Evaluation of a Bead-Free Coimmunoprecipitation Technique for Identification of Virus–Host Protein Interactions Using High-Resolution Mass Spectrometry

Abstract

Protein interactions between virus and host are essential for viral propagation and movement, as viruses lack most of the proteins required to thrive on their own. Precision methods aimed at disrupting virus-host interactions represent new approaches to disease management but require in-depth knowledge of the identity and binding specificity of host proteins within these interaction networks. Protein coimmunoprecipitation (co-IP) coupled with mass spectrometry (MS) provides a high-throughput way to characterize virus-host interactomes in a single experiment. Common co-IP methods use antibodies immobilized on agarose or magnetic beads to isolate virus-host complexes in solutions of host tissue homogenate. Although these workflows are well established, they can be fairly laborious and expensive. Therefore, we evaluated the feasibility of using antibody-coated microtiter plates coupled with MS analysis as an easy, less expensive way to identify host proteins that interact with Potato leafroll virus (PLRV), an insect-borne RNA virus that infects potatoes. With the use of the bead-free platform, we were able to detect 36 plant and 1 nonstructural viral protein significantly coimmunoprecipitating with PLRV. Two of these proteins, a 14-3-3 signal transduction protein and malate dehydrogenase 2 (mMDH2), were detected as having a weakened or lost association with a structural mutant of the virus, demonstrating that the bead-free method is sensitive enough to detect quantitative differences that can be used to pin-point domains of interaction. Collectively, our analysis shows that the bead-free platform is a low-cost alternative that can be used by core facilities and other investigators to identify plant and viral proteins interacting with virions and/or the viral structural proteins.

Targeted disruption of aphid transmission: a vision for the management of crop diseases caused by Luteoviridae members

“…CIPK7 could therefore be part of a plant defense mechanism and, in this regard, regulating its expression could reduce virus accumulation and thus virus transmission by aphids. DeBlasio, Cilia and colleagues [55][56][57][58][59] used affinity purification coupled to mass spectrometry (AP-MS) to identify the host-virus interactome for the PLRV structural proteins during infection of Nicotiana benthamiana and Solanum tuberosum (potato). They characterized three phosphorylated positional isomers of the RTP [56], suggesting possible residue targets for CIPK7.…”

The Interaction Dynamics of Two Potato Leafroll Virus Movement Proteins Affects Their Localization to the Outer Membranes of Mitochondria and Plastids

“…The false discovery rate was less than 1% on both the protein and peptide level. The relative levels of PLRV CP [ 32 ], P3a (YP_009179365.2), P17 (Genbank: NP_056750.1), P1 polyprotein (NP_056747.1), Protein A (AAA56730.1), and IgG (ABD64612.1) were measured across the different AP conditions using Skyline 64-bit version 4.1.0.11714 [ 33 ] through integration of the precursor ion (MS1) intensity peak areas for protein specific peptides ( Supplemental Table S1 ). For quantification of protein abundance, the integrated peak areas minus the background (minimum intensity at peak boundaries) for three precursor isotope ions (M, M + 1, and M + 2) were first added to get an abundance value for each peptide than values for each peptide in a protein were averaged together to get a protein abundance value ( Supplemental Table S1 ).…”