Identification of protein interactions of grapevine fanleaf virus RNA-dependent RNA polymerase during infection of Nicotiana benthamiana by affinity purification and tandem mass spectrometry

J Gen Virol. 2021 May;102(5):001607. doi: 10.1099/jgv.0.001607.

Abstract

The RNA-dependent RNA polymerase (1EPol) is involved in replication of grapevine fanleaf virus (GFLV, Nepovirus, Secoviridae) and causes vein clearing symptoms in Nicotiana benthamiana. Information on protein 1EPol interaction with other viral and host proteins is scarce. To study protein 1EPol biology, three GFLV infectious clones, i.e. GHu (a symptomatic wild-type strain), GHu-1EK802G (an asymptomatic GHu mutant) and F13 (an asymptomatic wild-type strain), were engineered with protein 1EPol fused to a V5 epitope tag at the C-terminus. Following Agrobacterium tumefaciens-mediated delivery of GFLV clones in N. benthamiana and protein extraction at seven dpi, when optimal 1EPol:V5 accumulation was detected, two viral and six plant putative interaction partners of V5-tagged protein 1EPol were identified for the three GFLV clones by affinity purification and tandem mass spectrometry. This study provides insights into the protein interactome of 1EPol during GFLV systemic infection in N. benthamiana and lays the foundation for validation work.

Keywords: Nepovirus; Nicotiana benthamiana; RNA-dependent RNA polymerase; Secoviridae; affinity purification; grapevine fanleaf virus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrobacterium tumefaciens / genetics
  • Chromatography, Affinity
  • Host-Pathogen Interactions
  • Mutation
  • Nepovirus / physiology*
  • Nicotiana / virology*
  • Plant Diseases / virology
  • Plant Proteins / metabolism
  • Protein Interaction Maps*
  • Proteomics
  • RNA-Dependent RNA Polymerase / genetics
  • RNA-Dependent RNA Polymerase / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Tandem Mass Spectrometry
  • Viral Proteins / genetics
  • Viral Proteins / isolation & purification
  • Viral Proteins / metabolism*

Substances

  • Plant Proteins
  • Recombinant Fusion Proteins
  • Viral Proteins
  • RNA-Dependent RNA Polymerase

Supplementary concepts

  • Grapevine fanleaf virus