Summary
Toll-like receptor (TLR) signalling involves five TIR adapter proteins, which couple to downstream protein kinases that ultimately lead to the activation of transcription factors such as nuclear factor-κB (NF-κB) and members of the interferon regulatory factor (IRF) family. TLRs play a crucial role in host defence against invading microorganisms, and highlighting their importance in the immune system is the fact that TLRs are targeted by viral immune evasion strategies. Identifying the target host proteins of such viral inhibitors is very important because valuable insights into how host cells respond to infection may be obtained. Also, viral proteins may have potential as therapeutic agents. Luciferase reporter gene assays are a very useful tool for the analysis of TLR signalling pathways, as the effect of a putative viral inhibitor on a large amount of signals can be examined in one experiment. A basic reporter gene assay involves the transfection of cells with a luciferase reporter gene, along with an activating expression plasmid, with or without a plasmid expressing a viral inhibitor. Induction of a signalling pathway leads to luciferase protein expression, which is measured using a luminometer. Results from these assays can be informative for deciding which host proteins to test for interactions with a viral inhibitor. Successful assays for measuring protein–protein interactions include co-immunoprecipitations (Co-IPs) and glutathione- S-transferase (GST)-pulldowns. Co-IP experiments involve precipitating a protein out of a cell lysate using a specific antibody bound to Protein A/G sepharose. Additional molecules complexed to that protein are captured as well and can be detected by Western blot analysis. GST-pulldown experiments are similar in principle to Co-IPs, but a bait GST-fusion protein complexed to glutathione-sepharose (GSH) beads is used to pull down interaction partners instead of an antibody. Again, complexes recovered from the beads are analysed by Western blotting.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Bowie, A. G. (2007). Translational mini-review series on Toll-like receptors: recent advances in understanding the role of Toll-like receptors in anti-viral immunity. Clin Exp Immunol 147, 217–26.
Li, K., Foy, E., Ferreon, J. C., Nakamura, M., Ferreon, A. C., Ikeda, M., Ray, S. C., Gale, M. and Lemon S.M. (2005). Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor adapter protein TRIF. Proc Natl Acad Sci U S A 102, 2992–7.
3.Li, X.-D., Sun, L., Seth, R. B., Pineda, G. and Chen, Z. J. From the cover: hepatitis C virus protease NS3/4A cleaves mitochondrial anti-viral signalling protein off the mitochondria to evade innate immunity. Proc Natl Acad Sci U S A102, 17717–22.
Bowie, A., Kiss-Toth, E., Symons, J. A., Smith, G. L., Dower, S. K. and O’Neill, L. A. (2000). A46R and A52R from vaccinia virus are antagonists of host IL-1 and toll-like receptor signaling. Proc Natl Acad Sci U S A 97, 10162–7.
Stack, J., Haga, I. R., Schroder, M., Bartlett, N. W., Maloney, G., Reading, P. C., Fitzgerald, K. A., Smith, G. L. and Bowie, A. G. (2005). Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence. J Exp Med 201, 1007–18.
McCoy, S. L., Kurtz, S. E., Macarthur, C. J., Trune, D. R. and Hefeneider, S. H. (2005). Identification of a peptide derived from vaccinia virus A52R protein that inhibits cytokine secretion in response to TLR-dependent signaling and reduces in vivo bacterial-induced inflammation. J Immunol 174, 3006–14.
Tsung, A., McCoy, S. L., Klune, J. R., Geller, D. A., Billiar, T. R. and Hefeneider, S. H. (2007). A novel inhibitory peptide of Toll-like receptor signalling limits lipopolysaccharide-induced production of inflammatory mediators and enhances survival in mice. Shock 27, 364–369.
Smith, G. L., Chan, Y. S. and Howard, S. T. (1991). J Gen Virol 72, 1349–1376.
Applequist, S. E., Wallin, R. P. and Ljunggren, H. G. (2002). Variable expression of Toll-like receptor in murine innate and adaptive immune cell lines. Int Immunol 14, 1065–74.
Fitzgerald, K. A., Rowe, D. C., Barnes, B. J., Caffrey, D. R., Visintin, A., Latz, E., Monks, B., Pitha, P. M. and Golenbock, D. T. (2003). LPS-TLR4 Signaling to IRF-3/7 and NF-kB involves the Toll adapters TRAM and TRIF. J Exp Med 198, 1043–55.
Schoenemeyer, A., Barnes, B. J., Mancl, M. E., Latz, E., Goutagny, N., Pitha, P. M., Fitzgerald, K. A. and Golenbock, D. T. (2005). The interferon regulatory factor, IRF5, is a central mediator of TLR7 signaling. J Biol Chem 280, 17005–12.
Jefferies, C., Bowie, A., Brady, G., Cooke, E. L., Li, X. and O’Neill, L. A. (2001). Transactivation by the p65 subunit of NF-kappaB in response to interleukin-1 (IL-1) involves MyD88, IL-1 receptor-associated kinase 1, TRAF-6, and Rac1. Mol Cell Biol 21, 4544–52.
Sato, M., Suemori, H., Hata, N., Asagiri, M., Ogasawara, K., Nakao, K., Nakaya, T., Katsuki, M., Noguchi, S., Tanaka, N. and Taniguchi, T. (2000). Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-alpha/beta gene induction. Immunity 13, 539–48.
Kiss-Toth, E., Guesdon, F. M., Wyllie, D. H., Qwarnstrom, E. E. and Dower, S. K. (2000). A novel mammalian expression screen exploiting green fluorescent protein-based transcription detection in single cells. J Immunol Methods 239, 125–35.
Lin, R., Heylbroeck, C., Genin, P., Pitha, P.M. and Hiscott, J. (1999). Essential role of interferon regulatory factor 3 in direct activation of RANTES chemokine transcription. Mol Cell Biol 19, 959–66.
Medzhitov, R., Preston-Hurlburt, P. and Janeway, C. A., Jr. (1997). A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388, 394–7.
Fitzgerald, K. A., Palsson-McDermott, E. M., Bowie, A. G., Jefferies, C. A., Mansell, A. S., Brady, G., Brint, E., Dunne, A., Gray, P., Harte, M. T., McMurray, D., Smith, D. E., Sims, J. E., Bird, T. A. and O’Neill, L. A. (2001). Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction. Nature 413, 78–83.
Muzio, M., Ni, J., Feng, P. and Dixit, V. M. (1997). IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. Science 278, 1612–5.
Sato, S., Sugiyama, M., Yamamoto, M., Watanabe, Y., Kawai, T., Takeda, K. and Akira, S. (2003). Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF) associates with TNF receptor-associated factor 6 and TANK-binding kinase 1, and activates two distinct transcription factors, NF-kappa B and IFN-regulatory factor-3, in the Toll-like receptor signaling. J Immunol 171, 4304–10.
Liberati, N. T., Fitzgerald, K. A., Kim, D. H., Feinbaum, R., Golenbock, D. T. and Ausubel, F. M. (2004). Requirement for a conserved Toll/interleukin-1 resistance domain protein in the Caenorhabditis elegans immune response. Proc Natl Acad Sci U S A 101, 6593–8.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science + Business Media, LLC
About this protocol
Cite this protocol
Stack, J., Bowie, A.G. (2009). Characterisation of Viral Proteins that Inhibit Toll-Like Receptor Signal Transduction. In: McCoy, C.E., O’Neill, L.A.J. (eds) Toll-Like Receptors. Methods in Molecular Biology™, vol 517. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-541-1_14
Download citation
DOI: https://doi.org/10.1007/978-1-59745-541-1_14
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-72-5
Online ISBN: 978-1-59745-541-1
eBook Packages: Springer Protocols