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Investigating the Interactions of the Cucumber Mosaic Virus 2b Protein with the Viral 1a Replicase Component and the Cellular RNA Silencing Factor Argonaute 1.

Published version
Peer-reviewed

Repository DOI


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Authors

Crawshaw, Sam 
Nietlispach, Daniel  ORCID logo  https://orcid.org/0000-0003-4364-9291

Abstract

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient, it induces reinforcement of antiviral silencing by AGO2 and triggers increased resistance against aphids, CMV's insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein, which sequesters sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated, and red and green fluorescent protein fusions were used to investigate subcellular colocalization with AGO1 and the 1a protein. The effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein's interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein's ability to interact with AGO1, the 1a protein, and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

Description

Peer reviewed: True


Acknowledgements: We thank Adrienne E. Pate for expert technical assistance and Tomás Canto and Lewis G. Watt for useful discussions and the provision of DNA constructs.


Publication status: Published

Keywords

RNA silencing suppressor regulation, intrinsically disordered protein, protein condensate, protein folding, protein phase separation, Argonaute Proteins, Cucumovirus, Arabidopsis, Arabidopsis Proteins, Protein Binding, Viral Proteins, Host-Pathogen Interactions, Viral Replicase Complex Proteins, Plant Diseases, RNA-Dependent RNA Polymerase, Methyltransferases

Journal Title

Viruses

Conference Name

Journal ISSN

1999-4915
1999-4915

Volume Title

16

Publisher

MDPI AG
Sponsorship
BBSRC (BB/W510609/1)
Royal Society (ICA\R1\201221)
Isaac Newton Trust (23.07(a))
Leverhulme Trust (RPG-2022-134)
Biotechnology and Biological Sciences Research Council (2273242)
Biotechnology and Biological Sciences Research Council (BB/M011194/1)
SC received a studentship from the UK Biotechnological and Biological Sciences Research Council (BBSRC)-Cambridge University Doctoral Training Program (BB/M011194/1). AMM was supported by grants from the BBSRC (21ROMITIGATIONFUND CAMBRIDGE BB/W510609/1), The Royal Society (ICA\R1\201221) and from The Leverhulme Trust (RPG-2022-134). We thank the Isaac Newton Trust for funding to PJER.
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