Single-cell mutation rate of turnip crinkle virus (-)-strand replication intermediates.

Viruses with single-stranded, positive-sense (+) RNA genomes incur high numbers of errors during replication, thereby creating diversified genome populations from which new, better adapted viral variants can emerge. However, a definitive error rate is known for a relatively few (+) RNA plant viruses, due to challenges to account for perturbations caused by natural selection and/or experimental set-ups. To address these challenges, we developed a new approach that exclusively profiled errors in the (-)-strand replication intermediates of turnip crinkle virus (TCV), in singly infected cells. A series of controls and safeguards were devised to ensure errors inherent to the experimental process were accounted for. This approach permitted the estimation of a TCV error rate of 8.47 X 10-5 substitution per nucleotide site per cell infection. Importantly, the characteristic error distribution pattern among the 50 copies of 2,363-base-pair cDNA fragments predicted that nearly all TCV (-) strands were products of one replication cycle per cell. Furthermore, some of the errors probably elevated error frequencies by lowering the fidelity of TCV RNA-dependent RNA polymerase, and/or permitting occasional re-replication of progeny genomes. In summary, by profiling errors in TCV (-)-strand intermediates incurred during replication in single cells, this study provided strong support for a stamping machine mode of replication employed by a (+) RNA virus.

Turnip crinkle virus-encoded suppressor of RNA silencing suppresses mRNA decay by interacting with Arabidopsis XRN4.

Plant cells employ intricate defense mechanisms, including mRNA decay pathways, to counter viral infections. Among the RNA quality control (RQC) mechanisms, nonsense-mediated decay (NMD), no-go decay (NGD), and nonstop decay (NSD) pathways play critical roles in recognizing and cleaving aberrant mRNA molecules. Turnip crinkle virus (TCV) is a plant virus that triggers mRNA decay pathways, but it has also evolved strategies to evade this antiviral defense. In this study, we investigated the activation of mRNA decay during TCV infection and its impact on TCV RNA accumulation. We found that TCV infection induced the upregulation of essential mRNA decay factors, indicating their involvement in antiviral defense and the capsid protein (CP) of TCV, a well-characterized viral suppressor of RNA silencing (VSR), also compromised the mRNA decay-based antiviral defense by targeting AtXRN4. This interference with mRNA decay was supported by the observation that TCV CP stabilized a reporter transcript with a long 3' untranslated region (UTR). Moreover, TCV CP suppressed the decay of known NMD target transcripts, further emphasizing its ability to modulate host RNA control mechanisms. Importantly, TCV CP physically interacted with AtXRN4, providing insight into the mechanism of viral interference with mRNA decay. Overall, our findings reveal an alternative strategy employed by TCV, wherein the viral coat protein suppresses the mRNA decay pathway to facilitate viral infection.

CASC3 Biomolecular Condensates Restrict Turnip Crinkle Virus by Limiting Host Factor Availability.

The exon-junction complex (EJC) plays a role in post-transcriptional gene regulation and exerts antiviral activity towards several positive-strand RNA viruses. However, the spectrum of RNA viruses that are targeted by the EJC or the underlying mechanisms are not well understood. EJC components from Arabidopsis thaliana were screened for antiviral activity towards Turnip crinkle virus (TCV, Tombusviridae). Overexpression of the accessory EJC component CASC3 inhibited TCV accumulation > 10-fold in Nicotiana benthamiana while knock-down of endogenous CASC3 resulted in a > 4-fold increase in TCV accumulation. CASC3 forms cytoplasmic condensates and deletion of the conserved SELOR domain reduced condensate size 7-fold and significantly decreased antiviral activity towards TCV. Mass spectrometry of CASC3 complexes did not identify endogenous stress granule or P-body markers and CASC3 failed to co-localize with an aggresome-specific dye suggesting that CASC3 condensates are distinct from well-established membraneless compartments. Mass spectrometry and bimolecular fluorescence complementation assays revealed that CASC3 sequesters Heat shock protein 70 (Hsp70-1) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), two host factors with roles in tombusvirus replication. Overexpression of Hsp70-1 or GAPDH reduced the antiviral activity of CASC3 2.1-fold and 2.8-fold, respectively, and suggests that CASC3 inhibits TCV by limiting host factor availability. Unrelated Tobacco mosaic virus (TMV) also depends on Hsp70-1 and CASC3 overexpression restricted TMV accumulation 4-fold and demonstrates that CASC3 antiviral activity is not TCV-specific. Like CASC3, Auxin response factor 19 (ARF19) forms poorly dynamic condensates but ARF19 overexpression failed to inhibit TCV accumulation and suggests that CASC3 has antiviral activities that are not ubiquitous among cytoplasmic condensates.

Molecular characterization of a novel gammacarmovirus infecting cucurbits in India.

In this study, we describe the identification of a new gammacarmovirus infecting Cucurbita pepo plants showing a range of mosaic, stunting, yellowing, and wilting symptoms. The virus had a narrow host range and mostly produced chlorotic and necrotic local lesions in the majority of the tested plants. However, Nicotiana benthamiana showed systemic symptoms under laboratory conditions. Using a combination of Sanger sequencing and rapid amplification of cDNA ends (RACE), the complete genome sequence of the virus was determined to be 4274 nucleotides (nt) in length. Its genome organization is similar to that of members of the genus Gammacarmovirus in the family Tombusviridae, consisting of five overlapping open reading frames (ORFs) encoding p28, replicase, p7A, p7B, and coat protein (CP), respectively. The genome is flanked by short 5' and 3' non-coding regions (NCR) at either end. In pairwise comparisons of replicase and CP sequences, the virus showed the highest amino acid sequence identity of 71.55% and 54.86%, respectively, to melon necrotic spot virus (MNSV), the type member of the genus Gammacarmovirus. Since the sequence identity values are below the species demarcation threshold suggested by the International Committee on Taxonomy of Viruses (ICTV), the virus from Cucurbita pepo plants, for which the name "cucurbit carmovirus" (CuCV) is proposed, represents a new species. In phylogenetic analysis based on the replicase and CP amino acid sequences, CuCV clustered with MNSV but formed a distinct branch, further confirming that the virus is a distinct member of the genus Gammacarmovirus.

Turnip crinkle virus-encoded suppressor of RNA silencing interacts with Arabidopsis SGS3 to enhance virus infection.

Most plant viruses encode suppressors of RNA silencing (VSRs) to protect themselves from antiviral RNA silencing in host plants. The capsid protein (CP) of Turnip crinkle virus (TCV) is a well-characterized VSR, whereas SUPPRESSOR OF GENE SILENCING 3 (SGS3) is an important plant-encoded component of the RNA silencing pathways. Whether the VSR activity of TCV CP requires it to engage SGS3 in plant cells has yet to be investigated. Here, we report that TCV CP interacts with SGS3 of Arabidopsis in both yeast and plant cells. The interaction was identified with the yeast two-hybrid system, and corroborated with bimolecular fluorescence complementation and intracellular co-localization assays in Nicotiana benthamiana cells. While multiple partial TCV CP fragments could independently interact with SGS3, its hinge domain connecting the surface and protruding domains appears to be essential for this interaction. Conversely, SGS3 enlists its N-terminal domain and the XS rice gene X and SGS3 (XS) domain as the primary CP-interacting sites. Interestingly, SGS3 appears to stimulate TCV accumulation because viral RNA levels of a TCV mutant with low VSR activities decreased in the sgs3 knockout mutants, but increased in the SGS3-overexpressing transgenic plants. Transgenic Arabidopsis plants overexpressing TCV CP exhibited developmental abnormalities that resembled sgs3 knockout mutants and caused similar defects in the biogenesis of trans-acting small interfering RNAs. Our data suggest that TCV CP interacts with multiple RNA silencing pathway components that include SGS3, as well as previously reported DRB4 (dsRNA-binding protein 4) and AGO2 (ARGONAUTE protein 2), to achieve efficient suppression of RNA silencing-mediated antiviral defence.

Novel 3' Proximal Replication Elements in Umbravirus Genomes.

The 3' untranslated regions (UTRs) of positive-strand RNA plant viruses commonly contain elements that promote viral replication and translation. The ~700 nt 3'UTR of umbravirus pea enation mosaic virus 2 (PEMV2) contains three 3' cap-independent translation enhancers (3'CITEs), including one (PTE) found in members of several genera in the family Tombusviridae and another (the 3'TSS) found in numerous umbraviruses and several carmoviruses. In addition, three 3' terminal replication elements are found in nearly every umbravirus and carmovirus. For this report, we have identified a set of three hairpins and a putative pseudoknot, collectively termed "Trio", that are exclusively found in a subset of umbraviruses and are located just upstream of the 3'TSS. Modification of these elements had no impact on viral translation in wheat germ extracts or in translation of luciferase reporter constructs in vivo. In contrast, Trio hairpins were critical for viral RNA accumulation in Arabidopsis thaliana protoplasts and for replication of a non-autonomously replicating replicon using a trans-replication system in Nicotiana benthamiana leaves. Trio and other 3' terminal elements involved in viral replication are highly conserved in umbraviruses possessing different classes of upstream 3'CITEs, suggesting conservation of replication mechanisms among umbraviruses despite variation in mechanisms for translation enhancement.

Replication-Dependent Biogenesis of Turnip Crinkle Virus Long Noncoding RNAs.

Long noncoding RNAs (lncRNAs) of virus origin accumulate in cells infected by many positive-strand (+) RNA viruses to bolster viral infectivity. Their biogenesis mostly utilizes exoribonucleases of host cells that degrade viral genomic or subgenomic RNAs in the 5'-to-3' direction until being stalled by well-defined RNA structures. Here, we report a viral lncRNA that is produced by a novel replication-dependent mechanism. This lncRNA corresponds to the last 283 nucleotides of the turnip crinkle virus (TCV) genome and hence is designated tiny TCV subgenomic RNA (ttsgR). ttsgR accumulated to high levels in TCV-infected Nicotiana benthamiana cells when the TCV-encoded RNA-dependent RNA polymerase (RdRp), also known as p88, was overexpressed. Both (+) and (-) strand forms of ttsgR were produced in a manner dependent on the RdRp functionality. Strikingly, templates as short as ttsgR itself were sufficient to program ttsgR amplification, as long as the TCV-encoded replication proteins p28 and p88 were provided in trans. Consistent with its replicational origin, ttsgR accumulation required a 5' terminal carmovirus consensus sequence (CCS), a sequence motif shared by genomic and subgenomic RNAs of many viruses phylogenetically related to TCV. More importantly, introducing a new CCS motif elsewhere in the TCV genome was alone sufficient to cause the emergence of another lncRNA. Finally, abolishing ttsgR by mutating its 5' CCS gave rise to a TCV mutant that failed to compete with wild-type TCV in Arabidopsis. Collectively, our results unveil a replication-dependent mechanism for the biogenesis of viral lncRNAs, thus suggesting that multiple mechanisms, individually or in combination, may be responsible for viral lncRNA production. IMPORTANCE Many positive-strand (+) RNA viruses produce long noncoding RNAs (lncRNAs) during the process of cellular infections and mobilize these lncRNAs to counteract antiviral defenses, as well as coordinate the translation of viral proteins. Most viral lncRNAs arise from 5'-to-3' degradation of longer viral RNAs being stalled at stable secondary structures. Here, we report a viral lncRNA that is produced by the replication machinery of turnip crinkle virus (TCV). This lncRNA, designated ttsgR, shares the terminal characteristics with TCV genomic and subgenomic RNAs and overaccumulates in the presence of moderately overexpressed TCV RNA-dependent RNA polymerase (RdRp). Furthermore, templates that are of similar sizes as ttsgR are readily replicated by TCV replication proteins (p28 and RdRp) provided from nonviral sources. In summary, this study establishes an approach for uncovering low abundance viral lncRNAs, and characterizes a replicating TCV lncRNA. Similar investigations on human-pathogenic (+) RNA viruses could yield novel therapeutic targets.

Introducing SELEX via a semester-long course-based undergraduate research experience (CURE).

With the growing importance of the field of RNA biology, undergraduates need to perform RNA-related research. Systematic evolution of ligands by exponential enrichment (SELEX) has become an important method in RNA biology. The principles of SELEX were applied to a semester-long course-based undergraduate research experience (CURE) in which two rounds of in vivo functional selection of regions of a viral RNA were performed. As the labwork had an unknown outcome, students indicated that they were excited by the work and became invested in the experience. By completing two rounds of SELEX, the students repeated molecular methods (e.g., RNA extraction, RT-PCR, agarose gel electrophoresis, DNA purification, cloning, and sequence analysis) and reported that repetition reinforced their learning and helped them build confidence in their lab abilities. Students also appreciated that they did not learn a "technique-per-week" without context, but rather they understood why certain methods were used for certain molecular tasks. Results from a 19-question multiple-choice assessment indicated increased comprehension of theory underlying methods performed. Details regarding experimental methods and timeline, and assessment and attitudinal results from three student cohorts, are described herein.

Molecular modeling and interaction between Arabidopsis sulfite oxidase and the GW motif of Turnip crinkle virus coat protein.

Previous study has shown that Hibiscus sulfite oxidase (SO) interacts with Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) and triggers sulfur enhanced defense (SED). In this study, we show the interaction of Arabidopsis SO (AtSO) and Turnip crinkle virus (TCV) CP in Arabidopsis thaliana plants. We identified the binding sites of TCV CP (W274) and AtSO (D223) using bioinformatics and confirmed it experimentally. Mutation of binding site W274 to A274 in TCV CP resulted in failure of TCV infection. TCV accumulation in SO over-expression (SO_OE) plants was lower than that in wild-type (WT) and SO knock-out (SO_KO) plants at 7 dpi but reached a level similar to that of WT and SO_KO plants at 10 dpi. AtSO competed with Argonaute 1 (AGO1) for TCV CP binding in vitro. AtSO may serve as an anti-viral factor through sequestering TCV CP for binding with AGO1 and confers virus resistance.

Phospholipase pPLAIIIα Increases Germination Rate and Resistance to Turnip Crinkle Virus when Overexpressed.

Patatin-related phospholipase As (pPLAs) are major hydrolases acting on acyl-lipids and play important roles in various plant developmental processes. pPLAIII group members, which lack a canonical catalytic Ser motif, have been less studied than other pPLAs. We report here the characterization of pPLAIIIα in Arabidopsis (Arabidopsis thaliana) based on the biochemical and physiological characterization of pPLAIIIα knockouts, complementants, and overexpressors, as well as heterologous expression of the protein. In vitro activity assays on the purified recombinant protein showed that despite lack of canonical phospholipase motifs, pPLAIIIα had a phospholipase A activity on a wide variety of phospholipids. Overexpression of pPLAIIIα in Arabidopsis resulted in a decrease in many lipid molecular species, but the composition in major lipid classes was not affected. Fluorescence tagging indicated that pPLAIIIα localizes to the plasma membrane. Although Arabidopsis pplaIIIα knockout mutants showed some phenotypes comparable to other pPLAIIIs, such as reduced trichome length and increased hypocotyl length, control of seed size and germination were identified as distinctive pPLAIIIα-mediated functions. Expression of some PLD genes was strongly reduced in the pplaIIIα mutants. Overexpression of pPLAIIIα caused increased resistance to turnip crinkle virus, which associated with a 2-fold higher salicylic acid/jasmonic acid ratio and an increased expression of the defense gene pathogenesis-related protein1. These results therefore show that pPLAIIIα has functions that overlap with those of other pPLAIIIs but also distinctive functions, such as the control of seed germination. This study also provides new insights into the pathways downstream of pPLAIIIα.

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

RNA secondary structures play diverse roles in positive-sense (+) RNA virus infections, but those located with the replication protein coding sequence can be difficult to investigate. Structures that regulate the translation of replication proteins pose particular challenges, as their potential involvement in post-translational steps cannot be easily discerned independent of their roles in regulating translation. In the current study, we attempted to overcome these difficulties by providing viral replication proteins in trans. Specifically, we modified the plant-infecting turnip crinkle virus (TCV) into variants that are unable to translate one (p88) or both (p28 and p88) replication proteins, and complemented their replication with the corresponding replication protein(s) produced from separate, non-replicating constructs. This approach permitted us to re-examine the p28/p88 coding region for potential RNA elements needed for TCV replication. We found that, while more than a third of the p88 coding sequence could be deleted without substantially affecting viral RNA levels, two relatively small regions, known as RSE and IRE, were essential for robust accumulation of TCV genomic RNA, but not subgenomic RNAs. In particular, the RSE element, found previously to be required for regulating the translational read-through of p28 stop codon to produce p88, contained sub-elements needed for efficient replication of the TCV genome. Application of this new approach in other viruses could reveal novel RNA secondary structures vital for viral multiplication.

A conserved motif in three viral movement proteins from different genera is required for host factor recruitment and cell-to-cell movement.

Due to their minimal genomes, plant viruses are forced to hijack specific cellular pathways to ensure host colonization, a condition that most frequently involves physical interaction between viral and host proteins. Among putative viral interactors are the movement proteins, responsible for plasmodesma gating and genome binding during viral transport. Two of them, DGBp1 and DGBp2, are required for alpha-, beta- and gammacarmovirus cell-to-cell movement, but the number of DGBp-host interactors identified at present is limited. By using two different approaches, yeast two-hybrid and bimolecular fluorescence complementation assays, we found three Arabidopsis factors, eIF3g1, RPP3A and WRKY36, interacting with DGBp1s from each genus mentioned above. eIF3g1 and RPP3A are mainly involved in protein translation initiation and elongation phases, respectively, while WRKY36 belongs to WRKY transcription factor family, important regulators of many defence responses. These host proteins are not expected to be associated with viral movement, but knocking out WRKY36 or silencing either RPP3A or eIF3g1 negatively affected Arabidopsis infection by Turnip crinkle virus. A highly conserved FNF motif at DGBp1 C-terminus was required for protein-protein interaction and cell-to-cell movement, suggesting an important biological role.

Superinfection Exclusion by p28 of Turnip Crinkle Virus Is Separable from Its Replication Function.

We recently reported that the p28 auxiliary replication protein encoded by turnip crinkle virus (TCV) is also responsible for eliciting superinfection exclusion (SIE) against superinfecting TCV. However, it remains unresolved whether the replication function of p28 could be separated from its ability to elicit SIE. Here, we report the identification of two single amino acid mutations that decouple these two functions. Using an Agrobacterium infiltration-based delivery system, we transiently expressed a series of p28 deletion and point mutants, and tested their ability to elicit SIE against a cointroduced TCV replicon. We found that substituting alanine (A) for valine (V) and phenylalanine (F) at p28 positions 181 and 182, respectively, modestly compromised SIE in transiently expressed p28 derivatives. Upon incorporation into TCV replicons, V181A and F182A decoupled TCV replication and SIE diametrically. Although V181A impaired SIE without detectably compromising replication, F182A abolished TCV replication but had no effect on SIE once the replication of the defective replicon was restored through complementation. Both mutations diminished accumulation of p28 protein, suggesting that p28 must reach a concentration threshold in order to elicit a strong SIE. Importantly, the severe reduction of F182A protein levels correlated with a dramatic loss in the number of intracellular p28 foci formed by p28-p28 interactions. Together, these findings not only decouple the replication and SIE functions of p28 but also unveil a concentration dependence for p28 coalescence and SIE elicitation. These data further highlight the role of p28 multimerization in driving the exclusion of secondary TCV infections.

Interviral Recombination between Plant, Insect, and Fungal RNA Viruses: Role of the Intracellular Ca2+/Mn2+ Pump.

Recombination is one of the driving forces of viral evolution. RNA recombination events among similar RNA viruses are frequent, although RNA recombination could also take place among unrelated viruses. In this paper, we have established efficient interviral recombination systems based on yeast and plants. We show that diverse RNA viruses, including the plant viruses tomato bushy stunt virus, carnation Italian ringspot virus, and turnip crinkle virus-associated RNA; the insect plus-strand RNA [(+)RNA] viruses Flock House virus and Nodamura virus; and the double-stranded L-A virus of yeast, are involved in interviral recombination events. Most interviral recombinants are minus-strand recombinant RNAs, and the junction sites are not randomly distributed, but there are certain hot spot regions. Formation of interviral recombinants in yeast and plants is accelerated by depletion of the cellular SERCA-like Pmr1 ATPase-driven Ca2+/Mn2+ pump, regulating intracellular Ca2+ and Mn2+ influx into the Golgi apparatus from the cytosol. The interviral recombinants are generated by a template-switching mechanism during RNA replication by the viral replicase. Replication studies revealed that a group of interviral recombinants is replication competent in cell-free extracts, in yeast, and in the plant Nicotiana benthamiana We propose that there are major differences among the viral replicases to generate and maintain interviral recombinants. Altogether, the obtained data promote the model that host factors greatly contribute to the formation of recombinants among related and unrelated viruses. This is the first time that a host factor's role in affecting interviral recombination is established.IMPORTANCE Viruses with RNA genomes are abundant, and their genomic sequences show astonishing variation. Genetic recombination in RNA viruses is a major force behind their rapid evolution, enhanced pathogenesis, and adaptation to their hosts. We utilized a previously identified intracellular Ca2+/Mn2+ pump-deficient yeast to search for interviral recombinants. Noninfectious viral replication systems were used to avoid generating unwanted infectious interviral recombinants. Altogether, interviral RNA recombinants were observed between plant and insect viruses, and between a fungal double-stranded RNA (dsRNA) virus and an insect virus, in the yeast host. In addition, interviral recombinants between two plant virus replicon RNAs were identified in N. benthamiana plants, in which the intracellular Ca2+/Mn2+ pump was depleted. These findings underline the crucial role of the host in promoting RNA recombination among unrelated viruses.

Antiviral ARGONAUTEs Against Turnip Crinkle Virus Revealed by Image-Based Trait Analysis.

RNA-based silencing functions as an important antiviral immunity mechanism in plants. Plant viruses evolved to encode viral suppressors of RNA silencing (VSRs) that interfere with the function of key components in the silencing pathway. As effectors in the RNA silencing pathway, ARGONAUTE (AGO) proteins are targeted by some VSRs, such as that encoded by Turnip crinkle virus (TCV). A VSR-deficient TCV mutant was used to identify AGO proteins with antiviral activities during infection. A quantitative phenotyping protocol using an image-based color trait analysis pipeline on the PlantCV platform, with temporal red, green, and blue imaging and a computational segmentation algorithm, was used to measure plant disease after TCV inoculation. This process captured and analyzed growth and leaf color of Arabidopsis (Arabidopsis thaliana) plants in response to virus infection over time. By combining this quantitative phenotypic data with molecular assays to detect local and systemic virus accumulation, AGO2, AGO3, and AGO7 were shown to play antiviral roles during TCV infection. In leaves, AGO2 and AGO7 functioned as prominent nonadditive, anti-TCV effectors, whereas AGO3 played a minor role. Other AGOs were required to protect inflorescence tissues against TCV. Overall, these results indicate that distinct AGO proteins have specialized, modular roles in antiviral defense across different tissues, and demonstrate the effectiveness of image-based phenotyping to quantify disease progression.

Repression of turnip crinkle virus replication by its replication protein p88.

We recently reported that p28, one of the two turnip crinkle virus (TCV) replication proteins, trans-complemented a defective TCV lacking p28, yet repressed the replication of another TCV replicon encoding wild-type p28 (Zhang et al., 2017). Here we show that p88, the TCV-encoded RNA-dependent RNA polymerase, likewise trans-complemented a p88-defective TCV replicon, but repressed one encoding wild-type p88. Surprisingly, lowering p88 protein levels enhanced trans-complementation, but weakened repression. Repression by p88 was not simply due to protein over-expression, as deletion mutants missing 127 or 224 N-terminal amino acids accumulated to higher levels but were poor repressors. Finally, both trans-complementation and repression by p88 were accompanied by preferential accumulation of subgenomic RNA2, and a novel class of small TCV RNAs. Our results suggest that repression of TCV replication by p88 may manifest a viral mechanism that regulates the ratio of genomic and subgenomic RNAs based on p88 abundance.

RNA virus evasion of nonsense-mediated decay.

Nonsense-mediated decay (NMD) is a host RNA control pathway that removes aberrant transcripts with long 3' untranslated regions (UTRs) due to premature termination codons (PTCs) that arise through mutation or defective splicing. To maximize coding potential, RNA viruses often contain internally located stop codons that should also be prime targets for NMD. Using an agroinfiltration-based NMD assay in Nicotiana benthamiana, we identified two segments conferring NMD-resistance in the carmovirus Turnip crinkle virus (TCV) genome. The ribosome readthrough structure just downstream of the TCV p28 termination codon stabilized an NMD-sensitive reporter as did a frameshifting element from umbravirus Pea enation mosaic virus. In addition, a 51-nt unstructured region (USR) at the beginning of the TCV 3' UTR increased NMD-resistance 3-fold when inserted into an unrelated NMD-sensitive 3' UTR. Several additional carmovirus 3' UTRs also conferred varying levels of NMD resistance depending on the construct despite no sequence similarity in the analogous region. Instead, these regions displayed a marked lack of RNA structure immediately following the NMD-targeted stop codon. NMD-resistance was only slightly reduced by conversion of 19 pyrimidines in the USR to purines, but resistance was abolished when a 2-nt mutation was introduced downstream of the USR that substantially increased the secondary structure in the USR through formation of a stable hairpin. The same 2-nt mutation also enhanced the NMD susceptibility of a subgenomic RNA expressed independently of the genomic RNA. The conserved lack of RNA structure among most carmoviruses at the 5' end of their 3' UTR could serve to enhance subgenomic RNA stability, which would increase expression of the encoded capsid protein that also functions as the RNA silencing suppressor. These results demonstrate that the TCV genome has features that are inherently NMD-resistant and these strategies could be widespread among RNA viruses and NMD-resistant host mRNAs with long 3' UTRs.

SELEX and SHAPE reveal that sequence motifs and an extended hairpin in the 5' portion of Turnip crinkle virus satellite RNA C mediate fitness in plants.

Noncoding RNAs use their sequence and/or structure to mediate function(s). The 5' portion (166 nt) of the 356-nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) was previously modeled to contain a central region with two stem-loops (H6 and H7) and a large connecting hairpin (H2). We now report that in vivo functional selection (SELEX) experiments assessing sequence/structure requirements in H2, H6, and H7 reveal that H6 loop sequence motifs were recovered at nonrandom rates and only some residues are proposed to base-pair with accessible complementary sequences within the 5' central region. In vitro SHAPE of SELEX winners indicates that the central region is heavily base-paired, such that along with the lower stem and H2 region, one extensive hairpin exists composing the entire 5' region. As these SELEX winners are highly fit, these characteristics facilitate satRNA amplification in association with TCV in plants.

Production of Mosaic Turnip Crinkle Virus-Like Particles Derived by Coinfiltration of Wild-Type and Modified Forms of Virus Coat Protein in Plants.

When the coat protein reading frame of turnip crinkle virus (TCV) is transiently expressed in leaves, virus-like particles (VLPs) are readily formed. However, after introducing genetic modifications to the full-length coat protein sequence, such as the introduction of an epitope-specific sequence within the coat protein sequence or the in-frame carboxyl terminal fusion of GFP, the formation of such modified VLPs is poor. However, by coexpression of one of these modified forms with wild-type TCV coat protein by the coinfiltration of appropriate Agrobacterium suspensions, VLP generation is enhanced through the formation of "mosaics," that is, individual VLPs consisting of both modified and wild-type subunits (also known as phenotypically mixed VLPs). Here we describe methods for the introduction of genetic modifications into the TCV coat protein sequence, the production of mosaic TCV VLPs and their characterization.

Shannon Entropy to Evaluate Substitution Rate Variation Among Viral Nucleotide Positions in Datasets of Viral siRNAs.

Next-generation sequencing has opened the door to the reconstruction of viral populations and examination of the composition of mutant spectra in infected cells, tissues, and host organisms. In this chapter we present details on the use of the Shannon entropy method to estimate the site-specific nucleotide relative variability of turnip crinkle virus, a positive (+) stranded RNA plant virus, in a large dataset of short RNAs of Cicer arietinum L., a natural reservoir of the virus. We propose this method as a viral metagenomics tool to provide a more detailed description of the viral quasispecies in infected plant tissue. Viral replicative fitness relates to an optimal composition of variants that provide the molecular basis of virus behavior in the complex environment of natural infections. A complete description of viral quasispecies may have implications in determining fitness landscapes for host-virus coexistence and help to design specific diagnostic protocols and antiviral strategies.