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Combating herpesvirus encephalitis by potentiating a TLR3–mTORC2 axis

Nature Immunology volume 19, pages 1071–1082 (2018)Cite this article

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Abstract

TLR3 is a sensor of double-stranded RNA that is indispensable for defense against infection with herpes simplex virus type 1 (HSV-1) in the brain. We found here that TLR3 was required for innate immune responses to HSV-1 in neurons and astrocytes. During infection with HSV-1, TLR3 recruited the metabolic checkpoint kinase complex mTORC2, which led to the induction of chemokines and trafficking of TLR3 to the cell periphery. Such trafficking enabled the activation of molecules (including mTORC1) required for the induction of type I interferons. Intracranial infection of mice with HSV-1 was exacerbated by impairment of TLR3 responses with an inhibitor of mTOR and was significantly ‘rescued’ by potentiation of TLR3 responses with an agonistic antibody to TLR3. These results suggest that the TLR3–mTORC2 axis might be a therapeutic target through which to combat herpes simplex encephalitis.

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Fig. 1: Indispensable roles of TLR3 in the defense against HSV-1 in the CNS.
Fig. 2: mTORC2 and mTORC1 are required for the TLR3 response to infection with HSV-1.
Fig. 3: Trafficking of TLR3 to the cell periphery in a manner dependent on mTORC2 and PKC.
Fig. 4: Rab7a is associated with TLR3 and is required for its trafficking.
Fig. 5: The production of type interferons during infection with HSV-1 depends on the trafficking of TLR3 to TRAF3.
Fig. 6: Innate immune defense against infection with HSV-1 in the CNS depends on the TLR3–mTOR axis.
Fig. 7: Antibody-mediated potentiation of TLR3 increased mice surviving intracranial infection with HSV-1.

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Data availability

Further details of the experimental procedures are provided in the Life Sciences Reporting Summary. The data that support the findings of the study are available from the corresponding author upon request.

References

  1. Kawai, T. & Akira, S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11, 373–384 (2010).

    Article  CAS  PubMed  Google Scholar 

  2. Préhaud, C., Mégret, F., Lafage, M. & Lafon, M. Virus infection switches TLR-3-positive human neurons to become strong producers of beta interferon. J. Virol. 79, 12893–12904 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lafaille, F. G. et al. Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells. Nature 491, 769–773 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zhang, S. Y. et al. Human Toll-like receptor-dependent induction of interferons in protective immunity to viruses. Immunol. Rev. 220, 225–236 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Zhang, S.-Y. & Casanova, J.-L. Inborn errors underlying herpes simplex encephalitis: from TLR3 to IRF3. J. Exp. Med. 212, 1342–1343 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Guo, Y. et al. Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity. J. Exp. Med. 208, 2083–2098 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Gao, D. et al. Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses. Science 341, 903–906 (2013).

    Article  CAS  PubMed  Google Scholar 

  8. Li, X.-D. et al. Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science 341, 1390–1394 (2013).

    Article  CAS  PubMed  Google Scholar 

  9. Reinert, L. S. et al. Sensing of HSV-1 by the cGAS-STING pathway in microglia orchestrates antiviral defence in the CNS. Nat. Commun. 7, 13348 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Yordy, B., Iijima, N., Huttner, A., Leib, D. & Iwasaki, A. A neuron-specific role for autophagy in antiviral defense against herpes simplex virus. Cell Host Microbe 12, 334–345 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Chen, C. Y., Liu, H. Y. & Hsueh, Y. P. TLR3 downregulates expression of schizophrenia gene Disc1 via MYD88 to control neuronal morphology. EMBO Rep. 18, 169–183 (2017).

    Article  CAS  PubMed  Google Scholar 

  12. Uematsu, S. et al. Interleukin-1 receptor-associated kinase-1 plays an essential role for Toll-like receptor (TLR)7- and TLR9-mediated interferon-α induction. J. Exp. Med. 201, 915–923 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hoshino, K. et al. IκB kinase-α is critical for interferon-α production induced by Toll-like receptors 7 and 9. Nature 440, 949–953 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. Oganesyan, G. et al. Critical role of TRAF3 in the Toll-like receptor-dependent and -independent antiviral response. Nature 439, 208–211 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Häcker, H. et al. Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6. Nature 439, 204–207 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Sasai, M., Linehan, M. M. & Iwasaki, A. Bifurcation of Toll-like receptor 9 signaling by adaptor protein 3. Science 329, 1530–1534 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Honda, K. et al. Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature 434, 1035–1040 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Guiducci, C. et al. Properties regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation. J. Exp. Med. 203, 1999–2008 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 168, 960–976 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Caron, A., Richard, D. & Laplante, M. The roles of mTOR complexes in lipid metabolism. Annu. Rev. Nutr. 35, 321–348 (2015).

    Article  CAS  PubMed  Google Scholar 

  21. Cao, W. et al. Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3)K-mTOR-p70S6K pathway. Nat. Immunol. 9, 1157–1164 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Bodur, C. et al. The IKK-related kinase TBK1 activates mTORC1 directly in response to growth factors and innate immune agonists. EMBO J. 37, 19–38 (2018).

    Article  CAS  PubMed  Google Scholar 

  23. Schmitz, F. et al. Mammalian target of rapamycin (mTOR) orchestrates the defense program of innate immune cells. Eur. J. Immunol. 38, 2981–2992 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Saitoh, S. I. et al. TLR7 mediated viral recognition results in focal type I interferon secretion by dendritic cells. Nat. Commun. 8, 1592 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Murakami, Y. et al. Roles of the cleaved N-terminal TLR3 fragment and cell surface TLR3 in double-stranded RNA sensing. J. Immunol. 193, 5208–5217 (2014).

    Article  CAS  PubMed  Google Scholar 

  26. Vilela, M. C. et al. The chemokine CCL5 is essential for leukocyte recruitment in a model of severe Herpes simplex encephalitis. Ann. NY Acad. Sci. 1153, 256–263 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. Benjamin, D., Colombi, M., Moroni, C. & Hall, M. N. Rapamycin passes the torch: a new generation of mTOR inhibitors. Nat. Rev. Drug Discov. 10, 868–880 (2011).

    Article  CAS  PubMed  Google Scholar 

  28. Kim, Y. M., Brinkmann, M. M., Paquet, M. E. & Ploegh, H. L. UNC93B1 delivers nucleotide-sensing toll-like receptors to endolysosomes. Nature 452, 234–238 (2008).

    Article  CAS  PubMed  Google Scholar 

  29. Sato, S. et al. Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) associates with TNF receptor-associated factor 6 and TANK-binding kinase 1, and activates two distinct transcription factors, NF-κB and IFN-regulatory factor-3, in the Toll-like receptor signaling. J. Immunol. 171, 4304–4310 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Gan, X. et al. PRR5L degradation promotes mTORC2-mediated PKC-δ phosphorylation and cell migration downstream of Gα12. Nat. Cell Biol. 14, 686–696 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Mrakovic, A., Kay, J. G., Furuya, W., Brumell, J. H. & Botelho, R. J. Rab7 and Arl8 GTPases are necessary for lysosome tubulation in macrophages. Traffic 13, 1667–1679 (2012).

    Article  CAS  PubMed  Google Scholar 

  32. Sancak, Y. et al. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290–303 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ebner, M., Sinkovics, B., Szczygieł, M., Ribeiro, D. W. & Yudushkin, I. Localization of mTORC2 activity inside cells. J. Cell Biol. 216, 343–353 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Johnson, J. et al. Protein kinase Calpha is involved in interferon regulatory factor 3 activation and type I interferon-β synthesis. J. Biol. Chem. 282, 15022–15032 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. Pankiv, S. et al. FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport. J. Cell Biol. 188, 253–269 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Sato, A., Linehan, M. M. & Iwasaki, A. Dual recognition of herpes simplex viruses by TLR2 and TLR9 in dendritic cells. Proc. Natl. Acad. Sci. USA 103, 17343–17348 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wang, J. P. et al. Role of specific innate immune responses in herpes simplex virus infection of the central nervous system. J. Virol. 86, 2273–2281 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Menasria, R. et al. Both TRIF and IPS-1 adaptor proteins contribute to the cerebral innate immune response against herpes simplex virus 1 infection. J. Virol. 87, 7301–7308 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Dalpke, A. H. et al. Immunostimulatory CpG-DNA activates murine microglia. J. Immunol. 168, 4854–4863 (2002).

    Article  CAS  PubMed  Google Scholar 

  40. Aravalli, R. N., Hu, S., Rowen, T. N., Palmquist, J. M. & Lokensgard, J. R. Cutting edge: TLR2-mediated proinflammatory cytokine and chemokine production by microglial cells in response to herpes simplex virus. J. Immunol. 175, 4189–4193 (2005).

    Article  CAS  PubMed  Google Scholar 

  41. Temizoz, B. et al. TLR9 and STING agonists synergistically induce innate and adaptive type-II IFN. Eur. J. Immunol. 45, 1159–1169 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kawamura, N. et al. Delivery of endosomes to lysosomes via microautophagy in the visceral endoderm of mouse embryos. Nat. Commun. 3, 1071 (2012).

    Article  CAS  PubMed  Google Scholar 

  43. Magee, J. A. et al. Temporal changes in PTEN and mTORC2 regulation of hematopoietic stem cell self-renewal and leukemia suppression. Cell Stem Cell 11, 415–428 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Barber, G. N. Innate immune DNA sensing pathways: STING, AIMII and the regulation of interferon production and inflammatory responses. Curr. Opin. Immunol. 23, 10–20 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Floden, A. M. & Combs, C. K. Microglia repetitively isolated from in vitro mixed glial cultures retain their initial phenotype. J. Neurosci. Methods 164, 218–224 (2007).

    Article  CAS  PubMed  Google Scholar 

  46. Fukui, R. et al. Cleavage of Toll-Like receptor 9 ectodomain is required for in vivo responses to single strand DNA. Front. Immunol. 9, 1491 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Ejercito, P. M., Kieff, E. D. & Roizman, B. Characterization of herpes simplex virus strains differing in their effects on social behaviour of infected cells. J. Gen. Virol. 2, 357–364 (1968).

    Article  CAS  PubMed  Google Scholar 

  48. Wisner, T. W., Sugimoto, K., Howard, P. W., Kawaguchi, Y. & Johnson, D. C. Anterograde transport of herpes simplex virus capsids in neurons by both separate and married mechanisms. J. Virol. 85, 5919–5928 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank P.W. Kincade for critical reading of the manuscript; K. Kontani (Meiji Pharmaceutical University) for rabbit polyclonal antibody to mouse Arl8; T. Kitamura (The University of Tokyo) for retroviral pMXpuro vectors; S. Akira (Osaka University) for C57BL/6 Tlr3–/– mice; and N. Tanimura, C. Sato and Y. Motoi for advice on the experiments. Supported by Grant-in-Aid for Scientific Research (S) (16H06388 to K.M.); Grant-in-Aid for Scientific Research (B) (17H04080 to Y.K.); Grant-in-Aid for Scientific Research (C) (16K08827 and 17K09369 to T.I.; 17K08851 to J.A.; and 18K07169 to R.F.); Grant for Joint Research Project of the Institute of Medical Science; the US National Institutes of Health (NIAID/AI079336 to G.N.B.); the University of Tokyo; a Grant-in-Aid for Young Scientists (A) (17H05069 to A.K.; and 18K15133 to Y.M.); Grant-in-Aid for Challenging Exploratory Research (17K19548 to K.M.; and 17K19549 to A.K.); Grant-in-Aid for Scientific Research on Innovative Areas (16H06433, 16H06429 and 16K21723 to Y.K.; 18H04666 to K.M.; 18H04968 to A.K.; and 18H04856 to S.-I.S.); and a contract research fund from the Japan Initiative for Global Research Network on Infectious Diseases (JP18fm0108006 to Y.K.).

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Author notes

  1. These authors contributed equally: Ryota Sato, Akihisa Kato.

Authors and Affiliations

  1. Division of Innate Immunity, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    Ryota Sato, Shin-Ichiroh Saitoh, Takuma Shibata, Yusuke Murakami, Ryutaro Fukui, Kaiwen Liu, Yun Zhang & Kensuke Miyake

  2. Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    Akihisa Kato, Jun Arii & Yasushi Kawaguchi

  3. Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    Akihisa Kato, Jun Arii & Yasushi Kawaguchi

  4. Division of Neuronal Network, Department of Basic Medical Sciences, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    Takahiko Chimura & Toshiya Manabe

  5. Department of Biochemistry, Faculty of Pharmaceutical Sciences, Doshisha Women’s College, Kyoto, Japan

    Ge-Hong Sun-Wada

  6. Division of Biological Science, Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan

    Yoh Wada

  7. Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    Tsuneo Ikenoue

  8. UM/Sylvester Cancer Center, University of Miami Miller School of Medicine, Miami, FL, USA

    Glen N. Barber

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Contributions

R.S., A.K., T.C., S.-I.S., T.S., Y.M, R.F., K.L, Y.Z. and J.A. conducted the experiments; G.-H.S.-W., Y.W., T.I., and G.N.B. provided reagents; T.M. and Y.K. discussed the results; and R.S. and K. M. wrote the manuscript.

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Correspondence to Yasushi Kawaguchi or Kensuke Miyake.

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Supplementary Figure 1 TLR3 responses in primary neurons and fibroblasts.

(a) WT mice (n = 8 per group) were intracranially infected with indicated doses of HSV-1 and mice survival was evaluated. (b) Primary neurons, astrocytes, and microglia were stained with antibodies to a neuron marker NeuN, an astrocyte marker GFAP, and a microglia marker CD11b (red histograms). Black histograms show staining with the second reagent alone. (c) Primary neurons were left unstimulated or stimulated with poly(I:C) (PIC) at 25 μg/ml, cGAMP at 7 μM with Lipofectoamine or ISD 1 μg/ml with Lipofectoamine for 24 h. Real-time PCR was conducted to measure Ccl5 mRNA. These results are normalized by HPRT mRNA and represented by the mean value ± s. d. of triplicate wells. Statistical analysis was performed by the two-tailed Student’s t test. ***P < 0.001. (d) WT and Tlr3–/– primary neurons were left uninfected (U) or infected (HSV-1) with HSV-1 at an MOI of 10. Real-time PCR was conducted to measure Ifnb1 mRNA. The results were analyzed as in (c). (e) Membrane permeabilized staining of TLR3 and Unc93B1-Flag in NIH3T3 cells or those expressing the indicated molecules (red histograms). Black histograms indicate labeling with the secondary antibody alone. (f) NIH3T3 cells or those expressing the indicated molecules were left unstimulated (U) or stimulated with poly(I:C) at 5 (PIC 5) or 25 (PIC 25) μg/ml for 24 h. The concentration of CCL5 in the supernatant was determined by ELISA. The results are represented by the mean value ± s. d. of triplicate wells. (g, h) NIH3T3-TLR3-Unc93B1 cells were treated with vehicle, Rapamycin at 50 or 250 nM or Torin 1 at 50 or 250 nM for 2 h. The treated cells were left unstimulated (U) or stimulated with poly(I:C) at 5 (P5) or 25 μg/ml (P25) for 24 h (g) or at 25 μg/ml for the indicated periods of time (h). Immunoblotting of the indicated molecules was conducted (h). Grb2 is shown as a loading control. The concentration of CCL5 in the supernatant was determined by ELISA. The results are represented by the mean value ± s. d. of triplicate wells. Statistical analysis was performed by the two-tailed Student’s t test. ***P < 0.001. These experiments were repeated more than three times. (i) The deletion of RICTOR is shown by immunoblot of the whole cell lysate. Grb2, a loading control. Shown on the right is membrane permeabilized staining of TLR3 in WT and RictorΔ/Δ fibroblasts (red histograms). Black histograms indicate labeling with the secondary antibody alone

Supplementary Figure 2 Innate immune responses to HSV-1 in fibroblasts and macrophages.

(a) WT MEF cells or those expressing the indicated molecules were stained with the anti-TLR3 mAb (PaT3) or -flag mAb for Unc93B1. The results are shown in red histograms, and the black histograms show the staining with the secondary reagent alone. (b, c) MEF-TLR3-Unc93B1 cells were left unstimulated (U), stimulated with poly(I:C) at 25 μg/ml, or with HSV-1 at an MOI of 10 for indicated periods of times. Real-time PCR was conducted to evaluate Ifnb1 mRNA expression. The results are normalized by Hprt mRNA and represented by the mean value ± s. d. from triplicates. (d) MEF cells expressing indicated molecules were left uninfected (U) or infected with HSV-1 at an MOI of 10 for 24 h. IFN-β production was determined by ELISA. The results are represented by the mean value ± s. d. of triplicate wells. (e) MEF-TLR3-Unc93B1 cells were infected with HSV-1 at an MOI of 10. After the indicated periods of times, cell lysate was prepared, and immunoblotting of the indicated molecules was conducted. ICP4, an HSV-1 regulatory protein, is shown as a marker indicating HSV-1 infection. Grb2, a loading control. (f) MEF-TLR3-Unc93B1 cells were infected with HSV-1 at an MOI of 10 for indicated periods of times. Immunoblotting of indicated molecules in the whole lysates was conducted. (g) MEF cells expressing hCas9 with or without TRAF3 gRNA were left uninfected (U) or infected with HSV-1 at an MOI of 10. After 24 h, IFN-β production was measured by ELISA. The deletion of TRAF3 is shown by immunostaining of the whole cell lysate. Grb2, a loading control. (h) Bone marrow derived macrophages were stimulated with poly(I:C) at 25 μg/ml for indicated periods of times. Immunostaining of the indicated molecules in the whole lysate was conducted. *P < 0.05, ***P < 0.001

Supplementary Figure 3 Cytoskeletal changes and TLR3 trafficking are mediated by PKC and mTORC2.

(a, b) NIH3T3-TLR3-Unc93B1 cells were left untreated or treated with Rapamycin at 400 nM (R), Torin 1 at 250 nM (T), or Go 6976 at 250 nM (G) for 2 h. Cells were left unstimulated or stimulated (PIC) with poly(I:C) at 25 μg/ml for 18 h. Polymerized α-tubulin and actin were stained. The mean fluorescence intensity of α-tubulin and actin was statistically analyzed (b). Statistical analysis was performed by one-way ANOVA and Tukey’s test. ***P < 0.001. Scale bar, 20 μm. These experiments were repeated more than three times. (c) The wildtype and RictorΔ/Δ fibroblasts were transduced to express TLR3 and Unc93B1. These cells were left unstimulated (U), stimulated (PIC) with poly(I:C) at 25 μg/ml for 16 h or infected (HSV-1) with HSV-1 at an MOI of 10 for 6 h. TLR3 (green), LAMP-1 (red) and nuclei (blue) were stained and analyzed by confocal microscopy. The statistical analyses of the peripheral TLR3 is also shown. Statistical analysis was performed by one-way ANOVA and Tukey’s test. ***P < 0.001, Scale bar, 20 μm. These experiments were repeated more than three times

Supplementary Figure 4 TLR3 trafficking to the cell periphery depends on microtubule polymerization.

MEF/TLR3/Unc93B1 cells were untreated (UT) or treated for 2 h with a Nocodazole at 10 µM. Cells were left unstimulated (U), stimulated (PIC) with poly(I:C) at 25 μg/ml for 16 h or infected (HSV-1) with HSV-1 at an MOI of 10 for 6 h. TLR3 (green), LAMP-1 (red) and nuclei (blue) were stained and analyzed by confocal microscopy. The statistical analyses of the peripheral TLR3 and LAMP-1 are also shown. Statistical analysis was performed by one-way ANOVA and Tukey’s test. ***P < 0.001, Scale bar, 20 μm. These experiments were repeated more than three times

Supplementary Figure 5 Cytoskeletal changes and TLR3 trafficking in HSV-1-infected fibroblasts depend on mTOR, PKC and Rab7a.

(a, b) MEF-TLR3-Unc93B1 cells were left untreated (-) or treated with Go 6976 at 250 nM (G) or Torin 1 at 250 nM for 2 h (T). Cells were left uninfected (-) or infected with HSV-1 at an MOI of 10. After 6 h, polymerized α-tubulin and actin were stained (a). The statistical analyses of the mean fluorescence intensity of α-tubulin and actin are shown (b). ***P < 0.001. These experiments were repeated more than three times. (c) MEF-TLR3-Unc93B1 cells were untreated (U) or treated for 2 h with Go 6976 at 250 nM or Torin 1 at 1 μM. The treated cells were left uninfected (U) or infected with HSV-1 at an MOI of 10 for 6 h. TLR3 (green) and nuclei (blue) were stained. The percentages of peripheral TLR3 are also shown. (d) WT and Rab7aΔ/Δ MEF-TLR3-Unc93B1 cells were left uninfected (U) or infected with HSV-1 at an MOI of 10 for 6 h. TLR3 (green) and nuclei (blue) were stained, and the percentages of peripheral TLR3 were statistically analyzed. Statistical analyses were performed by one-way ANOVA and Tukey’s test. ***P < 0.001, Scale bar, 20 μm. These experiments were repeated more than three times

Figure Supplementary 6 The effect of inhibitors, molecular deficiency, and anti-TLR3 mAb on HSV-1 infection.

(a) Progeny virus yields (PFU/ml) in supernatants from MEF-TLR3-Unc93B1 cells untreated (-) or treated for 2 h with Go 6976 (G) at 250 nM or Torin 1 (T) at 1 μM before the HSV-1 infection for 24 h at an MOI of 10. The progeny virus yields were determined with Vero cells. (b) WT and Rab7aΔ/Δ MEF-TLR3-Unc93B1 cells were infected with the HSV-1 gB-RFP (red fluorescent protein) at an MOI of 10. RFP expression enables detection of HSV-1 in infected cells by confocal microscopy. After 6 h post-infection, the cells were immunostained with anti-TLR3 mAb, and the subcellular distribution of HSV-1 gB-RFP and TLR3 was analyzed. Statistical analyses of TLR3 colocalization with gB-RFP are shown. These experiments were repeated more than three times. (c) MEF-TLR3-Unc93B1 cells were treated with DMSO (Vehicle) or Torin 1 at 1 μM for 24 h and stained with Annexin V to determine cell viability. (d) WT mice (n = 10 per group) were intracranially administered with DMSO (Vehicle) or 6 μg of Torin1. Mice survival is shown. (e) WT mice were intracranially treated with 6 μg of Torin1. After 2 h, cell lysate was prepared, and immunoblotting of the indicated molecules was conducted. Grb2, a loading control. These experiments were repeated more than three times. (f) MEF-TLR3-Unc93B1 cells were left untreated or treated with control IgG or anti-TLR3 mAb (PaT3) at 4 or 20 μg/ml for 2 h. The treated cells were left unstimulated (U), stimulated (PIC) with poly(I:C) at 25 μg/ml for 24 h, or infected (HSV) with HSV-1 at an MOI of 10 for 24 h. The concentration of CCL5 and IFN-β in the supernatants was determined by ELISA. The results are represented by the mean value ± s.d. of triplicate wells. (g) WT mice (n = 8 per group) were intracranially infected with indicated doses of HSV-1 and mice survival was evaluated. (h, i) WT and Ifnar1−/− mice were intracranially infected with 103 PFU of HSV-1 (h) Mice survival is shown. n = 8 per group. (i) Virus yields in the brain at 2 days post infection. n > 3 per group. Statistical analysis was performed by the log-rank test (h) or the two-tailed Student’s t test (i). ***P < 0.001

Supplementary Figure 7 TLR3-mTORC2 axis is required for protection against HSV-1 infection.

Prior to its activation, TLR3 is associated with the GTPase Rab7a and is localized in late endosomes/lysosomes around the nucleus. Upon Herpes Simplex Virus-1 (HSV-1) infection, TLR3 is activated and recruits mTORC2, leading to TRAF6-dependent NF-κB activation to produce proinflammatory cytokines such as the chemokine CCL5. TLR3-dependent mTORC2 activation also induces microtubule elongation to the cell periphery by activating PKCs. Rab7a links TLR3-containing lysosomes with elongated microtubules for TLR3 trafficking to the cell periphery. This trafficking is required for the interaction between TLR3 and type I interferon (type I IFN) signaling molecules, such as TRAF3 and mTORC1. A monoclonal antibody to TLR3 shows the rescuing effect against Herpes Simplex Encephalitis by enhancing TLR3 responses. N, nucleus

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Sato, R., Kato, A., Chimura, T. et al. Combating herpesvirus encephalitis by potentiating a TLR3–mTORC2 axis. Nat Immunol 19, 1071–1082 (2018). https://doi.org/10.1038/s41590-018-0203-2

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