Co-immunoudfældning af musen Mx1 Protein med influenza A-virus nukleoprotein

Published: April 21st, 2015

DOI:

10.3791/52871

Judith Verhelst^1,2, Dorien De Vlieger^1,2, Xavier Saelens^1,2

¹Inflammation Research Center, VIB, ²Department of Biomedical Molecular Biology, Ghent University

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP–Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

Myxovirus modstand (Mx) proteiner er en vigtig del af det medfødte immunforsvar mod virale patogener. Disse proteiner er store dynamin-lignende GTPaser, der induceres af type I og type III interferoner. De tilsvarende Mx gener er til stede i næsten alle hvirveldyr i en eller flere kopier og deres genprodukter inhibere en bred vifte af vira, herunder Orthomyxoviridae (f.eks., Influenza virus), Rhabdoviridae (f.eks., Vesikulær stomatitis virus), Bunyaviridae (f.eks. , La Crosse-virus) og Retroviridae (fx humant immundefektvirus-1) ^1-4. Det er uklart, hvordan disse proteiner genkender en så bred vifte af vira, uden nogen ti....

Bemærk: Følgende transfektion og co-immunopræcipitering protokol er udfærdiget til en 9 cm petriskål format. Andre formater er også muligt efter skalering protokollen.

1. podning af den humane embryonale nyre (HEK) 293T-celler

Pode HEK293T celler en dag før transfektion ved 1,2 x 10 ⁶ celler pr 9 cm petriskål i 12 ml Dulbeccos modificerede Eagles medium (DMEM) suppleret med 10% føtalt kalveserum, 2 mM L-glutamin, 0,4 mM Na-pyruvat, 0,1 mM ikke-essentielle amino.......

N-ethylmaleimid er en organisk forbindelse, der kan anvendes til irreversibelt at ændre frie thiolgrupper, fx at inhibere cysteinproteaser (figur 1).

Den antivirale Mx1 protein inhiberer influenza A virus replikation ved at interagere med den virale nukleoprotein. Den optimerede co-immunpræcipitering protokollen beskrevet her tillader at undersøge dette NP-Mx1 interaktion. HEK293T celler blev transficeret med ekspressionsvektorer for den antivirale Mx1 protein i .......

Studer interaktionen mellem antivirale proteiner og deres virale mål er meget vigtigt at forstå detaljerne i den antivirale mekanisme af disse proteiner. Dette kan give ny indsigt i, hvordan vira og deres værter co-udviklet og danne grundlag for udvikling af nye antivirale strategier. Den optimerede co-immunpræcipitering protokollen beskrevet her tillader at studere interaktionen mellem muse Mx1 protein og dets virale mål, influenza NP protein. Det vigtigste aspekt af denne protokol er tilsætningen af NEM i lysepu.......

Dette arbejde blev støttet af FWO-Vlaanderen, IBS projektet IOF10 / StarTT / 027 og Ghent University Special Research Grant BOF12 / GOA / 014.

....

Name	Company	Catalog Number	Comments
DMEM high glucose	Gibco	52100-047
N-Ethylmaleimide	Sigma	E-3876	Toxic
Igepal CA-630	Sigma	I-30212	also known as NP40
Protease Inhibitor Cocktail	Roche	11 873 580 001
anti-NP monoclonal antibody	NIH Biodefense and Emerging Infections Research Resources Repository	NR-4282	ascites blend of clones A1 and A3
anti-RNP polyclonal serum	NIH Biodefense and Emerging Infections Research Resources Repository	NR-3133	directed against A/Scotland/840/74 (H3N2)
Protein G Sepharose 4FF	GE Healthcare	17-0618-01
Hyperfilm ECL 18 x 24 cm	GE Healthcare	28-9068-36
ECL Western Blotting Substrate	Pierce	32106

Verhelst, J., Hulpiau, P., Saelens, X. Mx proteins: antiviral gatekeepers that restrain the uninvited. Microbiol Mol Biol Rev. 77 (4), 551-566 (2013).
Goujon, C., et al. Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection. Nature. 502 (7472), 559-562 (2013).
Kane, M., et al. MX2 is an interferon-induced inhibitor of HIV-1 infection. Nature. 502 (7472), 563-566 (2013).
Liu, Z., et al. The interferon-inducible MxB protein inhibits HIV-1 infection. Cell Host Microbe. 14 (4), 398-410 (2013).
Kochs, G., Haller, O. Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids. Proc Natl Acad Sci U S A. 96 (5), 2082-2086 (1999).
Flohr, F., Schneider-Schaulies, S., Haller, O., Kochs, G. The central interactive region of human MxA GTPase is involved in GTPase activation and interaction with viral target structures. FEBS Lett. 463 (1-2), 24-28 (1999).
Kochs, G., Haller, O. GTP-bound human MxA protein interacts with the nucleocapsids of Thogoto virus (Orthomyxoviridae). J Biol Chem. 274 (7), 4370-4376 (1999).
Mitchell, P. S., et al. Evolution-guided identification of antiviral specificity determinants in the broadly acting interferon-induced innate immunity factor MxA. Cell Host Microbe. 12 (4), 598-604 (2012).
Patzina, C., Haller, O., Kochs, G. Structural requirements for the antiviral activity of the human MxA protein against Thogoto and influenza A virus. J Biol Chem. 289 (9), 6020-6027 (2014).
Turan, K., et al. Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome. Nucleic Acids Res. 32 (2), 643-652 (2004).
Dittmann, J., et al. Influenza A virus strains differ in sensitivity to the antiviral action of Mx-GTPase. J Virol. 82 (7), 3624-3631 (2008).
Zimmermann, P., Manz, B., Haller, O., Schwemmle, M., Kochs, G. The viral nucleoprotein determines Mx sensitivity of influenza A viruses. J Virol. 85 (16), 8133-8140 (2011).
Manz, B., et al. Pandemic influenza A viruses escape from restriction by human MxA through adaptive mutations in the nucleoprotein. PLoS Pathog. 9 (3), e1003279 (2013).
Verhelst, J., Parthoens, E., Schepens, B., Fiers, W., Saelens, X. Interferon-inducible protein Mx1 inhibits influenza virus by interfering with functional viral ribonucleoprotein complex assembly. J Virol. 86 (24), 13445-13455 (2012).
Brewer, C. F., Riehm, J. P. Evidence for possible nonspecific reactions between N-ethylmaleimide and proteins. Anal Biochem. 18 (2), 248-255 (1967).
Wu, C. Y., Jeng, K. S., Lai, M. M. The SUMOylation of matrix protein M1 modulates the assembly and morphogenesis of influenza A virus. J Virol. 85 (13), 6618-6628 (2011).
Chen, Z., Zhou, J., Zhang, Y., Bepler, G. Modulation of the ribonucleotide reductase M1-gemcitabine interaction in vivo by N-ethylmaleimide. Biochem Biophys Res Commun. 413 (2), 383-388 (2011).
Rennie, M. L., McKelvie, S. A., Bulloch, E. M., Kingston, R. L. Transient dimerization of human MxA promotes GTP hydrolysis, resulting in a mechanical power stroke. Structure. 22 (10), 1433-1445 (2014).
Gifford, J. L., Walsh, M. P., Vogel, H. J. Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs. Biochem J. 405 (2), 199-221 (2007).
Gallagher, S., Chakavarti, D. Immunoblot analysis. J Vis Exp. (16), (2008).
Eslami, A., Lujan, J. Western blotting: sample preparation to detection. J Vis Exp. (44), (2010).
Pavlovic, J., Haller, O., Staeheli, P. Human and mouse Mx proteins inhibit different steps of the influenza virus multiplication cycle. J Virol. 66 (4), 2564-2569 (1992).

This article has been published

Video Coming Soon

Keep me updated: