ICTV Virus Taxonomy Profile: Potyviridae 2022.

The family Potyviridae includes plant viruses with single-stranded, positive-sense RNA genomes of 8-11 kb and flexuous filamentous particles 650-950 nm long and 11-20 nm wide. Genera in the family are distinguished by the host range, genomic features and phylogeny of the member viruses. Most genomes are monopartite, but those of members of the genus Bymovirus are bipartite. Some members cause serious disease epidemics in cultivated plants. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Potyviridae, which is available at ictv.global/report/potyviridae.

Coinfection of Tomato Plants with Tomato yellow leaf curl virus and Tomato chlorosis virus Affects the Interaction with Host and Whiteflies.

Susceptible plants infected by single or multiple viruses can differ in symptoms and other alterations influencing virus dissemination. Furthermore, behavior of viruliferous vectors may be altered in certain cases to favor acquisition and inoculation processes conductive to virus transmission. We explored single and mixed infections frequently occurring in tomato crops, caused by two viruses transmitted by the whitefly Bemisia tabaci: Tomato yellow leaf curl virus (TYLCV, Begomovirus, Geminiviridae) and Tomato chlorosis virus (ToCV, Crinivirus, Closteroviridae). Coinfection of both viruses in tomato plants showed more severe symptoms at late stages compared with single infections, although at earlier stages the interaction began with attenuation. This asymmetric synergism correlated with the dynamics of ToCV accumulation and expression of the salicylic acid responsive gene PR-P6. Visual and olfactory cues in whitefly preference were evaluated under controlled conditions in choice assays, testing viruliferous and nonviruliferous adult whiteflies. In experiments allowing both visual and olfactory cues, whiteflies preferred symptomatic leaflets from plants infected either with TYLCV alone or with TYLCV and ToCV, over those infected with ToCV alone or noninfected leaflets, suggesting that TYLCV drove host selection. Odor cues tested in Y-tube olfactometer assays showed neutral effects on whiteflies' preference, and bioassays comparing the attractiveness of colored sticky cards confirmed preference for sectors colored to mimic TYLCV symptomatic leaves compared with asymptomatic leaves. Our results show that the presence of coinfecting viruses affect the host and could alter the behavior of insect vectors.

Specificity of Resistance and Tolerance to Cucumber Vein Yellowing Virus in Melon Accessions and Resistance Breaking with a Single Mutation in VPg.

Cucumber vein yellowing virus (CVYV) is an emerging virus on cucurbits in the Mediterranean Basin, against which few resistance sources are available, particularly in melon. The melon accession PI 164323 displays complete resistance to isolate CVYV-Esp, and accession HSD 2458 presents a tolerance, i.e., very mild symptoms despite virus accumulation in inoculated plants. The resistance is controlled by a dominant allele Cvy-11, while the tolerance is controlled by a recessive allele cvy-2, independent from Cvy-11. Before introducing the resistance or tolerance in commercial cultivars through a long breeding process, it is important to estimate their specificity and durability. Upon inoculation with eight molecularly diverse CVYV isolates, the resistance was found to be isolate-specific because many CVYV isolates induced necrosis on PI 164323, whereas the tolerance presented a broader range. A resistance-breaking isolate inducing severe mosaic on PI 164323 was obtained. This isolate differed from the parental strain by a single amino acid change in the VPg coding region. An infectious CVYV cDNA clone was obtained, and the effect of the mutation in the VPg cistron on resistance to PI 164323 was confirmed by reverse genetics. This represents the first determinant for resistance-breaking in an ipomovirus. Our results indicate that the use of the Cvy-11 allele alone will not provide durable resistance to CVYV and that, if used in the field, it should be combined with other control methods such as cultural practices and pyramiding of resistance genes to achieve long-lasting resistance against CVYV.

When Viruses Play Team Sports: Mixed Infections in Plants.

The pathological importance of mixed viral infections in plants might be underestimated except for a few well-characterized synergistic combinations in certain crops. Considering that the host ranges of many viruses often overlap and that most plant species can be infected by several unrelated viruses, it is not surprising to find more than one virus simultaneously in the same plant. Furthermore, dispersal of the majority of plant viruses relies on efficient transmission mechanisms mediated by vector organisms, mainly but not exclusively insects, which can contribute to the occurrence of multiple infections in the same plant. Recent work using different experimental approaches has shown that mixed viral infections can be remarkably frequent, up to the point that they could be considered the rule more than the exception. The purpose of this review is to describe the impact of multiple infections not only on the participating viruses themselves but also on their vectors and on the common host. From this standpoint, mixed infections arise as complex events that involve several cross-interacting players, and they consequently require a more general perspective than the analysis of single-virus/single-host approaches for a full understanding of their relevance.

Assessing the Impact on Virus Transmission and Insect Vector Behavior of a Viral Mixed Infection in Melon.

Mixed viral infections in plants are common, and can result in synergistic or antagonistic interactions. Except in complex diseases with severe symptoms, mixed infections frequently remain unnoticed, and their impact on insect vector transmission is largely unknown. In this study, we considered mixed infections of two unrelated viruses commonly found in melon plants, the crinivirus cucurbit yellow stunting disorder virus (CYSDV) and the potyvirus watermelon mosaic virus (WMV), and evaluated their vector transmission by whiteflies and aphids, respectively. Their dynamics of accumulation was analyzed until 60 days postinoculation (dpi) in mixed-infected plants, documenting reduced titers of WMV and much higher titers of CYSDV compared with single infections. At 24 dpi, corresponding to the peak of CYSDV accumulation, similar whitefly transmission rates were obtained when comparing either individual or mixed-infected plants as CYSDV sources, although its secondary dissemination was slightly biased toward plants previously infected with WMV, regardless of the source plant. However, at later time points, mixed-infected plants partially recovered from the initially severe symptoms, and CYSDV transmission became significantly higher. Interestingly, aphid transmission rates both at early and late time points were unaltered when WMV was acquired from mixed-infected plants despite its reduced accumulation. This lack of correlation between WMV accumulation and transmission could result from compensatory effects observed in the analysis of the aphid feeding behavior by electrical penetration graphs. Thus, our results showed that mixed-infected plants could provide advantages for both viruses, directly favoring CYSDV dissemination while maintaining WMV transmission.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) proteomic profiling of cerebrospinal fluid in the diagnosis of enteroviral meningitis: a proof-of-principle study.

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for diagnosing viral infections by directly testing clinical specimens has not previously been explored. In this proof-of-principle study, we tested the hypothesis that proteomic profiling of cerebrospinal fluid (CSF) by mass spectrometry may be useful in the diagnosis of enteroviral (EV) meningitis. A total of 114 cryopreserved CSF samples were analyzed, of which 47 were positive for EV and 67 were negative. Total CSF proteins were precipitated and subjected to MALDI-TOF-MS analysis in a low (2-20 kDa) molecular weight range using a MicroFlex LT mass spectrometer. The whole data set was randomly split into a training set (n = 76 specimens) and a validation set (n = 38 samples). Backward/forward stepwise logistic regression analyses identified 30 peaks that were differentially present in EV-positive and EV-negative specimens. These were used to build a model which displayed an overall classification accuracy of 93%. The discriminative ability of the model was confirmed by using a validation sample set (overall accuracy 83%). In fact, the model was able to correctly classify 61 out of 67 EV-negative samples and 42 out of 47 EV-positive specimens. EV meningitis is associated with a distinctive protein profile that may be directly detectable in CSF specimens by MALDI-TOF-MS.

ICTV Virus Taxonomy Profile: Potyviridae.

The Potyviridae is the largest family of RNA plant viruses, members of which have single-stranded, positive-sense RNA genomes and flexuous filamentous particles 680-900 nm long and 11-20 nm wide. There are eight genera, distinguished by the host range, genomic features and phylogeny of the member viruses. Genomes range from 8.2 to 11.3 kb, with an average size of 9.7 kb. Most genomes are monopartite but those of members of the genus Bymovirus are bipartite. Some members cause serious disease epidemics in cultivated plants. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Potyviridae, which is available at www.ictv.global/report/potyviridae.

The P1N-PISPO trans-Frame Gene of Sweet Potato Feathery Mottle Potyvirus Is Produced during Virus Infection and Functions as an RNA Silencing Suppressor.

UNLABELLED: The positive-sense RNA genome of Sweet potato feathery mottle virus (SPFMV) (genus Potyvirus, family Potyviridae) contains a large open reading frame (ORF) of 3,494 codons translatable as a polyprotein and two embedded shorter ORFs in the -1 frame: PISPO, of 230 codons, and PIPO, of 66 codons, located in the P1 and P3 regions, respectively. PISPO is specific to some sweet potato-infecting potyviruses, while PIPO is present in all potyvirids. In SPFMV these two extra ORFs are preceded by conserved G2A6 motifs. We have shown recently that a polymerase slippage mechanism at these sites could produce transcripts bringing these ORFs in frame with the upstream polyprotein, thus leading to P1N-PISPO and P3N-PIPO products (B. Rodamilans, A. Valli, A. Mingot, D. San Leon, D. B. Baulcombe, J. J. Lopez-Moya, and J.A. Garcia, J Virol 89:6965-6967, 2015, doi:10.1128/JVI.00337-15). Here, we demonstrate by liquid chromatography coupled to mass spectrometry that both P1 and P1N-PISPO are produced during viral infection and coexist in SPFMV-infected Ipomoea batatas plants. Interestingly, transient expression of SPFMV gene products coagroinfiltrated with a reporter gene in Nicotiana benthamiana revealed that P1N-PISPO acts as an RNA silencing suppressor, a role normally associated with HCPro in other potyviruses. Moreover, mutation of WG/GW motifs present in P1N-PISPO abolished its silencing suppression activity, suggesting that the function might require interaction with Argonaute components of the silencing machinery, as was shown for other viral suppressors. Altogether, our results reveal a further layer of complexity of the RNA silencing suppression activity within the Potyviridae family. IMPORTANCE: Gene products of potyviruses include P1, HCPro, P3, 6K1, CI, 6K2, VPg/NIaPro, NIb, and CP, all derived from the proteolytic processing of a large polyprotein, and an additional P3N-PIPO product, with the PIPO segment encoded in a different frame within the P3 cistron. In sweet potato feathery mottle virus (SPFMV), another out-of-frame element (PISPO) was predicted within the P1 region. We have shown recently that a polymerase slippage mechanism can generate the transcript variants with extra nucleotides that could be translated into P1N-PISPO and P3N-PIPO. Now, we demonstrate by mass spectrometry analysis that P1N-PISPO is indeed produced in SPFMV-infected plants, in addition to P1. Interestingly, while in other potyviruses the suppressor of RNA silencing is HCPro, we show here that P1N-PISPO exhibited this activity in SPFMV, revealing how the complexity of the gene content could contribute to supply this essential function in members of the Potyviridae family.

Pyrene-tagged carbohydrate-based mixed P/S ligand: spacer effect on the Rh(i)-catalyzed hydrogenation of methyl α-acetamidocinnamate.

The post-functionalization of a chiral catalyst offers the advantage of providing it with additional physical characteristics that, together with its enantioselective capacity, increase its overall synthetic value. Taking advantage of the modularity and polyfunctionality of carbohydrate-derived ligands, herein we report the synthesis of two mixed P/S catalysts functionalized with a pyrene group through the 6 position of the sugar by carbon chains of different lengths. Using the hydrogenation of methyl (Z)-α-acetamidocinnamate as the model reaction has shown that the proximity of the pyrenyl group to the catalytic center is detrimental to the activity and enantioselectivity of the hydrogenation process, the most efficient catalyst being the complex derived from pyrenebutyric acid 12. The study of the supramolecular π-π interaction of the most active complex 12 with SWCNTs by UV-Vis spectroscopy shows, that in ethyl acetate complex 12 is totally adsorbed onto the SWCNT surface, while in methylene chloride there is an equilibrium between the adsorbed and the free form of the complex, allowing the use of complex 12 and SWCNTs in a catch and release process. Interestingly, it has been determined that the nanocatalyst 12/SWCNT is more enantioselective than complex 12 alone, affording (S)-N-acetylphenyl alanine 16 in quantitative yield and 96% ee.

Small RNA profiling reveals regulation of Arabidopsis miR168 and heterochromatic siRNA415 in response to fungal elicitors.

BACKGROUND: Small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), have emerged as important regulators of eukaryotic gene expression. In plants, miRNAs play critical roles in development, nutrient homeostasis and abiotic stress responses. Accumulating evidence also reveals that sRNAs are involved in plant immunity. Most studies on pathogen-regulated sRNAs have been conducted in Arabidopsis plants infected with the bacterial pathogen Pseudomonas syringae, or treated with the flagelin-derived elicitor peptide flg22 from P. syringae. This work investigates sRNAs that are regulated by elicitors from the fungus Fusarium oxysporum in Arabidopsis. RESULTS: Microarray analysis revealed alterations on the accumulation of a set of sRNAs in response to elicitor treatment, including miRNAs and small RNA sequences derived from massively parallel signature sequencing. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. Promoter analysis in transgenic Arabidopsis plants revealed transcriptional activation of MIR168 by fungal elicitors. Furthermore, transgenic plants expressing a GFP-miR168 sensor gene confirmed that the elicitor-induced miR168 is active. MiR823, targeting Chromomethylase3 (CMT3) involved in RNA-directed DNA methylation (RdDM) was also found to be regulated by fungal elicitors. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA. Studies on Arabidopsis mutants impaired in small RNA biogenesis demonstrated that this sRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. Hc-siRNAs are known to be involved in RNA-directed DNA methylation (RdDM). SiRNA415 is detected in several plant species. CONCLUSION: Results here presented support a transcriptional regulatory mechanism underlying MIR168 expression. This finding highlights the importance of miRNA functioning in adaptive processes of Arabidopsis plants to fungal infection. The results of this study also lay a foundation for the involvement of RdDM processes through the activity of siRNA415 and miR823 in mediating regulation of immune responses in Arabidopsis plants.

The transcriptomic and proteomic effects of ectopic overexpression of miR-30d in human endometrial epithelial cells.

miR-30d is known to be up-regulated during the acquisition of receptivity in the endometrium. In order to determine the transcriptomic and proteomic changes which occur after transient overexpression of miR-30d in primary endometrial epithelial cells, in vitro cultured human endometrial epithelial cells (hEECs) were studied experimentally. Two different miRNAs (scramble versus mimic; n = 15) were transiently transfected into primary hEECs from four different patients and were evaluated for mRNA and protein expression using Agilent's gene expression microarray and iTRAQ analysis techniques, respectively. A set of differentially expressed mRNAs were validated by qPCR and several differentially expressed proteins were validated by western blot. Finally, methylation differential immunoprecipitation (MeDIP) was used to validate the epigenetic changes in the H19 gene. The results showed that transient transfection with miR-30d miRNA induced the differential mRNA-expression of 176 genes (75 up-regulated and 101 down-regulated). Several of them have been associated with reproductive and endocrine system disorders, tissue development, and are implicated in epithelial cell proliferation. Also, the down-regulation of some genes such as H19 and N-methyltransferase (NNMT) may suggest that epigenetic alterations are induced. Furthermore, upstream effects of genes regulated by the estrogen receptor alpha 1 (ESR1) transcription factor have been predicted. Proteomic analysis identified 2290 proteins, of which 108 were differentially expressed (47 up-regulated and 61 down-regulated). Among these differentially expressed proteins DNA methyl transferase (DNMT)1 was found to be up-regulated; this protein participates in the maintenance of DNA methylation, supporting an epigenetic role for miR-30d. Finally MeDIP showed an increase in methylation in the H19 DMR region. In conclusion transient in vitro overexpression of the receptivity-up-regulated miRNA miR-30d in hEECs seems to activate genes which are associated with hormonal response and the epigenetic status of these cells.

Survival of patients with non-small cell lung cancer according to lymph node disease: single pN1 vs multiple pN1 vs single unsuspected pN2.

PURPOSE: This study was designed to describe the characteristics and survival of NSCLC patients treated with surgery and single pN1 disease, multiple pN1, and single unsuspected pN2. METHODS: In 2005-2009, we treated 378 lung cancer patients with surgery with radical intent; 152 cases were pN1 or pN2. We excluded patients with neoadjuvant treatment, incomplete resection, incomplete lymph node dissection, metastasis, cN2 disease, multiple pN2, SCLC, and lack of PET-CT. All patients were staged with TNM 2010. We included 72 patients: 21 single pN1, 26 multiple pN1, and 25 single unsuspected pN2. Statistical analysis included descriptive statistics, chi-square test, Kaplan-Meier, log-rank test, and Cox proportional hazard model. RESULTS: The sample included 62 men (86 %) and 10 women (14 %), mean age 64 ± 9 years. The three subgroups did not show statistically significant differences in the main characteristics. Adjuvant treatment was performed in 56 patients (78 %). The 5 year overall survival (OS) for single pN1 was 73 %; for multiple pN1, 34 %; and for single unsuspected pN2, 25 % (P = 0.15). The mean OS for single pN1 was 63 ± 6 months; median OS for multiple pN1 was 45 (range, 42-48) months and for single pN2 was 54 (range, 32-77) months. Multivariate analysis found the following negative prognostic factors of OS: for single pN1, age, female sex, and microscopic intratumoral lymphatic and vascular invasion; for multiple pN1, ≤10 lymph nodes resected. CONCLUSIONS: Patients with single pN1 had better OS than patients with multiple pN1. Patients with single unsuspected pN2 had OS similar to that of multiple pN1.

cmv1-Mediated Resistance to CMV in Melon Can Be Overcome by Mixed Infections with Potyviruses.

Resistance to cucumber mosaic virus (CMV) strain LS in melon is controlled by the gene cmv1, which restricts phloem entry. In nature, CMV is commonly found in mixed infections, particularly with potyviruses, where a synergistic effect is frequently produced. We have explored the possibility that this synergism could help CMV-LS to overcome cmv1-mediated resistance. We demonstrate that during mixed infection with a potyvirus, CMV-LS is able to overcome cmv1-controlled resistance and develop a systemic infection and that this ability does not depend on an increased accumulation of CMV-LS in mechanically inoculated cotyledons. Likewise, during a mixed infection initiated by aphids, the natural vector of both cucumoviruses and potyviruses that can very efficiently inoculate plants with a low number of virions, CMV-LS also overcomes cmv1-controlled resistance. This indicates that in the presence of a potyvirus, even a very low amount of inoculum, can be sufficient to surpass the resistance and initiate the infection. These results indicate that there is an important risk for this resistance to be broken in nature as a consequence of mixed infections, and therefore, its deployment in elite cultivars would not be enough to ensure a long-lasting resistance.

Microbial community on industrial salty bovine hides: From the slaughterhouse to the salting.

The leather-making industry is an age-old industry and desiccation with salt has been one of the most used methodologies for obtaining valuable skins. However, halophiles may proliferate and affect the integrity of the hide-collagen structure, as well as leading to undesirable red colorations or less-frequent purple stains. To understand the basis of these industrial hide contaminations, the microbial community from raw hide samples, salt-cured samples and four different industrial salts, was analyzed by 16S rRNA gene metabarcoding together with standard cultivation methods. Comparison of raw hides and correctly cured hides revealed a core microbiome that was absent from contaminated hides. In addition, archaea were missing from well-cured hides, whereas Psychrobacter and Acinetobacter were highly represented (23 % and 17.4 %, respectively). In damaged hides, only a few operational taxonomic units (OTUs), from among the hundreds detected, were able to proliferate and, remarkably, a single Halomonas OTU represented 57.66 % of the reads. Halobacteria, mainly Halovenus, Halorubrum and Halovivax, increased by up to 36.24-39.5 % in the red- and purple-affected hides. The major contaminants were isolated and hide infections, together with collagenase activity, were evaluated. The results showed that hides enriched with the non-pigmented isolate Halomonas utahensis COIN160 damaged the collagen fibers similarly to Halorubrum, and together they were considered to be one of the major causes. Putative degrading inhibitors were also identified from among the Alkalibacillus isolates. It was concluded that hide contaminations were driven by clonal outbreaks of a few specific microbes, which may have been non-pigmented collagen degraders. Acinetobacter and Alkalibacillus, members of the core microbiome of raw and well-cured salted hides, are suggested as hide contaminant inhibitors that need further analysis.

Enhanced Susceptibility to Tomato Chlorosis Virus (ToCV) in Hsp90- and Sgt1-Silenced Plants: Insights from Gene Expression Dynamics.

The emerging whitefly-transmitted crinivirus tomato chlorosis virus (ToCV) causes substantial economic losses by inducing yellow leaf disorder in tomato crops. This study explores potential resistance mechanisms by examining early-stage molecular responses to ToCV. A time-course transcriptome analysis compared naïve, mock, and ToCV-infected plants at 2, 7, and 14 days post-infection (dpi). Gene expression changes were most notable at 2 and 14 dpi, likely corresponding to whitefly feeding and viral infection. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed key genes and pathways associated with ToCV infection, including those related to plant immunity, flavonoid and steroid biosynthesis, photosynthesis, and hormone signaling. Additionally, virus-derived small interfering RNAs (vsRNAs) originating from ToCV predominantly came from RNA2 and were 22 nucleotides in length. Furthermore, two genes involved in plant immunity, Hsp90 (heat shock protein 90) and its co-chaperone Sgt1 (suppressor of the G2 allele of Skp1) were targeted through viral-induced gene silencing (VIGS), showing a potential contribution to basal resistance against viral infections since their reduction correlated with increased ToCV accumulation. This study provides insights into tomato plant responses to ToCV, with potential implications for developing effective disease control strategies.

CryoEM and stability analysis of virus-like particles of potyvirus and ipomovirus infecting a common host.

Sweet potato feathery mottle virus (SPFMV) and Sweet potato mild mottle virus (SPMMV) are members of the genera Potyvirus and Ipomovirus, family Potyviridae, sharing Ipomoea batatas as common host, but transmitted, respectively, by aphids and whiteflies. Virions of family members consist of flexuous rods with multiple copies of a single coat protein (CP) surrounding the RNA genome. Here we report the generation of virus-like particles (VLPs) by transient expression of the CPs of SPFMV and SPMMV in the presence of a replicating RNA in Nicotiana benthamiana. Analysis of the purified VLPs by cryo-electron microscopy, gave structures with resolutions of 2.6 and 3.0 Å, respectively, showing a similar left-handed helical arrangement of 8.8 CP subunits per turn with the C-terminus at the inner surface and a binding pocket for the encapsidated ssRNA. Despite their similar architecture, thermal stability studies reveal that SPMMV VLPs are more stable than those of SPFMV.

A novel model of human implantation: 3D endometrium-like culture system to study attachment of human trophoblast (Jar) cell spheroids.

There is an urgent need to develop optimized experimental models to examine human implantation. These studies aimed to (i) establish a human endometrium-like three-dimensional (3D) culture system, and (ii) examine the attachment of trophoblast-like Jar spheroids to the culture. In the present work, 3D endometrial cultures were constructed with fibrin-agarose as matrix scaffold, and using epithelial and stromal cells from both human primary cultures and established cell lines. An attachment assay between trophoblast cells and the 3D culture was developed. Epithelial cells (cytokeratin(+)) concentrated on top of the matrix forming a monolayer, and stromal cells (vimentin(+)) resided within the matrix, resembling the normal endometrial structure. The capability of primary epithelial cells to form glands spontaneously was observed. Human trophoblast cells (Jar cells) were hCG(+) by immunostaining, allowed to form spheroids, and confirmed to secrete hCG into the medium. Time-dependent experiments demonstrated a high rate of attachment of Jar spheroids to the epithelium, and adhesion was strongly related to the various cell types present in the 3D culture. An architecturally and functionally competent 3D endometrial culture system was established, that coupled with Jar spheroids mimicking trophoblast cells, provides a unique in vitro model for the study of certain aspects of human implantation.

Viral protein inhibits RISC activity by argonaute binding through conserved WG/GW motifs.

RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins. These viral proteins can target one or more key points in the silencing machinery. Here we show that in Sweet potato mild mottle virus (SPMMV, type member of the Ipomovirus genus, family Potyviridae), the role of silencing suppressor is played by the P1 protein (the largest serine protease among all known potyvirids) despite the presence in its genome of an HC-Pro protein, which, in potyviruses, acts as the suppressor. Using in vivo studies we have demonstrated that SPMMV P1 inhibits si/miRNA-programmed RISC activity. Inhibition of RISC activity occurs by binding P1 to mature high molecular weight RISC, as we have shown by immunoprecipitation. Our results revealed that P1 targets Argonaute1 (AGO1), the catalytic unit of RISC, and that suppressor/binding activities are localized at the N-terminal half of P1. In this region three WG/GW motifs were found resembling the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are absolutely required for both binding and suppression of AGO1 function. In contrast to other viral silencing suppressors analyzed so far P1 inhibits both existing and de novo formed AGO1 containing RISC complexes. Thus P1 represents a novel RNA silencing suppressor mechanism. The discovery of the molecular bases of P1 mediated silencing suppression may help to get better insight into the function and assembly of the poorly explored multiprotein containing RISC.

P/S ligands derived from carbohydrates in Rh-catalyzed hydrosilylation of ketones.

Reported is the synthesis of a number of diastereomerically pure cationic Rh(I)-complexes I starting from phosphinite thioglycosides. These complexes were used in the asymmetric hydrosilylation of prochiral ketones. The reactivity and enantioselectivity of the reaction was shown to be dependent on the pyranose ring, the substituent at the sulfur atom, the hydroxylic protective groups and most significantly on the alkene co-ligand.