Skip to main content
SpringerLink
Log in

Methods to Visualize MAVS Subcellular Localization

  • Protocol
  • First Online:
Book cover Innate Antiviral Immunity

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1656))

Abstract

The mitochondrial antiviral signaling (MAVS) protein is a central adaptor protein required for antiviral innate immune signaling. To facilitate its roles in innate immunity, MAVS localizes to multiple intracellular membranous compartments, including the mitochondria, the mitochondrial-associated ER membrane (MAM), and peroxisomes. Studies of MAVS function therefore often require an analysis of MAVS localization. To detect MAVS protein on intracellular membranes, biochemical fractionation to isolate MAMs, mitochondria, or peroxisomes can be used. Further, immunofluorescence with antibodies against specific membrane markers can be used to visualize MAVS distribution throughout the cell. Here, we describe the biochemical fractionation and immunofluorescence protocols used to detect MAVS subcellular localization.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Vazquez C, Horner SM (2015) MAVS coordination of antiviral innate immunity. J Virol 89(14):6974–6977. doi:10.1128/JVI.01918-14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Seth RB, Sun L, Ea CK, Chen ZJ (2005) Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell 122(5):669–682. doi:S0092-8674(05)00816-0 [pii]10.1016/j.cell.2005.08.012

    Article  CAS  Google Scholar 

  3. Kawai T, Takahashi K, Sato S, Coban C, Kumar H, Kato H, Ishii KJ, Takeuchi O, Akira S (2005) IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat Immunol 6(10):981–988. doi:ni1243 [pii]10.1038/ni1243

    Article  CAS  Google Scholar 

  4. Horner SM, Liu HM, Park HS, Briley J, Gale M Jr (2011) Mitochondrial-associated endoplasmic reticulum membranes (MAM) form innate immune synapses and are targeted by hepatitis C virus. Proc Natl Acad Sci U S A 108(35):14590–14595. doi:10.1073/pnas.1110133108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Dixit E, Boulant S, Zhang Y, Lee AS, Odendall C, Shum B, Hacohen N, Chen ZJ, Whelan SP, Fransen M, Nibert ML, Superti-Furga G, Kagan JC (2010) Peroxisomes are signaling platforms for antiviral innate immunity. Cell 141(4):668–681. doi:S0092-8674(10)00435-6 [pii] 10.1016/j.cell.2010.04.018

    Article  CAS  Google Scholar 

  6. Li K, Foy E, Ferreon JC, Nakamura M, Ferreon AC, Ikeda M, Ray SC, Gale M Jr, Lemon SM (2005) Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the toll-like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci U S A 102(8):2992–2997. doi:0408824102 [pii]10.1073/pnas.0408824102

    Article  CAS  Google Scholar 

  7. Li XD, Sun L, Seth RB, Pineda G, Chen ZJ (2005) Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity. Proc Natl Acad Sci U S A 102(49):17717–17722. doi:0508531102 [pii] 10.1073/pnas.0508531102

    Article  CAS  Google Scholar 

  8. Meylan E, Curran J, Hofmann K, Moradpour D, Binder M, Bartenschlager R, Tschopp J (2005) Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437(7062):1167–1172. doi:nature04193 [pii]10.1038/nature04193

    Article  CAS  Google Scholar 

  9. Loo YM, Owen DM, Li K, Erickson AK, Johnson CL, Fish PM, Carney DS, Wang T, Ishida H, Yoneyama M, Fujita T, Saito T, Lee WM, Hagedorn CH, Lau DT, Weinman SA, Lemon SM, Gale M Jr (2006) Viral and therapeutic control of IFN-beta promoter stimulator 1 during hepatitis C virus infection. Proc Natl Acad Sci U S A 103(15):6001–6006. doi:0601523103 [pii] 10.1073/pnas.0601523103

    Article  CAS  Google Scholar 

  10. Horner SM, Wilkins C, Badil S, Iskarpatyoti J, Gale M Jr (2015) Proteomic analysis of mitochondrial-associated ER membranes (MAM) during RNA virus infection reveals dynamic changes in protein and organelle trafficking. PLoS One 10(3):e0117963. doi:10.1371/journal.pone.0117963

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bozidis P, Williamson CD, Colberg-Poley AM (2007) Isolation of endoplasmic reticulum, mitochondria, and mitochondria-associated membrane fractions from transfected cells and from human cytomegalovirus-infected primary fibroblasts. Curr Protoc Cell Biol Chapter 3:Unit 3 27. doi:10.1002/0471143030.cb0327s37

    Article  Google Scholar 

  12. Graham JM (2001) Isolation of peroxisomes from tissues and cells by differential and density gradient centrifugation. Curr Protoc Cell Biol Chapter 3:Unit 3 5. doi:10.1002/0471143030.cb0305s06

    Google Scholar 

Download references

Acknowledgment

We thank the Duke University Light Microscopy Core Facility for assistance with imaging and analysis. Research in the Horner laboratory is supported by funds from the National Institutes of Health (NIH) (R01AI125416 and R21AI124100) and a Duke School of Medicine Whitehead Scholarship. Additional funding sources include the Ford Foundation (CV) and NIH T32CA009111 (DCB).

Author information

Authors and Affiliations

  1. Department of Molecular Genetics and Microbiology, Duke University Medical Center, 213 Research Dr., Box 3053, Durham, NC, 27710, USA

    Christine Vazquez, Dia C. Beachboard & Stacy M. Horner

  2. Department of Medicine, Duke University Medical Center, 213 Research Dr., Box 3053, Durham, NC, 27710, USA

    Stacy M. Horner

Authors
  1. Christine Vazquez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dia C. Beachboard
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stacy M. Horner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stacy M. Horner .

Editor information

Editors and Affiliations

  1. Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada

    Karen Mossman

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Vazquez, C., Beachboard, D.C., Horner, S.M. (2017). Methods to Visualize MAVS Subcellular Localization. In: Mossman, K. (eds) Innate Antiviral Immunity. Methods in Molecular Biology, vol 1656. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7237-1_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7237-1_7

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7236-4

  • Online ISBN: 978-1-4939-7237-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation