Size Selective Recognition of siRNA by an RNA Silencing Suppressor

RNA silencing in plants likely exists as a defense mechanism against molecular parasites such as RNA viruses, retrotransposons, and transgenes. As a result, many plant viruses have adapted mechanisms to evade and suppress gene silencing. Tombusviruses express a 19 kDa protein (p19), which has been shown to suppress RNA silencing in vivo and bind silencing-generated and synthetic small interferingRNAs (siRNAs) in vitro. Here we report the 2.5 A° crystal structure of p19 from the Carnation Italian ringspot virus (CIRV) bound to a 21 nt siRNA and demonstrate in biochemical and in vivo assays that CIRV p19 protein acts as molecular caliper to specifically select siRNAs based on the length of the duplex region of the RNA

Dissecting the multifunctional role of the N-terminal disordered domain of a plant virus coat protein in RNA packaging, viral movement and interference with antiviral plant defense

[EN] The coat protein (CP) of Melon necrotic spot virus (MNSV) is structurally composed of three major domains. The middle S-domain builds a robust protein shell around the viral genome, whereas the C-terminal protruding domain, or P-domain, is involved in the attachment of virions to the transmission vector. Here, we have shown that the N-terminal domain, or R-domain, and the arm region, which connects the R-domain and S-domain, are involved in different key steps of the viral cycle, such as cell-to-cell movement and the suppression of RNA silencing and pathogenesis through their RNA-binding capabilities. Deletion mutants revealed that the CP RNA-binding ability was abolished only after complete, but not partial, deletion of the R-domain and the arm region. However, a comparison of the apparent dissociation constants for the CP RNA-binding reaction of several partial deletion mutants showed that the arm region played a more relevant role than the R-domain in in vitro RNA binding. Similar results were obtained in in vivo assays, although, in this case, full-length CPs were required to encapsidate full-length genomes. We also found that the R-domain carboxyl portion and the arm region were essential for efficient cell-to-cell movement, for enhancement of Potato virus X pathogenicity, for suppression of systemic RNA silencing and for binding of small RNAs. Therefore, unlike other carmovirus CPs, the R-domain and the arm region of MNSV CP have acquired, in addition to other essential functions such as genome binding and encapsidation functions, the ability to suppress RNA silencing by preventing systemic small RNA transport.This work was funded by grant BIO2014-54862-R from the Spanish Ministry of Science and Innovation and the Prometeo Program GV2014/010 from the Generalitat Valenciana. J.A.N. and M.S.-S. are the recipients of a postdoctoral contract and a PhD fellowship, respectively, from the Ministerio de Educacion y Ciencia of Spain. We thank L. Corachan for technical assistance. The authors have no conflicts of interest to declare.Serra Soriano, M.; Navarro Bohigues, JA.; Pallás Benet, V. (2017). Dissecting the multifunctional role of the N-terminal disordered domain of a plant virus coat protein in RNA packaging, viral movement and interference with antiviral plant defense. Molecular Plant Pathology. 18(6):837-849. https://doi.org/10.1111/mpp.12448S83784918

The Tnt1 Retrotransposon Escapes Silencing in Tobacco, Its Natural Host

Retrotransposons' high capacity for mutagenesis is a threat that genomes need to control tightly. Transcriptional gene silencing is a general and highly effective control of retrotransposon expression. Yet, some retrotransposons manage to transpose and proliferate in plant genomes, suggesting that, as shown for plant viruses, retrotransposons can escape silencing. However no evidence of retrotransposon silencing escape has been reported. Here we analyze the silencing control of the tobacco Tnt1 retrotransposon and report that even though constructs driven by the Tnt1 promoter become silenced when stably integrated in tobacco, the endogenous Tnt1 elements remain active. Silencing of Tnt1-containing transgenes correlates with high DNA methylation and the inability to incorporate H2A.Z into their promoters, whereas the endogenous Tnt1 elements remain partially methylated at asymmetrical positions and incorporate H2A.Z upon induction. Our results show that the promoter of Tnt1 is a target of silencing in tobacco, but also that endogenous Tnt1 elements can escape this control and be expressed in their natural host

A Discontinuous RNA Platform Mediates RNA Virus Replication: Building an Integrated Model for RNA–based Regulation of Viral Processes

Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA–RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA–RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA–based interaction spanning ∼3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes

Two Plant Viral Suppressors of Silencing Require the Ethylene-Inducible Host Transcription Factor RAV2 to Block RNA Silencing

RNA silencing is a highly conserved pathway in the network of interconnected defense responses that are activated during viral infection. As a counter-defense, many plant viruses encode proteins that block silencing, often also interfering with endogenous small RNA pathways. However, the mechanism of action of viral suppressors is not well understood and the role of host factors in the process is just beginning to emerge. Here we report that the ethylene-inducible transcription factor RAV2 is required for suppression of RNA silencing by two unrelated plant viral proteins, potyvirus HC-Pro and carmovirus P38. Using a hairpin transgene silencing system, we find that both viral suppressors require RAV2 to block the activity of primary siRNAs, whereas suppression of transitive silencing is RAV2-independent. RAV2 is also required for many HC-Pro-mediated morphological anomalies in transgenic plants, but not for the associated defects in the microRNA pathway. Whole genome tiling microarray experiments demonstrate that expression of genes known to be required for silencing is unchanged in HC-Pro plants, whereas a striking number of genes involved in other biotic and abiotic stress responses are induced, many in a RAV2-dependent manner. Among the genes that require RAV2 for induction by HC-Pro are FRY1 and CML38, genes implicated as endogenous suppressors of silencing. These findings raise the intriguing possibility that HC-Pro-suppression of silencing is not caused by decreased expression of genes that are required for silencing, but instead, by induction of stress and defense responses, some components of which interfere with antiviral silencing. Furthermore, the observation that two unrelated viral suppressors require the activity of the same factor to block silencing suggests that RAV2 represents a control point that can be readily subverted by viruses to block antiviral silencing

Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts