Carnation Italian Ringspot Virus Research Articles

First report of carnation Italian ringspot virus in Gypsophila in the United Kingdom

First report of carnation Italian ringspot virus in Gypsophila in the United Kingdom

Co-opted cytosolic proteins form condensate substructures within membranous replication organelles of a positive-strand RNA virus.

Positive-strand RNA viruses co-opt organellar membranes for biogenesis of viral replication organelles (VROs). Tombusviruses also co-opt pro-viral cytosolic proteins to VROs. It is currently not known what type of molecular organization keeps co-opted proteins sequestered within membranous VROs. In this study, we employed tomato bushy stunt virus (TBSV) and carnation Italian ringspot virus (CIRV) - Nicotiana benthamiana pathosystems to identify biomolecular condensate formation in VROs. We show that TBSV p33 and the CIRV p36 replication proteins sequester glycolytic and fermentation enzymes in unique condensate substructures associated with membranous VROs. We find that p33 and p36 form droplets in vitro driven by intrinsically disordered region. The replication protein organizes partitioning of co-opted host proteins into droplets. VRO-associated condensates are critical for local adenosine triphosphate production to support energy for virus replication. We find that co-opted endoplasmic reticulum membranes and actin filaments form meshworks within and around VRO condensates, contributing to unique composition and structure. We propose that p33/p36 organize liquid-liquid phase separation of co-opted concentrated host proteins in condensate substructures within membranous VROs. Overall, we demonstrate that subverted membranes and condensate substructures co-exist and are critical for VRO functions. The replication proteins induce and connect the two substructures within VROs.

Subversion of selective autophagy for the biogenesis of tombusvirus replication organelles inhibits autophagy.

Elaborate viral replication organelles (VROs) are formed to support positive-strand RNA virus replication in infected cells. VRO formation requires subversion of intracellular membranes by viral replication proteins. Here, we showed that the key ATG8f autophagy protein and NBR1 selective autophagy receptor were co-opted by Tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus. Knockdown of ATG8f or NBR1 in plants led to reduced tombusvirus replication, suggesting pro-viral function for selective autophagy. BiFC and proximity-labeling experiments showed that the TBSV p33 replication protein interacted with ATG8f and NBR1 to recruit them to VROs. In addition, we observed that several core autophagy proteins, such as ATG1a, ATG4, ATG5, ATG101 and the plant-specific SH3P2 autophagy adaptor proteins were also re-localized to TBSV VROs, suggesting that TBSV hijacks the autophagy machinery in plant cells. We demonstrated that subversion of autophagy components facilitated the recruitment of VPS34 PI3 kinase and enrichment of phospholipids, such as phosphatidylethanolamine and PI3P phosphoinositide in the VRO membranes. Hijacking of autophagy components into TBSV VROs led to inhibition of autophagic flux. We also found that a fraction of the subverted ATG8f and NBR1 was sequestered in biomolecular condensates associated with VROs. We propose that the VRO-associated condensates trap those autophagy proteins, taking them away from the autophagy pathway. Overall, tombusviruses hijack selective autophagy to provide phospholipid-rich membranes for replication and to regulate the antiviral autophagic flux.

Carnation Italian Ringspot Virus p36 Expression Induces Mitochondrial Fission and Respiratory Chain Complex Impairment in Yeast.

Positive-strand RNA virus replication invariably occurs in association with host cell membranes, which are induced to proliferate and rearrange to form vesicular structures where the virus replication complex is assembled. In particular, carnation Italian ringspot virus (CIRV) replication takes place on the mitochondrial outer membrane in plant and yeast cells. In this work, the model host Saccharomyces cerevisiae was used to investigate the effects of CIRV p36 expression on the mitochondrial structure and function through the determination of mitochondrial morphology, mitochondrial respiratory parameters, and respiratory chain complex activities in p36-expressing cells. CIRV p36 ectopic expression was shown to induce alterations in the mitochondrial network associated with a decrease in mitochondrial respiration and the activities of NADH-cyt c, succinate-cyt c (C II-III), and cytochrome c oxidase (C IV) complexes. Our results suggest that the decrease in respiratory complex activity could be due, at least in part, to alterations in mitochondrial dynamics. This yeast-based model will be a valuable tool for identifying molecular targets to develop new anti-viral strategies.

Exploring the diversity and evolution of tombus-like viruses: phylogenetic analysis, recombination events, and suppressor protein homologs.

This study focuses on the phylogenetic analysis of previously unclassified tombus-like viruses, which are characterized by the presence of homologs of the suppressor protein p19. The primary objectives of this research were to investigate the evolutionary relationships among these viruses and to explore the impact of suppressor proteins and recombination events on their evolution. A dataset comprising 94 viral sequences was analyzed to achieve these goals. The phylogenetic analysis revealed the presence of two distinct clusters within the tombus-like virus group. One cluster consisted of viruses that encoded p19-like RNA suppressors, while the other cluster comprised viruses encoding p14-like suppressors. Based on these findings, we propose the classification of PGT-pt108 as an isolate of carnation Italian ringspot virus (CIRV), and both Tombusviridae sp. s48-k141_139792 and Tombusviridae sp. s51-k141_185213 as isolates of tomato bushy stunt virus (TBSV). Furthermore, this study suggests the establishment of two new genera within the family Tombusviridae, based on the observed divergence and distinct characteristics of these tombus-like viruses. Through the analysis of recombination events, we provide insights into the interspecies movement of CIRV, which is reflected in its phylogenetic positioning. This research contributes to our understanding of the evolutionary dynamics and classification of tombus-like viruses, shedding light on the role of suppressor proteins and recombination events in their evolution and interspecies transmission.

Affordable paper-based SWNTs field-effect transistor biosensors for nucleic acid amplification-free and label-free detection of micro RNAs

Micro RNAs (miRNAs) are 19–23 nucleotides-long non-coding RNA which have been identified as important biomarkers for many diseases, including cancers. Here, we report an ultrasensitive, highly specific, label-free, and without nucleic acid amplification (NAA) detection of miRNAs using paper-based single-walled carbon nanotubes (SWNTs) field-effect transistor (FET) biosensors. The strategy involved a two-step protocol starting with direct hybridization of the target miRNA with a specific RNA probe immobilized on SWNTs networks deposited on the paper substrate to generate the first response, followed by the recognition of the resulting RNA/miRNA duplexes with the Carnation Italian ringspot virus p19 protein (p19) in a size-dependent manner, giving the second electrical response that magnified the biosensor sensitivity. As a demonstration, we detected the miRNA-122a, a promising biomarker for the diagnosis of early-stage hepatocellular carcinoma (HCC) in various sample matrices, including phosphate buffers, human serum, and synthetic saliva. We achieved the lowest detection of 0.1 aM and a wide dynamic range of 0.1 aM–1 fM, that demonstrated its potential for rapid, facile, low-cost, and point-of-care detection of miRNAs for early cancer diagnosis.

First report of Carnation Italian ringspot virus infecting daisy fleabane in Korea

First report of <i>Carnation Italian ringspot virus</i> infecting daisy fleabane in Korea

Tombusviruses Target a Major Crossroad in the Endocytic and Recycling Pathways via Co-opting Rab7 Small GTPase.

Positive-strand RNA viruses induce the biogenesis of unique membranous organelles called viral replication organelles (VROs), which perform virus replication in infected cells. Tombusviruses have been shown to rewire cellular trafficking and metabolic pathways, remodel host membranes, and recruit multiple host factors to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely related carnation Italian ringspot virus (CIRV) usurp Rab7 small GTPase to facilitate building VROs in the surrogate host yeast and in plants. Depletion of Rab7 small GTPase, which is needed for late endosome and retromer biogenesis, strongly inhibits TBSV and CIRV replication in yeast and in planta. The viral p33 replication protein interacts with Rab7 small GTPase, which results in the relocalization of Rab7 into the large VROs. Similar to the depletion of Rab7, the deletion of either MON1 or CCZ1 heterodimeric GEFs (guanine nucleotide exchange factors) of Rab7 inhibited TBSV RNA replication in yeast. This suggests that the activated Rab7 has proviral functions. We show that the proviral function of Rab7 is to facilitate the recruitment of the retromer complex and the endosomal sorting nexin-BAR proteins into VROs. We demonstrate that TBSV p33-driven retargeting of Rab7 into VROs results in the delivery of several retromer cargos with proviral functions. These proteins include lipid enzymes, such as Vps34 PI3K (phosphatidylinositol 3-kinase), PI4Kα-like Stt4 phosphatidylinositol 4-kinase, and Psd2 phosphatidylserine decarboxylase. In summary, based on these and previous findings, we propose that subversion of Rab7 into VROs allows tombusviruses to reroute endocytic and recycling trafficking to support virus replication. IMPORTANCE The replication of positive-strand RNA viruses depends on the biogenesis of viral replication organelles (VROs). However, the formation of membranous VROs is not well understood yet. Using tombusviruses and the model host yeast, we discovered that the endosomal Rab7 small GTPase is critical for the formation of VROs. Interaction between Rab7 and the TBSV p33 replication protein leads to the recruitment of Rab7 into VROs. TBSV-driven usurping of Rab7 has proviral functions through facilitating the delivery of the co-opted retromer complex, sorting nexin-BAR proteins, and lipid enzymes into VROs to create an optimal milieu for virus replication. These results open up the possibility that controlling cellular Rab7 activities in infected cells could be a target for new antiviral strategies.

Dynamic interplay between the co-opted Fis1 mitochondrial fission protein and membrane contact site proteins in supporting tombusvirus replication.

Plus-stranded RNA viruses have limited coding capacity and have to co-opt numerous pro-viral host factors to support their replication. Many of the co-opted host factors support the biogenesis of the viral replication compartments and the formation of viral replicase complexes on subverted subcellular membrane surfaces. Tomato bushy stunt virus (TBSV) exploits peroxisomal membranes, whereas the closely-related carnation Italian ringspot virus (CIRV) hijacks the outer membranes of mitochondria. How these organellar membranes can be recruited into pro-viral roles is not completely understood. Here, we show that the highly conserved Fis1 mitochondrial fission protein is co-opted by both TBSV and CIRV via direct interactions with the p33/p36 replication proteins. Deletion of FIS1 in yeast or knockdown of the homologous Fis1 in plants inhibits tombusvirus replication. Instead of the canonical function in mitochondrial fission and peroxisome division, the tethering function of Fis1 is exploited by tombusviruses to facilitate the subversion of membrane contact site (MCS) proteins and peroxisomal/mitochondrial membranes for the biogenesis of the replication compartment. We propose that the dynamic interactions of Fis1 with MCS proteins, such as the ER resident VAP tethering proteins, Sac1 PI4P phosphatase and the cytosolic OSBP-like oxysterol-binding proteins, promote the formation and facilitate the stabilization of virus-induced vMCSs, which enrich sterols within the replication compartment. We show that this novel function of Fis1 is exploited by tombusviruses to build nuclease-insensitive viral replication compartment.

The retromer is co-opted to deliver lipid enzymes for the biogenesis of lipid-enriched tombusviral replication organelles

Biogenesis of viral replication organelles (VROs) is critical for replication of positive-strand RNA viruses. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely related carnation Italian ringspot virus (CIRV) hijack the retromer to facilitate building VROs in the surrogate host yeast and in plants. Depletion of retromer proteins, which are needed for biogenesis of endosomal tubular transport carriers, strongly inhibits the peroxisome-associated TBSV and the mitochondria-associated CIRV replication in yeast and in planta. In vitro reconstitution revealed the need for the retromer for the full activity of the viral replicase. The viral p33 replication protein interacts with the retromer complex, including Vps26, Vps29, and Vps35. We demonstrate that TBSV p33-driven retargeting of the retromer into VROs results in delivery of critical retromer cargoes, such as 1) Psd2 phosphatidylserine decarboxylase, 2) Vps34 phosphatidylinositol 3-kinase (PI3K), and 3) phosphatidylinositol 4-kinase (PI4Kα-like). The recruitment of these cellular enzymes by the co-opted retromer is critical for de novo production and enrichment of phosphatidylethanolamine phospholipid, phosphatidylinositol-3-phosphate [PI(3)P], and phosphatidylinositol-4-phosphate [PI(4)P] phosphoinositides within the VROs. Co-opting cellular enzymes required for lipid biosynthesis and lipid modifications suggest that tombusviruses could create an optimized lipid/membrane microenvironment for efficient VRO assembly and protection of the viral RNAs during virus replication. We propose that compartmentalization of these lipid enzymes within VROs helps tombusviruses replicate in an efficient milieu. In summary, tombusviruses target a major crossroad in the secretory and recycling pathways via coopting the retromer complex and the tubular endosomal network to build VROs in infected cells.

Impediometric Electrochemical Sensor Based on The Inspiration of Carnation Italian Ringspot Virus Structure to Detect an Attommolar of miR

Electrochemical sensors are the tools to detect the accurate and sensitive miRs. There is the challenge to increase the power and sensitivity of the surface for the electrochemical sensor. We design a virus-like hallow structure of cuco2o4 that it holds the large amounts of p19 protein by mimicking of inherent virus (Carnation italian ringspot virus) to detect 21mir with the limit of detection (LOD = 1aM). The electrochemical measurements are performed between the potentials at −0.3 V and +0.3 V with 1 mM [Fe(CN)6] −3/−4. After dropping the cuco2o4 on the SCPE (screen carbon printed electrode), the sensor is turned on due to the high electrochemical properties. Then, p19 proteins move into the hallow structure and inhibit the exchange of electrochemical reactions between the shells and the sensor is turned off. Then, adding the duplexes of RNA/miRs cause to increase the electrochemical property of p19 due to the change of p19 conformation and the system is turned on, again. So, for the first time, a virus-like hallow structure has been used to detect the 21miR in the human serum, MCF-7, Hella cells, with high sensitivity, specificity, and reproducibility in few minutes.

Estimation of the functions of viral RNA silencing suppressors by apple latent spherical virus vector.

Apple latent spherical virus (ALSV) is a latent virus with wide host range of plant species. In the present study, we prepared ALSV vectors expressing RNA silencing suppressors (RSSs) from eight plant viruses: P19 of carnation Italian ring spot virus (tombusvirus), 2b of peanut stunt virus (cucumovirus), NSs of tomato spotted wilt virus (tospovirus), HC-Pro of bean yellow mosaic virus (potyvirus), γb of barley stripe mosaic virus (hordeivirus), P15 of peanut clump virus (pecluvirus), P1 of rice yellow mottle virus (sobemovirus), or P21 of beet yellows virus (closterovirus). These vectors were inoculated to Nicotiana benthamiana to investigate the effects of RSSs on the virulence and accumulation of ALSV. Among the vectors, ALSV expressing NSs (ALSV-NSs) developed severe mosaic symptoms in newly developed leaves followed by plant death. Infection of ALSV-γb induced characteristic concentric ringspot symptoms on leaves, and plants infected with ALSV-HC-Pro showed mosaic and dwarf symptoms. Infection of the other five ALSV vectors did not show symptoms. ELISA and immunoblot assay indicated that virus titer increased in leaves infected with ALSV-NSs, γb, HC-Pro, or P19. RT-qPCR indicated that the amount of ALSV in plants infected with ALSV-NSs was increased by approximately 45 times compared with that of wtALSV without expression of any RSS. When ALSV-P19, NSs, or HC-Pro was inoculated to Cucumis sativus plants, none of these ALSV vectors induced symptoms, but accumulation of ALSV in plants infected with ALSV-NSs was increased, suggesting that functions of RSSs on virulence and accumulation of ALSV depend on host species.

Structural insights into interactions between viral suppressor of RNA silencing protein p19 mutants and small RNAs.

Viral suppressors of RNA silencing (VSRSs) are a diverse group of viral proteins that have evolved to disrupt eukaryotic RNA silencing pathways, thereby contributing to viral pathogenicity. The p19 protein is a VSRS that selectively binds to short interfering RNAs (siRNAs) over microRNAs (miRNAs). Mutational analysis has identified single amino acid substitutions that reverse this selectivity through new high‐affinity interactions with human miR‐122. Herein, we report crystal structures of complexed p19‐T111S (2.6 Å), p19‐T111H (2.3 Å) and wild‐type p19 protein (2.2 Å) from the Carnation Italian ringspot virus with small interfering RNA (siRNA) ligands. Structural comparisons reveal that these mutations do not lead to major changes in p19 architecture, but instead promote subtle rearrangement of residues and solvent molecules along the p19 midline. These observations suggest p19 uses many small interactions to distinguish siRNAs from miRNAs and perturbing these interactions can create p19 variants with novel RNA‐recognition properties.DatabaseModel data are deposited in the PDB database under the accession numbers 6BJG, 6BJH and 6BJV.

Recruitment of Vps34 PI3K and enrichment of PI3P phosphoinositide in the viral replication compartment is crucial for replication of a positive-strand RNA virus.

Tombusviruses depend on subversions of multiple host factors and retarget cellular pathways to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus (CIRV) recruit the cellular Vps34 phosphatidylinositol 3-kinase (PI3K) into the large viral replication compartment. The kinase function of Vps34 is critical for TBSV replication, suggesting that PI(3)P phosphoinositide is utilized by TBSV for building of the replication compartment. We also observed increased expression of Vps34 and the higher abundance of PI(3)P in the presence of the tombusviral replication proteins, which likely leads to more efficient tombusvirus replication. Accordingly, overexpression of PI(3)P phosphatase in yeast or plants inhibited TBSV replication on the peroxisomal membranes and CIRV replication on the mitochondrial membranes. Moreover, the purified PI(3)P phosphatase reduced TBSV replicase assembly in a cell-free system. Detection of PI(3)P with antibody or a bioprobe revealed the enrichment of PI(3)P in the replication compartment. Vps34 is directly recruited into the replication compartment through interaction with p33 replication protein. Gene deletion analysis in surrogate yeast host unraveled that TBSV replication requires the vesicle transport function of Vps34. In the absence of Vps34, TBSV cannot efficiently recruit the Rab5-positive early endosomes, which provide PE-rich membranes for membrane biogenesis of the TBSV replication compartment. We found that Vps34 and PI(3)P needed for the stability of the p33 replication protein, which is degraded by the 26S proteasome when PI(3)P abundance was decreased by an inhibitor of Vps34. In summary, Vps34 and PI(3)P are needed for providing the optimal microenvironment for the replication of the peroxisomal TBSV and the mitochondrial CIRV.

Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus.

Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs). The VRCs built by Tomato bushy stunt virus (TBSV) are enriched with phosphatidylethanolamine (PE) through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP)-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5–positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment.

Heterologous expression of carnation Italian ringspot virus p36 protein enhances necrotic cell death in response to acetic acid in Saccharomyces cerevisiae

A universal feature of the replication of positive-strand RNA viruses is the association with intracellular membranes. Carnation Italian ringspot virus (CIRV) replication in plants occurs in vesicles derived from the mitochondrial outer membrane. The product encoded by CIRV ORF1, p36, is required for targeting the virus replication complex to the outer mitochondrial membrane both in plant and yeast cells. Here the yeast Saccharomyces cerevisiae was used as a model host to study the effect of CIRV p36 on cell survival and death. It was shown that p36 does not promote cell death, but decreases cell growth rate. In addition, p36 changed the nature of acetic acid-induced cell death in yeast by increasing the number of cells dying by necrosis with concomitant decrease of the number of cells dying by programmed cell death, as judged by measurements of phosphatidylserine externalization. The tight association of p36 to membranes was not affected by acetic acid treatment, thus confirming the peculiar and independent interaction of CIRV p36 with mitochondria in yeast. This work proved yeast as an invaluable model organism to study both the mitochondrial determinants of the type of cell death in response to stress and the molecular pathogenesis of (+)RNA viruses.

First Report of Carnation Italian ringspot virus in Chrysanthemum zawadskii var. latilobum in Korea

First Report of <i>Carnation Italian ringspot virus</i> in <i>Chrysanthemum zawadskii</i> var. <i>latilobum</i> in Korea

A Unique N-Terminal Sequence in the Carnation Italian ringspot virus p36 Replicase-Associated Protein Interacts with the Host Cell ESCRT-I Component Vps23

Like most positive-strand RNA viruses, infection by plant tombusviruses results in extensive rearrangement of specific host cell organelle membranes that serve as the sites of viral replication. The tombusvirus Tomato bushy stunt virus (TBSV) replicates within spherules derived from the peroxisomal boundary membrane, a process that involves the coordinated action of various viral and cellular factors, including constituents of the endosomal sorting complex required for transport (ESCRT). ESCRT is comprised of a series of protein subcomplexes (i.e., ESCRT-0 -I, -II, and -III) that normally participate in late endosome biogenesis and some of which are also hijacked by certain enveloped retroviruses (e.g., HIV) for viral budding from the plasma membrane. Here we show that the replication of Carnation Italian ringspot virus (CIRV), a tombusvirus that replicates at mitochondrial membranes also relies on ESCRT. In plant cells, CIRV recruits the ESCRT-I protein, Vps23, to mitochondria through an interaction that involves a unique region in the N terminus of the p36 replicase-associated protein that is not conserved in TBSV or other peroxisome-targeted tombusviruses. The interaction between p36 and Vps23 also involves the Vps23 C-terminal steadiness box domain and not its N-terminal ubiquitin E2 variant domain, which in the case of TBSV (and enveloped retroviruses) mediates the interaction with ESCRT. Overall, these results provide evidence that CIRV uses a unique N-terminal sequence for the recruitment of Vps23 that is distinct from those used by TBSV and certain mammalian viruses for ESCRT recruitment. Characterization of this novel interaction with Vps23 contributes to our understanding of how CIRV may have evolved to exploit key differences in the plant ESCRT machinery. Positive-strand RNA viruses replicate their genomes in association with specific host cell membranes. To accomplish this, cellular components responsible for membrane biogenesis and modeling are appropriated by viral proteins and redirected to assemble membrane-bound viral replicase complexes. The diverse pathways leading to the formation of these replication structures are poorly understood. We have determined that the cellular ESCRT system that is normally responsible for mediating late endosome biogenesis is also involved in the replication of the tombusvirus Carnation Italian ringspot virus (CIRV) at mitochondria. Notably, CIRV recruits ESCRT to the mitochondrial outer membrane via an interaction between a unique motif in the viral protein p36 and the ESCRT component Vps23. Our findings provide new insights into tombusvirus replication and the virus-induced remodeling of plant intracellular membranes, as well as normal ESCRT assembly in plants.

P19-mediated enrichment and detection of siRNAs.

p19 is an RNA binding protein originally isolated from the Carnation Italian ring-spot virus (CIRV). It has been shown that p19 is a plant RNA-silencing suppressor that binds small interfering RNA (siRNA) with high affinity. A bifunctional p19 fusion protein, with an N-terminal maltose binding protein (MBP) and a C-terminal chitin binding domain (CBD) allows protein purification and binding of p19 to chitin magnetic beads via the chitin binding domain. The fusion p19 protein recognizes and binds double-stranded RNAs (dsRNA) in the size range of 20-23 nucleotides, but does not bind single strand RNA (ssRNA) or dsDNA. Furthermore, p19 can also bind mRNA, if there is a 19 bp blunt RNA duplex at the exact end of the RNA. Binding specificity of the p19 fusion protein for small dsRNA allows for detection of siRNAs derived either from exogenous or endogenous long dsRNA or microRNAs when hybridized to a complementary RNA. Here we describe a robust method using p19 and radioactive RNA probes to detect siRNAs in the sub-femtomole range and in the presence of a million-fold excess of total RNA. Unlike most nucleic acid detection methods, p19 selects for RNA hybrids of correct length and structure. This chapter describes the potential of p19 fusion protein to detect miRNAs, isolate exogenous or endogenous siRNAs, and purify longer RNAs that contain a 19-bp terminal RNA duplex.

Electronic Detection of MicroRNA at Attomolar Level with High Specificity

Small RNA (19-23 nucleotides) molecules play an important role in gene regulation, embryonic differentiation, hematopoiesis, and a variety of cancers. Here, we present an ultrasensitive, extremely specific, label-free, and rapid electronic detection of microRNAs (miRNAs) using a carbon nanotubes field-effect transistor functionalized with the Carnation Italian ringspot virus p19 protein biosensor. miRNA-122a was chosen as the target, which was first hybridized to a probe molecule. The probe-miRNA duplex was then quantified by measuring the change in resistance of biosensor resulting from its binding to p19, which selects 21-23 bp RNA duplexes in a size-dependent but sequence-independent manner. The biosensor displayed a wide dynamic range up to 10(-14) M and was able to detect as low as 1 aM miRNA in the presence of a million-fold excess of total RNA, paving the way for simple, point-of-care, low-cost early detection of miRNA as a biomarker in diagnosis of many diseases, including cancer.