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

J Biomol Tech. 2017 Sep;28(3):111-121. doi: 10.7171/jbt.17-2803-002. Epub 2017 Jul 24.

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.

Keywords: Luteoviridae, polerovirus; insect-borne virus; molecular virology; phloem-limited pathogen; potato leafroll.

MeSH terms

  • Host-Pathogen Interactions / genetics*
  • Immunoprecipitation / methods*
  • Luteoviridae / chemistry
  • Luteoviridae / genetics
  • Mass Spectrometry
  • Plant Diseases / genetics
  • Plant Diseases / virology
  • Plant Proteins / chemistry
  • Plant Proteins / immunology
  • Plant Proteins / isolation & purification*
  • Symbiosis / genetics
  • Viral Proteins / chemistry
  • Viral Proteins / immunology
  • Viral Proteins / isolation & purification*
  • Virion / chemistry
  • Virion / genetics

Substances

  • Plant Proteins
  • Viral Proteins

Supplementary concepts

  • Potato leafroll virus