Differences in Virulence among PVY Isolates of Different Geographical Origins When Infecting an Experimental Host under Two Growing Environments Are Not Determined by HCPro

The contribution of the HCPro factors expressed by several PVY isolates of different geographical origins (one from Scotland, one from Spain, and several from Tunisia) to differences in their virulence in Nicotiana benthamiana plants was investigated under two growing conditions: standard (st; 26 °C and current ambient levels of CO2), and climate change-associated (cc; 31 °C and elevated levels of CO2). In all cases, relative infection symptoms and viral titers were determined. The viral HCPro cistrons were also sequenced and amino-acid features of the encoded proteins were established, as well as phylogenetic distances. Additionally, the abilities of the HCPros of several isolates to suppress silencing were assessed under either growing condition. Overall, viral titers and infection symptoms decreased under cc vs. st conditions. However, within each growing condition, relative titers and symptoms were found to be isolate-specific, with titers and symptom severities not always correlating. Crucially, isolates expressing identical HCPros displayed different symptoms. In addition, all HCPro variants tested displayed comparable silencing suppression strengths. Therefore, HCPro alone could not be the main determinant of the relative differences in pathogenicity observed among the PVY isolates tested in this host, under the environments considered.

Characterization of Pathogenicity-Associated V2 Protein of Tobacco Curly Shoot Virus

V2 proteins encoded by some whitefly-transmitted geminiviruses were reported to be functionally important proteins. However, the functions of the V2 protein of tobacco curly shoot virus (TbCSV), a monopartite begomovirus that causes leaf curl disease on tomato and tobacco in China, remains to be characterized. In our report, an Agrobacterium infiltration-mediated transient expression assay indicated that TbCSV V2 can suppress local and systemic RNA silencing and the deletion analyses demonstrated that the amino acid region 1–92 of V2, including the five predicted α-helices, are required for local RNA silencing suppression. Site-directed substitutions showed that the conserved basic and ring-structured amino acids in TbCSV V2 are critical for its suppressor activity. Potato virus X-mediated heteroexpression of TbCSV V2 in Nicotiana benthamiana induced hypersensitive response-like (HR-like) cell death and systemic necrosis in a manner independent of V2′s suppressor activity. Furthermore, TbCSV infectious clone mutant with untranslated V2 protein (TbCSV∆V2) could not induce visual symptoms, and coinfection with betasatellite (TbCSB) could obviously elevate the viral accumulation and symptom development. Interestingly, symptom recovery occurred at 15 days postinoculation (dpi) and onward in TbCSV∆V2/TbCSB-inoculated plants. The presented work contributes to understanding the RNA silencing suppression activity of TbCSV V2 and extends our knowledge of the multifunctional role of begomovirus-encoded V2 proteins during viral infections.

Structure and dsRNA-binding activity of the Birnavirus Drosophila X Virus VP3 protein.

The Birnavirus multifunctional protein VP3 plays an essential role coordinating the virus life cycle, interacting with the capsid protein VP2, with the RNA-dependent RNA polymerase VP1 and with the dsRNA genome. Furthermore, the role of this protein in controlling host cell responses triggered by dsRNA and preventing gene silencing has been recently demonstrated. Here we report the X-ray structure and dsRNA-binding activity of the N-terminal domain of Drosophila X virus (DXV) VP3. The domain folds in a bundle of three α-helices and arranges as a dimer, exposing to the surface a well-defined cluster of basic residues. Site directed mutagenesis combined with Electrophoretic Mobility Shift Assays (EMSA) and Surface Plasmon Resonance (SPR) revealed that this cluster, as well as a flexible and positively charged region linking the first and second globular domains of DXV VP3, are essential for dsRNA-binding. Also, RNA silencing studies performed in insect cell cultures confirmed the crucial role of this VP3 domain for the silencing suppression activity of the protein. IMPORTANCE The Birnavirus moonlighting protein VP3 plays crucial roles interacting with the dsRNA genome segments to form stable ribonucleoprotein complexes and controlling host cell immune responses, presumably by binding to and shielding the dsRNA from recognition by the host silencing machinery. The structural, biophysical and functional data presented in this work has identified the N-terminal domain of VP3 as responsible for the dsRNA-binding and silencing suppression activities of the protein in Drosophila X virus.

A Newly Identified Virus in the Family Potyviridae Encodes Two Leader Cysteine Proteases in Tandem That Evolved Contrasting RNA Silencing Suppression Functions

ABSTRACT Potyviridae is the largest family of plant-infecting RNA viruses and includes many agriculturally and economically important viral pathogens. The viruses in the family, known as potyvirids, possess single-stranded, positive-sense RNA genomes with polyprotein processing as a gene expression strategy. The N-terminal regions of potyvirid polyproteins vary greatly in sequence. Previously, we identified a novel virus species within the family, Areca palm necrotic spindle-spot virus (ANSSV), which was predicted to encode two cysteine proteases, HCPro1 and HCPro2, in tandem at the N-terminal region. Here, we present evidence showing self-cleavage activity of these two proteins and define their cis-cleavage sites. We demonstrate that HCPro2 is a viral suppressor of RNA silencing (VSR), and both the variable N-terminal and conserved C-terminal (protease domain) moieties have antisilencing activity. Intriguingly, the N-terminal region of HCPro1 also has RNA silencing suppression activity, which is, however, suppressed by its C-terminal protease domain, leading to the functional divergence of HCPro1 and HCPro2 in RNA silencing suppression. Moreover, the deletion of HCPro1 or HCPro2 in a newly created infectious clone abolishes viral infection, and the deletion mutants cannot be rescued by addition of corresponding counterparts of a potyvirus. Altogether, these data suggest that the two closely related leader proteases of ANSSV have evolved differential and essential functions to concertedly maintain viral viability. IMPORTANCE The Potyviridae represent the largest group of known plant RNA viruses and account for more than half of the viral crop damage worldwide. The leader proteases of viruses within the family vary greatly in size and arrangement and play key roles during the infection. Here, we experimentally demonstrate the presence of a distinct pattern of leader proteases, HCPro1 and HCPro2 in tandem, in a newly identified member within the family. Moreover, HCPro1 and HCPro2, which are closely related and typically characterized with a short size, have evolved contrasting RNA silencing suppression activity and seem to function in a coordinated manner to maintain viral infectivity. Altogether, the new knowledge fills a missing piece in the evolutionary relationship history of potyvirids and improves our understanding of the diversification of potyvirid genomes.

Effect of RNA silencing suppression activity of chrysanthemum virus B p12 protein on small RNA species

Chrysanthemum virus B encodes a multifunctional p12 protein that acts as a transcriptional activator in the nucleus and as a suppressor of RNA silencing in the cytoplasm. Here, we investigated the impact of p12 on accumulation of major classes of small RNAs (sRNAs). The results show dramatic changes in the sRNA profiles characterised by an overall reduction in sRNA accumulation, changes in the pattern of size distribution of canonical siRNAs and in the ratio between sense and antisense strands, lower abundance of siRNAs with a U residue at the 5′-terminus, and changes in the expression of certain miRNAs, most of which were downregulated.

Investigation of the effects of P1 on HC-Pro-mediated gene silencing suppression through genetics and omics approaches

Background: Posttranscriptional gene silencing (PTGS) is one of the most important mechanisms for plants during viral infection. However, viruses have also developed viral suppressors to negatively control PTGS by inhibiting microRNA (miRNA) and short-interfering RNA (siRNA) regulation in plants. The first identified viral suppressor, P1/HC-Pro, is a fusion protein. Upon infecting plants, the P1 protein itself is released from HC-Pro by the self-cleaving activity of P1. P1 has an unknown function in enhancing HC-Pro-mediated PTGS suppression. We performed proteomics to identify P1-interacting proteins. We also performed transcriptomics that were generated from Col-0 and various P1/HC-Pro-related transgenic plants to identify novel genes. The results showed several novel genes were identified through the comparative network analysis that might be involved in P1/HC-Pro-mediated PTGS suppression. Results: First, we demonstrated that P1 enhances HC-Pro function and that the mechanism might work through P1 binding to VERNALIZATION INDEPENDENCE 3/SUPERKILLER 8 (VIP3/SKI8), a subunit of the exosome, to interfere with the 5'-fragment of the PTGS-cleaved RNA degradation product. Second, specifically the AGO1 was specifically posttranslationally degraded in transgenic Arabidopsis expressing P1/HC-Pro of turnip mosaic virus (TuMV) (P1/HCTu plant). Third, the comparative network highlighted potentially critical genes in PTGS, including miRNA targets, calcium signaling, hormone (JA, ET, and ABA) signaling, and defense response. Conclusion: Through these genetic and omics approaches, we revealed an overall perspective to identify many critical genes involved in PTGS. These new findings significantly impact in our understanding of P1/HC-Pro-mediated PTGS suppression.

Chilli veinal mottle virus HCPro interacts with catalase to facilitate virus infection in Nicotiana tabacum

Plant symptoms are derived from specific interactions between virus and host components. However, little is known about viral or host factors that participate in the establishment of systemic necrosis. Here, we showed that helper component proteinase (HCPro), encoded by Chilli veinal mottle virus (ChiVMV), could directly interact with catalase 1 (CAT1) and catalase 3 (CAT3) in the cytoplasm of tobacco (Nicotiana tabacum) plants to facilitate viral infection. In vitro, the activities of CAT1 and CAT3 were inhibited by the interaction between HCPro and CATs. The C-terminus of HCPro was essential for their interaction and was also required for the decrease of enzyme activities. Interestingly, the mRNA and protein level of CATs were up-regulated in tobacco plants in response to ChiVMV infection. Nicotiana tabacum plants with HCPro overexpression or CAT1 knockout were more susceptible to ChiVMV infection, which was similar to the case of H2O2-pre-treated plants, and the overexpression of CAT1 inhibited ChiVMV accumulation. Also, neither CAT1 nor CAT3 could affect the RNA silencing suppression (RSS) activity of HCPro. Our results showed that the interaction between HCPro and CATs promoted the development of plant systemic necrosis, revealing a novel role for HCPro in virus infection and pathogenicity.