Unravelling the involvement of cilevirus p32 protein in the viral transport

Abstract

Citrus leprosis (CL) is a severe disease that affects citrus orchards mainly in Latin America. It is caused by Brevipalpus-transmitted viruses from genera Cilevirus and Dichorhavirus. Currently, no reports have explored the movement machinery for the cilevirus. Here, we have performed a detailed functional study of the p32 movement protein (MP) of two cileviruses. Citrus leprosis-associated viruses are not able to move systemically in neither their natural nor experimental host plants. However, here we show that cilevirus MPs are able to allow the cell-to-cell and long-distance transport of movement-defective alfalfa mosaic virus (AMV). Several features related with the viral transport were explored, including: (i) the ability of cilevirus MPs to facilitate virus movement on a nucleocapsid assembly independent-manner; (ii) the generation of tubular structures from transient expression in protoplast; (iii) the capability of the N- and C- terminus of MP to interact with the cognate capsid protein (p29) and; (iv) the role of the C-terminus of p32 in the cell-to-cell and long-distance transport, tubule formation and the MP-plasmodesmata co-localization. The MP was able to direct the p29 to the plasmodesmata, whereby the C-terminus of MP is independently responsible to recruit the p29 to the cell periphery. Furthermore, we report that MP possess the capacity to enter the nucleolus and to bind to a major nucleolar protein, the fibrillarin. Based on our findings, we provide a model for the role of the p32 in the intra- and intercellular viral spread.

Spontaneous Mutation in the Movement Protein of Citrus Leprosis Virus C2, in a Heterologous Virus Infection Context, Increases Cell-to-Cell Transport and Generates Fitness Advantage

“…The C-terminus of the MPs assigned to the 30K superfamily has been described to be a multifunctional region intrinsically implicated with several aspects of viral movement, such as interaction with the cognate capsid protein (CP), formation of tubular structures, and efficiency in viral cell-to-cell and systemic transport [10,13,46,52,53]. In our recent study, the sequential C-terminal deletion of CiLV-C2 MP showed that, although the absence of this fragment (deletion of 70 residues) still enables cell-to-cell movement, its removal impairs the correct tubule polymerization and MP-plasmodesma association, affecting the viral cellto-cell and long-distance transport; furthermore, this region is independently responsible to recruit the p29 (cilevirus capsid protein) to the cell periphery [34]. Consequently, any MP modification in this region could affect one or several viral movement processes.…”

“…Consequently, any MP modification in this region could affect one or several viral movement processes. Given the positive effect in AMV transport by this amino acid change, it would be interesting to assess whether the presence of a putative alpha-helix structure at the carboxy-terminal of CiLV-C2 MP could favor the tubule formation and/or the MP association with plasmodesmata, since it has been shown that these processes were essential for an efficient AMV transport mediated by cilevirus MP [34].…”

“…We previously observed that the expression of the CiLV-C and CiLV-C2 MPs using AMV RNA 3 chimeric constructs in transgenic N. tabacum P12 plants revealed that CiLV-C MP generated a more efficient local movement but not a more systemic transport than the CiLV-C2 MP [34] (Supplementary Figure S1). To better understand this phenomenon, we quantified the AMV RNA accumulation on P12 protoplasts at 16 h post-transfection.…”

“…Cilevirus MPs belong to the 30K superfamily [31][32][33] and their role in the viral movement has recently been described [34]. In a previous study about cileviruses MP functionality using an AMV chimeric construct expressing the CiLV-C and CiLV-C2 MPs, it was observed that CiLV-C MP generated a more efficient local movement than the CiLV-C2 MP, whereas an inverse correlation was observed for systemic spread [34].…”

Molecular Epidemiology of Citrus Leprosis Virus C: A New Viral Lineage and Phylodynamic of the Main Viral Subpopulations in the Americas

“…P15, P61, and P24 are proteins without definitively associated functions, although the first two seem to be involved in the suppression of the RNA silencing mechanism ( Leastro et al, 2020 ) and the latter one is conserved among cileviruses, higreviruses, and an increasing number of arthropod-infecting viruses ( Kuchibhatla et al, 2014 ; Kondo et al, 2020 ; Ramos-González et al, 2020 ). The p32 encodes a movement protein (MP) of the 30K superfamily ( Mushegian and Elena, 2015 ; Leastro et al, 2021 ).…”