Revisiting the Roles of Tobamovirus Replicase Complex Proteins in Viral Replication and Silencing Suppression

Abstract

Tobamoviral replicase possesses an RNA-dependent RNA polymerase (RDR) domain and is translated from genomic (g)RNA via a stop codon readthrough mechanism at a one-to-ten ratio relative to a shorter protein lacking the RDR domain. The two proteins share methyltransferase and helicase domains and form a heterodimer implicated in gRNA replication. The shorter protein is also implicated in suppressing RNA silencing-based antiviral defenses. Using a stop codon mutant of Oilseed rape mosaic tobamovirus (ORMV), we demonstrate that the readthrough replicase (p182) is sufficient for gRNA replication and for subgenomic RNA transcription during systemic infection in Nicotiana benthamiana and Arabidopsis thaliana. However, the mutant virus displays milder symptoms and does not interfere with HEN1-mediated methylation of viral short interfering (si)RNAs or plant small (s)RNAs. The mutant virus tends to revert the stop codon, thereby restoring expression of the shorter protein (p125), even in the absence of plant Dicer-like activities that generate viral siRNAs. Plant RDR activities that generate endogenous siRNA precursors do not prevent replication or movement of the mutant virus, and double-stranded precursors of viral siRNAs representing the entire virus genome are likely synthesized by p182. Transgenic expression of p125 partially recapitulates the ORMV disease symptoms associated with overaccumulation of plant sRNAs. Taken together, the readthrough replicase p182 is sufficient for viral replication and transcription but not for silencing suppression. By contrast, the shorter p125 protein suppresses silencing, provokes severe disease symptoms, causes overaccumulation of unmethylated viral and plant sRNAs but it is not an essential component of the viral replicase complex.

Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV

“…2c). The three species are positive-sense single-strand RNA (ssRNA) viruses that belong to the Tobamovirus genus 32 .…”

Translation-Independent Roles of RNA Secondary Structures within the Replication Protein Coding Region of Turnip Crinkle Virus

“…As expected, without p88 provided in trans, none of the five mutant replicons produced viral gRNA to levels detectable by Northern blotting ( Figure 1B, lanes 3-7). In contrast, the presence of p88 enabled three mutants, ∆Fc (lanes 12 and 13), ∆1208-2197 (lanes [16][17], and ∆sg1Pro (lanes [18][19] to accumulate their corresponding gRNAs to easily detectable levels (approximately 50%-80% of TCV_sg2R plus p88; Figure 1C). Therefore, the regions deleted in these mutants, 141, 990, and 195 nt in respective lengths, were unlikely to contain cis-acting elements indispensable for TCV gRNA replication.…”

“…None of the new mutants could accumulate detectable levels of gRNA in the presence of p88 alone ( Figure 5C), illustrating that, unlike TMV [17], the TCV-encoded p28 ARP was absolutely needed for genome replication. The p28TS replicon did accumulate gRNA to detectable levels in the presence of both p28 and p88 ( Figure 5D, lanes 5-7).…”

Ribosome Shunting, Polycistronic Translation, and Evasion of Antiviral Defenses in Plant Pararetroviruses and Beyond

“…In plant RNAi-based antiviral defense, 21-, 22-, and 24-nt viral small interfering (si)RNAs are generated by four Dicer-like (DCL) enzymes from dsRNA precursors and then sorted by Argonaute (AGO) family proteins to form RNA-induced silencing complexes (RISCs) ( Blevins et al, 2006 , 2011 ; Bouché et al, 2006 ; Fusaro et al, 2006 ; Aregger et al, 2012 ; Malpica-López et al, 2018 ; reviewed in Carbonell and Carrington, 2015 ; Pooggin, 2016 ). The 21–22-nt siRNA-RISCs can potentially target viral RNA for cleavage and degradation or translational repression, both causing post-transcriptional gene silencing (PTGS), while 24-nt siRNA-RISCs can potentially target viral DNA for cytosine methylation and transcriptional gene silencing (TGS).…”