Chapter 18
Double-Stranded RNA Pull-Down to Characterize Viral
Replication Complexes in Plants
Marco Incarbone and Christophe Ritzenthaler
Abstract
Plant RNA viruses are obligate intracellular parasites that hijack specific cellular membranes to replicate
their genomes in what are commonly known as viral replication complexes (VRC). These contain host- and
virus-encoded proteins and viral RNA. Double-stranded RNA (dsRNA) is a mandatory intermediate of
RNA replication and a hallmark feature of VRCs. We have recently developed a method to isolate viral
dsRNA and its associated proteins through pull-down of an ectopically expressed dsRNA-binding protein
(B2:GFP) from infected Arabidopsis thaliana plants. After mass spectrometry analysis to identify the
dsRNA-associated proteins, resulting candidate proteins of interest are tagged with a red fluorescent
protein and their subcellular localization in relation to VRCs is assessed by transient expression within
leaves of B2:GFP-transgenic Nicotiana benthamiana plants. In this chapter we describe in detail these
experimental procedures to allow investigators to characterize the replication complexes of their plant RNA
virus of interest.
Key words Virus replication complex, dsRNA, Immunoprecipitation, Mass spectrometry, Northwestern blot, Laser confocal microscopy
1 Introduction
RNA replication is a key step in the infection cycle of RNA viruses.
This process involves both virus- and host-encoded proteins, and
takes place in endo-membrane invaginations/vesicles [1, 2]. While
the specific proteins and membranes involved in the formation of
virus replication complexes depend on the virus and host species,
the presence of dsRNA, as a product of the viral RNA-dependent
RNA polymerase, is a hallmark of infection. The precise molecular
composition of replication complexes remains poorly characterized
for most plant viruses, although the question has been approached
for a few model plant RNA viruses through different experimental
approaches. These include genetic screens on yeast as a surrogate
host [3] and in planta pull-down of viral proteins [4–6]. These
approaches yielded seminal results, but were limited to the use of
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_18, © Springer Science+Business Media, LLC, part of Springer Nature 2020
307
Double-Stranded RNA Pull-Down to Characterize Viral
Replication Complexes in Plants
Marco Incarbone and Christophe Ritzenthaler
Abstract
Plant RNA viruses are obligate intracellular parasites that hijack specific cellular membranes to replicate
their genomes in what are commonly known as viral replication complexes (VRC). These contain host- and
virus-encoded proteins and viral RNA. Double-stranded RNA (dsRNA) is a mandatory intermediate of
RNA replication and a hallmark feature of VRCs. We have recently developed a method to isolate viral
dsRNA and its associated proteins through pull-down of an ectopically expressed dsRNA-binding protein
(B2:GFP) from infected Arabidopsis thaliana plants. After mass spectrometry analysis to identify the
dsRNA-associated proteins, resulting candidate proteins of interest are tagged with a red fluorescent
protein and their subcellular localization in relation to VRCs is assessed by transient expression within
leaves of B2:GFP-transgenic Nicotiana benthamiana plants. In this chapter we describe in detail these
experimental procedures to allow investigators to characterize the replication complexes of their plant RNA
virus of interest.
Key words Virus replication complex, dsRNA, Immunoprecipitation, Mass spectrometry, Northwestern blot, Laser confocal microscopy
1 Introduction
RNA replication is a key step in the infection cycle of RNA viruses.
This process involves both virus- and host-encoded proteins, and
takes place in endo-membrane invaginations/vesicles [1, 2]. While
the specific proteins and membranes involved in the formation of
virus replication complexes depend on the virus and host species,
the presence of dsRNA, as a product of the viral RNA-dependent
RNA polymerase, is a hallmark of infection. The precise molecular
composition of replication complexes remains poorly characterized
for most plant viruses, although the question has been approached
for a few model plant RNA viruses through different experimental
approaches. These include genetic screens on yeast as a surrogate
host [3] and in planta pull-down of viral proteins [4–6]. These
approaches yielded seminal results, but were limited to the use of
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_18, © Springer Science+Business Media, LLC, part of Springer Nature 2020
307