Formation of large viroplasms and virulence of Cauliflower mosaic virus in turnip plants depend on the N-terminal EKI sequence of viral protein TAV.

Angèle Geldreich,Clément Bouton,Julie Kubina,Lyubov Ryabova,Gabrielle Haas,Maria Dimitrova,Mathieu Erhardt,Mélanie Tanguy,Mario Keller,Mikhail M Pooggin

doi:10.1371/journal.pone.0189062

Abstract

Cauliflower mosaic virus (CaMV) TAV protein (TransActivator/Viroplasmin) plays a pivotal role during the infection cycle since it activates translation reinitiation of viral polycistronic RNAs and suppresses RNA silencing. It is also the major component of cytoplasmic electron-dense inclusion bodies (EDIBs) called viroplasms that are particularly evident in cells infected by the virulent CaMV Cabb B-JI isolate. These EDIBs are considered as virion factories, vehicles for CaMV intracellular movement and reservoirs for CaMV transmission by aphids. In this study, focused on different TAV mutants in vivo, we demonstrate that three physically separated domains collectively participate to the formation of large EDIBs: the N-terminal EKI motif, a sequence of the MAV domain involved in translation reinitiation and a C-terminal region encompassing the zinc finger. Surprisingly, EKI mutant TAVm3, corresponding to a substitution of the EKI motif at amino acids 11–13 by three alanines (AAA), which completely abolished the formation of large viroplasms, was not lethal for CaMV but highly reduced its virulence without affecting the rate of systemic infection. Expression of TAVm3 in a viral context led to formation of small irregularly shaped inclusion bodies, mild symptoms and low levels of viral DNA and particles accumulation, despite the production of significant amounts of mature capsid proteins. Unexpectedly, for CaMV-TAVm3 the formation of viral P2-containing electron-light inclusion body (ELIB), which is essential for CaMV aphid transmission, was also altered, thus suggesting an indirect role of the EKI tripeptide in CaMV plant-to-plant propagation. This important functional contribution of the EKI motif in CaMV biology can explain the strict conservation of this motif in the TAV sequences of all CaMV isolates.

Highlights

Virus-infected cells often contain cytoplasmic and/or nuclear inclusion bodies mainly composed of viral proteins and called viroplasms
To further investigate the mechanism involved in viroplasm formation, we co-transfected tobacco BY-2 cells with recombinant plasmids encoding full-length TAV fused at its N-terminus to mRFP, and two truncated TAV proteins, fused at their N-termini to enhanced green fluorescent protein (EGFP) (EGFP-A and EGFP-TAVΔA), to verify by competition assays if, in addition to domain A, other TAV sequences are involved in the formation of electron-dense inclusion bodies (EDIBs) (Fig 2A and 2B)
Having already shown in a previous study that the fusion of EGFP did not modify the capacity of TAV to form EDIBs in BY-2 cells [9], we observed the cells using laser scanning confocal microscope (LSCM), 16 h and 24 h after transfection. mRFP-TAV protein assembled into large IBs when expressed alone (Fig 2B, panel 1), demonstrating that, as for EGFP, the fusion of mRFP at the N-terminus of TAV did not hinder TAV self-association

Summary

Introduction

Virus-infected cells often contain cytoplasmic and/or nuclear inclusion bodies mainly composed of viral proteins and called viroplasms. In cells infected by some animal viruses, viroplasms result from the assembly of small aggregates that are transported by dynein along microtubules to the microtubule organization centre at the periphery of the nucleus, where they recruit cellular proteins and mitochondria [2,3,4]. These viroplasms resemble aggresomes that naturally occur in cells to reduce the toxicity of misfolded proteins and make them susceptible to proteolysis by the proteasome and/or by autophagy [2]. Viroplasms are observed in cells infected by plant DNA viruses such as Cauliflower mosaic virus of the Caulimoviridae family [7,8,9]

Methods

Results

Conclusion