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Salivary factor LTRIN from Aedes aegypti facilitates the transmission of Zika virus by interfering with the lymphotoxin-β receptor

Abstract

Pathogens have co-evolved with mosquitoes to optimize transmission to hosts. Mosquito salivary-gland extract is known to modulate host immune responses and facilitate pathogen transmission, but the underlying molecular mechanisms of this have remained unknown. In this study, we identified and characterized a prominent 15-kilodalton protein, LTRIN, obtained from the salivary glands of the mosquito Aedes aegypti. LTRIN expression was upregulated in blood-fed mosquitoes, and LTRIN facilitated the transmission of Zika virus (ZIKV) and exacerbated its pathogenicity by interfering with signaling through the lymphotoxin-β receptor (LTβR). Mechanically, LTRIN bound to LTβR and ‘preferentially’ inhibited signaling via the transcription factor NF-κB and the production of inflammatory cytokines by interfering with the dimerization of LTβR during infection with ZIKV. Furthermore, treatment with antibody to LTRIN inhibited mosquito-mediated infection with ZIKV, and abolishing LTβR potentiated the infectivity of ZIKV both in vitro and in vivo. This study provides deeper insight into the transmission of mosquito-borne diseases in nature and supports the therapeutic potential of inhibiting the action of LTRIN to disrupt ZIKV transmission.

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Fig. 1: LTRIN targets LTβR.
Fig. 2: LTRIN inhibits LTβR signaling.
Fig. 3: LTRIN augments infection with ZIKV in vitro.
Fig. 4: LTRIN attenuates the activation of NF-κB and the production of proinflammatory cytokines.
Fig. 5: Effect of LTRIN on canonical and non-canonical NF-κB signaling triggered by infection with ZIKV.
Fig. 6: LTRIN promotes the pathogenesis of ZIKV in vivo.
Fig. 7: The early immune response trigged by infection with ZIKV is inhibited by LTRIN.
Fig. 8: Abolishing LTβR promotes the infectivity of ZIKV.

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Acknowledgements

We thank F. Shao (National Institute of Biological Sciences, Beijing, China) for Ifnar–/– mice, and Y.-X.Fu (UT Southwestern Medical Center) for LTβR-immunoglobulin. Supported by National Key Research and Development Program of China (2017YFD0500300 and 2016YFC1201000), National Natural Science Foundation of China (21761142002, 31600721, 31200590, 31630075 and 31701134), Chinese Academy of Sciences (XDB13000000, KSZD-EW-Z-007, CXJJ-17-M141, Y4ZK111B01 and Y602381081) and Yunnan Province (2012BC009).

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L.J., C.S., X.H., O.R., H.Z., M.Y. and R.L. performed LTRIN identification, LTRIN preparation and data analysis; L.J., X.G., P.S., P.L., T.X., C.H., C.-F.Q., J.G., H.P., M.Z., G.C. and X.Q. performed ZIKV infection and data analysis; X.Q. wrote the manuscript with input from R.L. and L.J.; and X.Q. and R.L. designed the study.

Corresponding authors

Correspondence to Gong Cheng or Xiaopeng Qi or Ren Lai.

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Integrated supplementary information

Supplementary Figure 1 LTRIN purification from SGE of mosquitoes

a, Representative salivary glands of mosquitoes. Salivary glands (SGs) from unfed (control) and fed A. aegypti were dissected for protein and RNA extraction. b, Salivary gland extract was separated by Sephadex G-75 gel filtration. Fraction I had immunosuppressive activities, which was selected for further purification. c, Fraction I was further separated by the Resource Q anion-exchange column. Peak IV (arrow indicated) was collected for SDS-PAGE analysis, and amino acid sequencing was performed by Edman degradation. d, Purified proteins from peak IV in (c) were separated on SDS-PAGE and stained with Coomassie Blue. The molecular weight of LTRIN was approximately 15-kDa. e, Protein sequence of A. aegypti LTRIN. Sequence of the peptide recovered from SGE of A. aegypti is given in red, and sequence of the recombinant protein purified from E. coli is underlined. f, Binding affinity analysis of LTRIN and LTβR. g, Binding affinity analysis of LTαβ2 and LTβR. Data are representative of three independent experiments. Scale bars, 300 μm for (a).

Supplementary Figure 2 LTRIN targets mouse LTβR

a, Homology analysis of human and mouse LTβRs. The extracellular domain is given in red. b, Dimerization analysis of mouse LTβR. Mouse BMDMs stimulated with mouse LTα and LTRIN for 1 h and cross-linked for 15 min with disuccinimidyl suberate, followed by lysing and western blot analysis. The ratio of mouse LTβR dimer to monomer was quantified (right). Data are representative of (left) or form (right) two independent experiments (b).

Supplementary Figure 3 Intracellular ZIKV staining in MSF, BMDM and HUVEC cells

a, Expression analysis of ZIKV in HUVEC and THP-1 cells during ZIKV infection (MOI 0.5) and ZIKV combined with LTRIN administration (ZV/IN, ZIKV at MOI 0.5 and LTRIN at 100 ng/mL) for 30 min. b, Confocal immunofluorescence analysis of ZIKV in MSFs and BMDMs during ZIKV infection (MOI 0.5) and ZIKV combined with LTRIN administration (ZIKV at MOI 0.5 and LTRIN at 100 ng/mL) for 12 hours. c, Confocal immunofluorescence analysis of ZIKV and AXL in LTRIN treated HUVEC cells and HUVEC cells with ZIKV infection (MOI 0.5) and ZIKV combined with LTRIN administration (ZIKV at MOI 0.5 and LTRIN at 100 ng/mL) for 12 hours. Scale bars, 20 μm for (b, c). *, P < 0.05; **, P < 0.01; ns, not significant (Student’s two-sided t-test without multiple-comparisons correction). Each symbol indicates an individual reaction in one experiment (a). Data are representative of (a, left in b and c) or from (right in b and c) three independent experiments.

Supplementary Figure 4 Effect of LTαβ on ZIKV infection

Expression analysis of ZIKV in THP-1 and HUVEC cells during ZIKV infection (MOI 0.5) and ZIKV combined with LTαβ2 administration (ZV/LTαβ2, ZIKV at MOI 0.5 and LTαβ2 at 1 nM) for indicated times. ns, not significant (Student’s two-sided t-test without multiple-comparisons correction). Each symbol indicates an individual reaction (technical replicate) in one experiment. Data are representative of two independent experiments.

Supplementary Figure 5 Effect of LTRIN on type I IFN response induced by ZIKV infection.

a,b, Expression analysis of ZIKV, Mx1, Mx2, Ifnb, Ifne, Cxcl9, Irf7, Isg15, Ifit1, Oasl2, and Usp18 in mouse BMDMs (a) and mouse MSFs (b) during ZIKV infection (MOI 0.5) and ZIKV combined with LTRIN administration (ZV/IN, ZIKV at MOI 0.5 and LTRIN at 100 ng/mL) for indicated times. c,d, Expression analysis of ZIKV, IFNB, and CXCL10 in THP-1 (c) and HUVEC cells (d) during ZIKV infection (MOI 0.5) and ZIKV combined with LTRIN administration (ZV/IN, ZIKV at MOI 0.5 and LTRIN at 100 ng/mL) for indicated times. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant (Student’s two-sided t-test without multiple-comparisons correction). Each symbol indicates an individual reaction (technical replicate) in one experiment. Data are representative of three independent experiments (a, b, c, d).

Supplementary Figure 6 LTRIN promoted ZIKV infection in testis and inflammatory response in Ifnar–/– mice in the late time of ZIKV infection.

a, Expression analysis of Il6, Tnf, Il1a, and Cxcl1 in the liver and kidney from WT and Ifnar–/– mice infected with ZIKV (500 PFUs) or ZIKV (500 PFUs) combined with LTRIN (2 μg per mouse) administration (ZV/IN) for 7 days. b, IL-6 and TNF production were measured in the sera and liver from Ifnar–/– mice infected with ZIKV (500 PFUs) or ZIKV (500 PFUs) combined with LTRIN (2 μg per mouse) administration (ZV/IN) for 7 days. c, Six- to seven-week-old male Ifnar–/– (n=3) mice were injected subcutaneously with 500 PFUs of ZIKV or ZIKV combined with LTRIN (2 μg per mouse) and weighed daily over time. d, Viral loads of ZIKV in the testis from mice in (c) at day 7 after infection were detected by qRT-PCR and plaque assay. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant (Student’s two-sided t-test without multiple-comparisons correction). Each symbol indicates an individual mouse in one experiment (a, b, d). Data are from two independent experiments (a, n=4; b, n=2), are representative of two independent experiments (c, d, n=3).

Supplementary Figure 7 Effect of LTRIN on dendritic cells, B cells and T cells recruitment trigged by ZIKV infection.

Four- to five-week-old WT mice were subcutaneously injected with 500 PFUs of ZIKV or 500 PFUs of ZIKV combined with LTRIN (2 μg per mouse) (ZV/IN) and immune cell infiltration was analyzed at day 1 post infection. a-d, Flow cytometry of dendritic cells (a), B cells (b), CD4+ T cells (c) and CD8+ T cells (d) in the spleen of mice infected with ZIKV or ZIKV combined with LTRIN (ZV/IN) for 1 day. The percentage of total cells was quantified (right). ns, not significant (Student’s two-sided t-test without multiple-comparisons correction). Each symbol indicates an individual mouse in one experiment (a, b, c, d). Data are representative of two independent experiments (n=4).

Supplementary Figure 8 Model of mosquito salivary protein LTRIN exhibited augmentation for ZIKV infectivity.

ZIKV was transmitted to mammalian host in the context of saliva during mosquito bites. After injection, one of the salivary proteins LTRIN directly and specifically binds to the LTβR and inhibits its activation leading to attenuated early inflammatory immune response, and enhances ZIKV transmission and dissemination in the mammalian host.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1-8

Life Sciences Reporting Summary

Supplementary Table 1

Human protein microarray analysis to identify proteins that interact with LTRIN

Supplementary Table 2

Top list of protein microarray analysis of LTRIN-binding proteins. R, confirmed times

Supplementary Table 3

Real Time qPCR Primer Sequences

Supplementary Data

Full scans of all blots

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Jin, L., Guo, X., Shen, C. et al. Salivary factor LTRIN from Aedes aegypti facilitates the transmission of Zika virus by interfering with the lymphotoxin-β receptor. Nat Immunol 19, 342–353 (2018). https://doi.org/10.1038/s41590-018-0063-9

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