and eye (14 mice for wild-type ZIKV, and 9 mice for E(K38R)- and E(K281R)- ZIKV). Unpaired, t-test, two-sided, *P < 0.05, **P < 0.01. Source data

Extended Data Fig. 4 TRIM7 interacts with and ubiquitinates E and promotes virus replication.

a, Differential expression of TRIM7 in mouse tissues by immunoblot. The predicted molecular weight of full-length TRIM7 is 56 kDa. b, c, TRIM7 knockdown (24 h) in JEG-3 (b) or HTB-15 (c) cells followed by infection with ZIKV (MOI 1). c, Viral RNA levels were determined by qRT–PCR at different time points (top). TRIM7 knockdown efficiency was confirmed by western blot (bottom). d, TRIM7-knockout A549 and wild-type parental cells were used for infections with ZIKV or DENV at an MOI of 0.5. Bottom, immunoblot of TRIM7. Plaque assays from supernatants collected at different time points are shown. e, f, Infections of wild-type and TRIM7-knockout JEG-3 cells with ZIKV (MOI 0.5) or poly(I:C) stimulation (f, transfection of 10 μg ml−1 with Lipofectamine 2000). Quantification of ZIKV RNA (e, top) and IFNB1 mRNA expression (e, bottom and f) by qPCR. g, Overexpression of TRIM7 enhances K63-linked polyubiquitination of wild-type E but not E(K38R) or E(K281R). HEK293T cells were transfected with vectors expressing wild-type E, E(K38R) or E(K281R) and different amounts of HA–TRIM7 (350 ng or 700 ng). Thirty hours after transfection, cells lysates were used for immunoprecipitation with anti-E 4G2 or isotype control. Immunoblots with indicated antibodies. h, Transfection of Huh7 cells with empty vector or vector expressing TRIM7. After 48 h cells were infected with wild-type or E(K38R)-mutant ZIKV. n = 3 technical replicates, mean ± s.e.m. Multiple t-test, two-sided, ***P < 0.001, ****P < 0.0001, NS, not significant (P > 0.05). i, Endogenous TRIM7 interacts with E in ZIKV-infected JEG-3 cells. Cells were infected with wild-type ZIKV (MOI 2). Thirty hours after infection, cells were lysed and whole-cell extracts were used for immunoprecipitation with anti-E (4G2) or isotype control. Representative of two independent experiments. Source data

Extended Data Fig. 5 TRIM7 colocalizes with E in the Golgi.

a, b, JEG-3 (a) or A549 (b) cells were mock-treated or infected with ZIKV (MOI 2). Twenty-four hours after infection, cells were fixed and stained for endogenous TRIM7 (red), Golgi (WGA–FITC, green) and E (4G2, purple) for confocal microscopy. Colocalization is shown in rectangles, and red–green–blue (RGB) profile graphs are on the right. All images were processed identically using the same conditions with ZEN 2.5.75.0 (Zeiss), and RGB profiles were obtained using ImageJ v1.52e (NIH). c, Cell fractionation of infected JEG-3 cells (20 h, MOI 2) for endoplasmic reticulum (ER) was performed following the manufacturer’s instructions (Sigma). Representative of two independent experiments.

Extended Data Fig. 6 Virions of ZIKV and DENV contain ubiquitinated E, and TRIM7 is important for ZIKV—but not DENV—replication.

a, Supernatants collected from JEG-3 ZIKV-infected cells stably expressing HA–Ub showed detectable levels of ubiquitinated E. Viruses were immunoprecipitated with anti-HA beads. Immunoblot for E and HA–Ub. The bottom panel shows the total HA–Ub levels expressed in these HA–Ub stable cell lines. b, Infection of wild-type A549 or TRIM7-knockout cells with ZIKV. c, d, Transfection of wild-type A549 or TRIM7-knockout cells with in vitro-transcribed ZIKV RNA. Viral RNA was quantified by qPCR from cell lysates (intracellular) (c) or from supernatants (extracellular) (d). Representative of two independent experiments. n = 3 technical replicates, mean ± s.e.m. e, K63-linked polyubiquitinated E was detected on wild-type ZIKV particles but reduced in E(K38R)- and E(K281R)-mutant ZIKV particles, after immunoprecipitation with anti-E 4G2 antibody. Although reduced, ubiquitination on E from ZIKV grown in mosquito C6/36 cells can also be detected. f, DENV from supernatants from infected BHK-21 cells was immunoprecipitated with anti-E antibody (4G2) or an IgG control. Immunoblot for K63-linked polyubiquitin and each viral protein are shown. Representative of two independent experiments. Source data

Extended Data Fig. 7 Proportion of ubiquitinated E in virions of ZIKV, and cryo-electron microscopy of ubiquitinated ZIKV.

a, b, ZIKV stocks were grown in Vero cells, wild-type JEG-3 or TRIM7-knockout JEG-3 cells, and used for immunoprecipitation using an anti-K63-linked ubiquitin antibody, or an IgG control to set the background levels. The immunoprecipitated virus, as well as a sample of input viruses, was lysed in Trizol for virus RNA quantification by qPCR (a). The virus RNA copy number was determined using a standard of purified ZIKV RNA and its known molecular weight. The proportion of ubiquitinated virus was calculated taking as 100% the input virus. n = 3 technical replicates, mean, unpaired two-sided t-test, ***P < 0.001. b, Cryo-electron microscopy of ubiquitinated ZIKV. Experimental approach. Supernatants from Vero cells infected with wild-type or E(K38R/K281R)-mutant ZIKV were washed and concentrated in Amicon filters followed by labelling with a primary antibody against K63-linked ubiquitin and secondary nano-gold-labelled antibody. Virus–antibody complexes were then purified by sucrose gradient. A visible band containing these complexes was recovered and passed through Amicon filters to remove sucrose, and concentrate the complexes. Samples were flash-frozen in liquid ethane cooled to liquid nitrogen temperatures on holey carbon grids and images were recorded in movie mode at 40,000× magnification using a 200 KV JEOL 2200FS transmission electron microscope. To facilitate visualization of virus particles, frames were further binned 3× to yield a pixel size of 4.398 Å per pixel. These binned micrographs were manually examined using EMAN2. To identify potentially gold-labelled ubiquitinated particles, we looked for spherical particles corresponding to the known approximately 500 Å (50 nm) size of mature ZIKV, and which were within 200 Å of the easily recognizable nano-gold clusters. Approximately 15% of visible approximately 500 Å wild-type ZIKV particles satisfied these criteria. None of the E(K38R/K281R)-mutant ZIKV was found labelled with gold particles (b). The cryo-electron microscopy experiments with gold particle labelling were performed only once, owing to the large amount of virus needed. Source data

Extended Data Fig. 8 Ubiquitination of E promotes virus–endosome membrane fusion.

These experiments were performed in JEG-3 cells. ac, Wild-type ZIKV, E(K38R)- and E(K281R)-mutant ZIKV were labelled with DiOC18. After filtration, viruses were incubated at 4 °C with JEG-3 cells at MOI 2, and after 30 min were washed and collected for controls as adsorbed viruses. Additional samples were then incubated at 37 °C for 1 h, in the presence or absence of NH4Cl to block acidification (as control), washed, fixed and visualized with a confocal microscope (a). The same experiment was repeated for quantification by fluorescence-activated cell sorting (FACS). Mean fluorescence intensity (MFI) is shown in b; the percentage of cells infected is shown in main Fig. 3a; and representative histograms are shown in c. n = 3 technical replicates, mean, unpaired two-sided t-test, *P < 0.05. Representative of two independent experiments. Source data

Extended Data Fig. 9 Ubiquitination of E promotes virus–endosome membrane fusion in A549 cells.

ad, Wild-type ZIKV, E(K38R)- and E(K281R)-mutant ZIKV were labelled with DiOC18. After filtration, viruses were incubated at 4 °C with A549 cells at MOI 2 and after 30 min were washed and collected for controls as adsorbed viruses. Additional samples were then incubated at 37 °C for 1 h, and NH4Cl was used as control to block acidification of the endosome. Cells were washed, fixed and visualized in a confocal microscope in a. Quantification by FACS is shown (mean fluorescence intensity) in b, c. d, Representative histograms showing green fluorescence (FL1: DiOC18) during ZIKV–endosome fusion. n = 3 technical replicates, mean, unpaired two-sided t-test, *P < 0.05, ***P < 0.05. Representative of two independent experiments. Source data

Extended Data Fig. 10 Ubiquitination of E on K38 promotes ZIKV attachment and enhanced replication in relevant human cells.

ae, Human brain microvascular endothelial cells (BMECs) (a, d), human astrocytes (b, e) and human primary induced-pluripotent neural stem cells (hiPS-NSCs) (c) were infected with wild-type or E(K38R)-mutant ZIKV (MOI 2), as described in Fig. 3b, c. Viral RNA was quantified by qPCR (a, b) and virus titres by plaque assay (ce). One experiment, n = 3 technical replicates, mean ± s.e.m. Unpaired two-sided t-test, *P < 0.05. f, Endoglycosidase analyses of E. Proteins from wild-type ZIKV and E(K38R)- and E(K281R)-mutant ZIKV were analysed by western blot. Viruses were treated with PNGase F for 1 h at 37 °C. g, Wild-type ZIKV or E(K38R)-mutant ZIKV grown in wild-type or TRIM7-knockout JEG-3 cells were used for attachment assays. Viruses were incubated at 4 °C for 30 min with JEG-3 cells and attachment was determined by measuring virus RNA by qPCR. The percentage of virus attachment was calculated by taking the input virus as 100%. hj, The deubiquitinase (DUB) domain of the OTU of the Crimean-Congo haemorrhagic fever (CCHF), which can cleave polyubiquitin chains (shown in h as control for activity), and a mutant (OTU (2A)) with reduced activity, were used to cleave ubiquitinated E of ZIKV. After incubation of ZIKV with purified recombinant OTU, the ability of the deubiquitinated virus to attach to cells and to replicate was tested by incubation with JEG-3 cells at 4 °C for 30 min and viral RNA quantified by qPCR (i), and replication by plaque assay (j). Representative of two independent experiments, n = 3 technical replicates, mean ± s.e.m., unpaired two-sided t-test, **P < 0.01. Source data

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Giraldo, M.I., Xia, H., Aguilera-Aguirre, L. et al. Envelope protein ubiquitination drives entry and pathogenesis of Zika virus. Nature 585, 414–419 (2020). https://doi.org/10.1038/s41586-020-2457-8

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