Cucurbit chlorotic yellows virus p22 suppressor of RNA silencing binds single-, double-stranded long and short interfering RNA molecules in vitro

Salavert, Ferran; Navarro, J. A.; Owen, Carolyn A.; Khechmar, Souheyla; Pallás, Vicente; Livieratos, Ioannis C.

doi:10.1016/j.virusres.2020.197887

Cited by 4 publications

(3 citation statements)

References 35 publications

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“…At this point, it is important to note that the non-radioactive EMSA described here (see section “Material and Methods”) requires the transfer to a nylon membrane step to detect both free RNA and the corresponding ribonucleoprotein (RNP) complex. When a large amount of molecules binds to the RNA, as in a non-sequence specific interaction, the transfer of the RNP complex to the nylon membrane and its posterior detection is difficult or even impossible ( Marcos et al, 1999 ; Herranz and Pallás, 2004 ; Salavert et al, 2020 ). In any case, the disappearance of the free RNA band is evidence of complex formation ( Carey, 1991 ) and was therefore quantified by film densitometry to calculate the apparent constant dissociation (Kd) of the RNA– GST: at ALBKH9Bwt interaction from the linear regression of the mean values from at least three technical replicates ( Marcos et al, 1999 ).…”

Section: Resultsmentioning

confidence: 99%

Mapping of Functional Subdomains in the atALKBH9B m6A-Demethylase Required for Its Binding to the Viral RNA and to the Coat Protein of Alfalfa Mosaic Virus

et al. 2021

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N6-methyladenosine (m6A) modification is a dynamically regulated RNA modification that impacts many cellular processes and pathways. This epitranscriptomic methylation relies on the participation of RNA methyltransferases (referred to as “writers”) and demethylases (referred to as “erasers”), respectively. We previously demonstrated that the Arabidopsis thaliana protein atALKBH9B showed m6A-demethylase activity and interacted with the coat protein (CP) of alfalfa mosaic virus (AMV), causing a profound impact on the viral infection cycle. To dissect the functional activity of atALKBH9B in AMV infection, we performed a protein-mapping analysis to identify the putative domains required for regulating this process. In this context, the mutational analysis of the protein revealed that the residues between 427 and 467 positions are critical for in vitro binding to the AMV RNA. The atALKBH9B amino acid sequence showed intrinsically disordered regions (IDRs) located at the N-terminal part delimiting the internal AlkB-like domain and at the C-terminal part. We identified an RNA binding domain containing an RGxxxRGG motif that overlaps with the C-terminal IDR. Moreover, bimolecular fluorescent experiments allowed us to determine that residues located between 387 and 427 are critical for the interaction with the AMV CP, which should be critical for modulating the viral infection process. Finally, we observed that atALKBH9B deletions of either N-terminal 20 residues or the C-terminal’s last 40 amino acids impede their accumulation in siRNA bodies. The involvement of the regions responsible for RNA and viral CP binding and those required for its localization in stress granules in the viral cycle is discussed.

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Section: Resultsmentioning

confidence: 99%

Mapping of Functional Subdomains in the atALKBH9B m6A-Demethylase Required for Its Binding to the Viral RNA and to the Coat Protein of Alfalfa Mosaic Virus

et al. 2021

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“…CCYV P22, a weak silencing suppressor, is able to suppress local RNA silencing induced by dsRNA ( Orfanidou et al, 2019 ). Using in vitro RNA binding analysis P22 is able to bind to ss and ds long RNAs, in addition to ss and ds small interfering (si) RNA molecules in vitro ( Salavert et al, 2020 ). A previous study reported that CCYV P22 interacts with SKP1 and F-box like domain of CCYV P22 is essential for the RNA silencing activity ( Chen et al, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

“…As RNA1 3′ ORFs are quite variable among criniviruses, both P6 and P22 show no significant similarity to corresponding proteins of other criniviruses ( Okuda et al, 2010 ). In addition to CCYV P22, P22 proteins of tomato chlorosis virus (ToCV) and sweet potato chlorotic stunt virus (SPCSV), P23 of lettuce chlorosis virus (LCV), and P25 of cucurbit yellow stunting disorder virus (CYSDV) have also been identified as RNA silencing suppressors ( Kreuze et al, 2005 ; Canizares et al, 2008 ; Kataya et al, 2009 ; Kubota and Ng, 2016 ; Orfanidou et al, 2019 ; Salavert et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Cucumber Ribosomal Protein CsRPS21 Interacts With P22 Protein of Cucurbit Chlorotic Yellows Virus

et al. 2021

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Cucurbit chlorotic yellows virus (CCYV) is a cucurbit-infecting crinivirus. RNA silencing can be initiated as a plant defense against viruses. Viruses encode various RNA silencing suppressors to counteract antiviral silencing. P22 protein encoded by RNA1 of CCYV is a silencing suppressor, but its mechanism of action remains unclear. In this study, the cucumber ribosomal-like protein CsRPS21 was found to interact with P22 protein in vitro and in vivo. A conserved CsRPS21 domain was indispensable for its nuclear localization and interaction with P22. Transient expression of CsRPS21 in Nicotiana benthamiana leaves interfered with P22 accumulation and inhibited P22 silencing suppressor activity. CsRPS21 expression in N. benthamiana protoplasts inhibited CCYV accumulation. Increasing numbers of ribosomal proteins are being found to be involved in viral infections of plants. We identified a P22-interacting ribosomal protein, CsRPS21, and uncovered its role in early viral replication and silencing suppressor activity. Our study increases knowledge of the function of ribosomal proteins during viral infection.

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Impact of viral silencing suppressors on plant viral synergism: a global agro-economic concern

Ghosh

Malavika

Chakraborty

2021

Appl Microbiol Biotechnol

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Cucurbit chlorotic yellows virus p22 suppressor of RNA silencing binds single-, double-stranded long and short interfering RNA molecules in vitro

Abstract: TitleCucurbit chlorotic yellows virus p22 suppressor of RNA silencing binds single-, double-stranded long and short interfering RNA molecules in vitro Article typeResearch Paper

Cited by 4 publications

References 35 publications

Mapping of Functional Subdomains in the atALKBH9B m6A-Demethylase Required for Its Binding to the Viral RNA and to the Coat Protein of Alfalfa Mosaic Virus

Mapping of Functional Subdomains in the atALKBH9B m6A-Demethylase Required for Its Binding to the Viral RNA and to the Coat Protein of Alfalfa Mosaic Virus

Cucumber Ribosomal Protein CsRPS21 Interacts With P22 Protein of Cucurbit Chlorotic Yellows Virus

Impact of viral silencing suppressors on plant viral synergism: a global agro-economic concern

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