Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Summary

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.

Abstract

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.

Introduction

Myxovirus resistance (Mx) proteins are an important part of the innate immune defense against viral pathogens. These proteins are large dynamin-like GTPases that are induced by type I and type III interferons. The corresponding Mx genes are present in nearly all vertebrates in one or multiple copies and their gene products inhibit a wide range of viruses, including Orthomyxoviridae (e.g., influenza virus), Rhabdoviridae (e.g., vesicular stomatitis virus), Bunyaviridae (e.g., la crosse virus) and Retroviridae (e.g., human immunodeficiency virus-1)^1-4. It is unclear how these proteins rec....

Protocol

Note: The following transfection and co-immunoprecipitation protocol is established for a 9 cm Petri dish format. Other formats are also possible after scaling the protocol.

1. Seeding the Human Embryonic Kidney (HEK) 293T Cells

1. Seed the HEK293T cells one day before transfection at 1.2 x 10⁶ cells per 9 cm Petri dish in 12 ml of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.4 mM Na-pyruvate, 0.1 mM non-essential amino acids, 100 U/ml penicillin and 0.1 mg/ml streptomycin.
2. Grow the cells 16 hr at 37 °C and 5% CO₂.
3. Vi....

Results

N-ethylmaleimide is an organic compound that can be used to irreversibly modify free thiol groups, e.g. to inhibit cysteine proteases (Figure 1).

The antiviral Mx1 protein inhibits influenza A virus replication by interacting with the viral nucleoprotein. The optimized co-immunoprecipitation protocol described here allows to study this NP–Mx1 interaction. HEK293T cells were transfected with expression vectors for the antiviral Mx1 protein in the absence or presence of.......

Discussion

Studying the interaction between antiviral proteins and their viral targets is very important to understand the details of the antiviral mechanism of these proteins. This can give new insights into how viruses and their hosts co-evolved and be the basis for the development of new antiviral strategies. The optimized co-immunoprecipitation protocol described here allows to study the interaction between the mouse Mx1 protein and its viral target, the influenza NP protein. The most important aspect of this protocol is the ad.......

Materials

List of materials used in this article

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