Turnip Crinkle Carmovirus Research Articles

Evidence for a Premature Termination Mechanism of Subgenomic mRNA Transcription in a Carmovirus

The transcriptional mechanism utilized by turnip crinkle carmovirus to synthesize subgenomic (sg) mRNAs was investigated by analyzing viral RNAs and their associated regulatory RNA elements. In vivo analyses revealed the following: (i) that minus-strand sg RNAs are detectable in infections, (ii) that minus-strand sg RNA accumulation can be partially uncoupled from that of their plus-strand sg mRNA counterparts, and (iii) that an RNA secondary structure located upstream of the sg mRNA start site mediates transcription by functioning in the plus strand of the viral genome. Collectively, these observations are consistent with this carmovirus using a premature termination mechanism for sg mRNA transcription.

A simplified method for cloning of short interfering RNAs from Brassica juncea infected with Turnip mosaic potyvirus and Turnip crinkle carmovirus

RNA silencing is a plant defense mechanism in which virus infected plants produce short interfering RNAs (siRNAs) derived from viral RNA, that attack the virus at the post-transcriptional level. In a previous study on Cymbidium ringspot tombusvirus (CymRSV) infection in Nicotiana benthamiana, siRNAs (determined by cloning and sequencing) predominantly originated from the sense (+) strand of the viral RNA, suggesting that the majority of siRNAs are produced through the direct cleavage of the virus single strand (ss) RNA by the plant Dicer-like enzyme. To test whether this asymmetry in strand polarity is a generic rule for all plant viruses, siRNAs from Brassica juncea, either singly infected by Turnip mosaic potyvirus (TuMV, the family Potyviridae), or doubly infected with TuMV and Turnip crinkle carmovirus (TCV, the family Tombusviridae) were investigated. A simplified siRNA cloning method was developed, using a single ligation reaction to attach both 5′ and 3′ adapters to the target short RNAs followed by one-step RT-PCR amplification. In the TCV infection, as for the CymRSV infection, siRNAs were produced predominantly (97.6%) from the +ss RNA. However, for TuMV infections, siRNAs were derived from both strands (+/−, 58.1–41.9%), indicating the presence of alternative siRNA production mechanisms.

Cap-independent translational enhancement of turnip crinkle virus genomic and subgenomic RNAs.

The presence of translational control elements and cap structures has not been carefully investigated for members of the Carmovirus genus, a group of small icosahedral plant viruses with positive-sense RNA genomes. In this study, we examined both the 5' and 3' untranslated regions (UTRs) of the turnip crinkle carmovirus (TCV) genomic RNA (4 kb) as well as the 5' UTR of the coat protein subgenomic RNA (1.45 kb) for their roles in translational regulation. All three UTRs enhanced translation of the firefly luciferase reporter gene to different extents. Optimal translational efficiency was achieved when mRNAs contained both 5' and 3' UTRs. The synergistic effect due to the 5'-3' cooperation was at least fourfold greater than the sum of the contributions of the individual UTRs. The observed translational enhancement of TCV mRNAs occurred in a cap-independent manner, a result consistent with the demonstration, using a cap-specific antibody, that the 5' end of the TCV genomic RNA was uncapped. Finally, the translational enhancement activity within the 5' UTR of 1.45-kb subgenomic RNA was shown to be important for the translation of coat protein in protoplasts and for virulent infection in Arabidopsis plants.

Mutational Analyses of the Putative Calcium Binding Site and Hinge of the Turnip Crinkle Virus Coat Protein

The turnip crinkle carmovirus (TCV) coat protein (CP) is folded into R (RNA-binding), S (shell), and P (protruding) domains. The S domain is an eight-stranded β barrel common to the coat protein subunits of most RNA viruses. A five-amino-acid hinge connects the S and P domains. In assembled particles, each pair of CP subunits is thought to bind a single calcium ion through interactions with three residues of one subunit and two residues of a neighboring subunit. These five residues comprise the putative calcium-binding site (CBS). The putative CBS and hinge are adjacent to one another. Mutations were introduced into the putative CBS or hinge in an effort to further determine the biological functions of TCV CP. One putative CBS mutant, TCV-M32, exhibited wild-type cell-to-cell movement but failed to move systemically in Nicotiana benthamiana, and particles were not detected. Another putative CBS mutant, TCV-M23, exhibited deficient cell-to-cell movement but particles accumulated in isolated protoplasts. Two other putative CBS mutants, TCV-M22 and -M33, showed wild-type cell-to-cell and systemic movement but elicited mild systemic symptoms that were somewhat delayed. All of the hinge mutants exhibited wild-type movement but some elicited non-wild-type symptoms. Point mutations in the putative CBS or hinge appear to alter virus–ion interactions, secondary structure, or particle conformation, thereby affecting interactions between the CP and plant hosts.

In VitroCharacterization of Late Steps of RNA Recombination in Turnip Crinkle Virus: I. Role of the Motif1-Hairpin Structure

Molecular mechanisms of RNA recombination were studied in turnip crinkle carmovirus (TCV), which has a uniquely high recombination frequency and nonrandom crossover site distribution among the recombining TCV-associated satellite RNAs. Anin vitrosystem has been developed that includes a partially purified TCV replicase preparation (RdRp) and chimeric RNAs that resemble the putativein vivorecombination intermediates (Nagy, P. D., Zhang, C., and Simon, A. E.EMBO J.17, 2392–2403, 1998). This system generates 3′-terminal extension products, which are analogous to the recombination end products. Efficient generation of 3′-terminal extension products depends on the presence of a hairpin structure (termed the motif1-hairpin) that possibly binds to the RdRp. Replacement of the motif1-hairpin with two separate randomized sequences resulted in a basal level of 3′-terminal extension. By using three separate constructs, each carrying similar mutations in the motif1-hairpin, we demonstrate that the role of the motif1-hairpin in 3′-terminal extension is complex and its function is influenced by flanking sequences. In addition to the mutagenesis approach, competition experiments between wild-type and mutated motif1-hairpin constructs suggest that the TCV RdRp likely recognizes the secondary and/or tertiary structure of the motif1-hairpin, while individual nucleotides play a less important role. Overall, the data shed new light into the mechanism of 3′-terminal extension by a viral RdRp that is analogous to the late steps of RNA recombination in TCV.

In VitroCharacterization of Late Steps of RNA Recombination in Turnip Crinkle Virus: II. The Role of the Priming Stem and Flanking Sequences

Turnip crinkle carmovirus (TCV) has a uniquely high recombination frequency and nonrandom cross-over site distribution among the recombining TCV-associated satellite RNAs. Anin vitrosystem has been developed that includes a partially purified TCV replicase preparation (RdRp) and chimeric RNAs that resemble the putativein vivorecombination intermediates (Nagy, P. D., Zhang, C., and Simon, A. E.,EMBO J.17, 2392–2403, 1998). This system mimics the strand transfer and primer extension steps of recombination events. We characterize in detail three RNA factors that, in addition to the previously characterized motif1-hairpin, can influence the efficient generation of 3′-terminal extension products: (i) a primer binding region, termed the priming stem; (ii) a spacer region; and (iii) a U-rich sequence located 5′ of the motif1-hairpin. The priming stem is formed between the acceptor RNA and the nascent RNA synthesized from the donor RNA template in the recombinants. The stability and location of the priming stem relative to the motif1-hairpin can influence both the efficiency and initiation site of 3′-terminal extension. A short flexible spacer region between the motif1-hairpin and the priming stem can increase the efficiency of 3′-terminal extensions. A U-rich sequence 5′ of the motif1-hairpin facilitates 3′-terminal extensions and its function partly overlaps with that of the spacer region. These RNA factors may also affect the late steps of RNA recombination in TCV.

Dissecting RNA recombination in vitro: role of RNA sequences and the viral replicase.

Molecular mechanisms of RNA recombination were studied in turnip crinkle carmovirus (TCV), which has a uniquely high recombination frequency and non-random crossover site distribution among the recombining TCV-associated satellite RNAs. To test the previously proposed replicase-driven template-switching mechanism for recombination, a partially purified TCV replicase preparation (RdRp) was programed with RNAs resembling the putative in vivo recombination intermediates. Analysis of the in vitro RdRp products revealed efficient generation of 3'-terminal extension products. Initiation of 3'-terminal extension occurred at or close to the base of a hairpin that was a recombination hotspot in vivo. Efficient generation of the 3'-terminal extension products depended on two factors: (i) a hairpin structure in the acceptor RNA region and (ii) a short base-paired region formed between the acceptor RNA and the nascent RNA synthesized from the donor RNA template. The hairpin structure bound to the RdRp, and thus is probably involved in its recruitment. The probable role of the base-paired region is to hold the 3' terminus near the RdRp bound to the hairpin structure to facilitate 3'-terminal extension. These regions were also required for in vivo RNA recombination between TCV-associated sat-RNA C and sat-RNA D, giving crucial and direct support for a replicase-driven template-switching mechanism of RNA recombination.

A novel 3'-end repair mechanism in an RNA virus.

Many positive-stranded RNA viruses contain short, single-stranded 3' ends that are vulnerable to degradation by host cellular RNases. Therefore, the existence of a 3'-end repair mechanism (analogous to cellular telomerases) must be required and/or advantageous for RNA viruses. Accordingly, we provide evidence suggesting that deletions of up to 6 nt from the 3' end of satellite (sat-) RNA C (a small parasitic RNA associated with turnip crinkle carmovirus) are repaired to the wild-type sequence in vivo and in vitro. The novel 3'-end repair mechanism involves the production of 4-8 nt oligoribonucleotides by abortive synthesis by the viral replicase using the 3' end of the viral genomic RNA as template. Based on our in vitro results, we postulate that the oligoribonucleotides are able to prime synthesis of wild-type negative-strand sat-RNA C in a reaction that does not require base pairing of the oligoribonucleotides to the mutant, positive-strand RNA template. The discovery of a 3'-end repair mechanism opens up new strategies for interfering with viral infections.

Several symptom-modulating mutations in the coat protein of turnip crinkle carmovirus result in particles with aberrant conformational properties

Particles of several symptom-modulating TCV coat protein (CP) mutants were pretreated at pH 5.5, 7.5 or 8.5 and their conformations compared by agarose gel electrophoresis to those of wild-type particles. Particles of two mutants were swollen under conditions in which wild-type particles remained contracted; particles of one mutant were contracted under conditions in which wild-type particles were swollen; a portion of the particles of one mutant was contracted and another portion swollen under conditions in which wild-type particles remained contracted; and particles of one mutant, that elicited wild-type symptoms, comigrated with wild-type particles under all conditions tested. The results of in vitro translation experiments with mutant particles were essentially similar to those with wild-type particles, despite conformational differences at pH 5.5 and 8.5. These results suggest that more than the swollen conformation is required for in vitro translation, and that particle conformation may play a role in symptom elicitation.

Cardamine Chlorotic Fleck Virus, a New Carmovirus From the Australian Alps.

A new carmovirus has been found in Cardamine lilacina in the Mt. Kosciusko alpine area of Australia. It causes chlorotic flecking of infected leaves, and we call it cardamine chlorotic fleck virus. It can be mechanically transmitted to C. lilacina, turnip (Brassica rapa) and candytuft (Iberis coronaria), and the latter is the best experimental host for propagating the virus. It has isometric particles 30 nm in diameter, and these contain a single-stranded RNA genome of approximately 4.1 kilobases. Partial sequencing of the genome has revealed regions with up to 82% nucleic acid homology with turnip crinkle carmovirus. However, no reaction was obtained between the particles of cardamine chlorotic fleck virus and an antiserum to turnip crinkle virus.

Use of Agarose Gel Electrophoresis to Monitor Conformational Changes of Some Small, Spherical Plant Viruses

Several small, spherical plant viruses undergo conformational changes (swell) under conditions that deprotonate carboxyl groups and/or remove divalent cations. In this study, swollen, ribonuclease A susceptible turnip crinkle carmovirus (TCV) and tomato bushy stunt tombusvirus (TBSV) particles migrated more slowly than contracted virions during agarose gel electrophoresis, and swollen TCV virions stained more intensely with ethidium bromide than did contracted virions (...)