Effect of template size on accumulation of defective interfering RNAs in protoplasts.

A turnip protoplast system has been used to study the effects of template size and sequence on the replication and/or stability of a small defective interfering (DI) RNA associated with turnip crinkle virus. Our results indicated that as little as a single base difference in the size of the molecule in some regions, rather than the specific sequence, affected the level of DI RNA accumulating in protoplasts.     

The virulent satellite RNA of turnip crinkle virus has a major domain homologous to the 3' end of the helper virus genome

RNA C (355 bases), RNA D (194 bases) and RNA F (230 bases) are small, linear satellite RNAs of turnip crinkle virus (TCV) which have been cloned as cDNAs and sequenced in this study. These RNAs produce dramatically different disease symptoms in infected plants. RNA C is a virulent satellite that intensifies virus symptoms when co-inoculated with its helper virus in turnip plants, while RNA D and RNA F are avirulent. RNA D and RNA F, the avirulent satellites, are closely related to each other except that RNA F has a 36-base insert near its 3' end, not found in RNA D. The 189 bases at the 5' end of RNA C, the virulent satellite, are homologous to the entire sequence of RNA D. However, the 3' half of RNA C, is composed of 166 bases which are nearly identical to two regions at the 3' end of the TCV helper virus genome. Hence, the virulent satellite is a composite molecule with one domain at its 5' end homologous to the other avirulent satellites and another domain at its 3' end homologous to the helper virus genome. All four TCV RNAs, RNAs C, D and F and the helper virus genome have identical 7 bases at their 3' ends. The secondary structure of RNA C deduced from the sequence can be folded into two separate domains — the domain of helper virus genome homology and the domain homologous to other TCV satellite RNAs. Comparative sequences of several different RNA C clones reveal that this satellite is a population of molecules with sequence and length heterogeneity.     

PROPERTIES AND HOST RANGE OF TURNIP CRINKLE, ROSETTE AND YELLOW MOSAIC VIRUSES

Three isolates of turnip yellow mosaic virus and two other flea-beetle transmitted viruses, turnip crinkle and turnip rosette, have many similar properties: thermal inactivation end-point between 80 and 90° C.; dilution end-point greater than 10^-4; longevity in vitro at about 20° C. at least 30 days. All were transmitted by mechanical inoculation to a wide range of cruciferous host plants, including many weeds. Turnip yellow mosaic virus infected only Reseda odorata outside the Cruciferae , whereas rosette virus infected a few and crinkle virus many non-cruciferous hosts.     

The complete nucleotide sequence of the maize chlorotic mottle virus genome.

The complete nucleotide sequence of the maize chlorotic mottle virus (MCMV) genome has been determined to be 4437 nucleotides. The viral genome has four long open reading frames (ORFs) which could encode polypeptides of 31.6, 50, 8.9 and 25.1 kd. If the termination codons, for the polypeptides encoded by the 50 and 8.9 kd ORFs are suppressed, readthrough products of 111 and 32.7 kd result. The 31.6 and 50 kd ORFs overlap for nearly the entire length of the 31.6 kd ORF. Striking amino acid homology has been observed between two potential polypeptides encoded by MCMV and polypeptides encoded by carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 25.1 kd ORF most likely encodes the capsid protein. The similar genome organization and amino acid sequence homology of MCMV with CarMV and TCV suggest an evolutionary relationship with these members of the carmovirus group.     

Encapsidation of turnip crinkle virus is defined by a specific packaging signal and RNA size.

A protoplast infection assay has been used to reliably examine the viral RNA encapsidation of turnip crinkle virus (TCV). Analysis of the encapsidation of various mutant viral RNAs revealed that a 186-nucleotide (nt) region at the 3' end of the coat protein (CP) gene, with a bulged hairpin loop of 28 nt as its most essential element, was indispensable for TCV RNA encapsidation. When RNA fragments containing the 186-nt region were used to replace the CP gene of a different virus, tomato bushy stunt virus, the resulting chimeric viral RNAs were encapsidated into TCV virions. Furthermore, analysis of the encapsidated chimeric RNA species established that the RNA size was an important determinant of the TCV assembly process.     

Internal structural anisotropy of spherical viruses studied with magnetic birefringence

Six so called spherical viruses (four plant and two animal) are shown to exhibit magnetically induced birefringence in solution. They must therefore be magnetically and optically anisotropic. This is attributed to static structural anisotropy of the interiors as neither natural shape nor field-induced deformations are likely causes. Thus at least part of these virus cores have a symmetry differing from that of their capsids. An estimate of the average orientation of the RNA bases is given for the plant viruses: turnip yellow mosaic, bromegrass mosaic, tomato bushy stunt and turnip crinkle. The packing geometry of the nucleic acid/protein cores of adenovirus and probably influenza virus are anisotropic but to an extent that cannot be quantified.     

A Single Amino Acid Change in Turnip Crinkle Virus Movement Protein p8 Affects RNA Binding and Virulence on Arabidopsis thaliana

Comparison of the symptoms caused by turnip crinkle virus strain M (TCV-M) and TCV-B infection of a resistant Arabidopsis thaliana line termed Di-17 demonstrates that TCV-B has a greater ability to spread in planta. This ability is due to a single amino acid change in the viral movement protein p8 and inversely correlates with p8 RNA binding affinity.     

Resolution of strawberry virus complexes; the isolation and some properties of virus 3

Aphids ( Capitophorus fragariae Theob.) allowed to feed for several days on a strawberry plant with severe crinkle transmitted two viruses. The isolation and properties of one (virus I) have already been described. The other (virus 3) was separated by transferring the aphids to fresh indicators after 24 hr.
Virus 3 was transmitted by aphids which had been allowed to feed on an infected plant for 6 days or more and persisted in the vector for several days. There was some evidence that the virus has a latent period in the vector. The symptoms produced by virus 3 on Fragaria vesca and Royal Sovereign strawberry are described.
On Royal Sovereign, viruses 1 and 3 together produced symptoms of severe crinkle and viruses z and 3 together produced yellow-edge. A form of severe crinkle is thus shown to be caused by a virus complex which can be resolved by means of the vector, and severe crinkle is shown to be etiologically distinct from mild crinkle.     

Natural Infection of Field-Grown Daikon Radish (Raphanus sativus L.) Crops by Turnip Crinkle Virus in North America

In 1993, stunted field-grown daikon radish plants were found with black lesions on the petioles and leaf blades, and new growth exhibited leaf crinkling and a mosaic pattern on distorted leaves. The plants had hollow roots and seed production was severely reduced. Symptomatic plants contained isometric virions identified as those of turnip crinkle virus (TCV). This is the first report of TCV causing serious economic damage and the first report of natural infection in North America.     

Structure and assembly of turnip crinkle virus. II. Mechanism of reassembly in vitro

Dissociation of turnip crinkle virus (TCV) at elevated pH and ionic strength produces free dimers of the coat protein and a ribonucleoprotein complex that contains the viral RNA, six coat-protein subunits, and the minor protein species, p80 (a covalently linked coat-protein dimer). This "rp-complex" is stable for several days in high salt at pH 8.5. Reassembly of TCV can be accomplished under physiological conditions, using isolated coat protein and either rp-complex or protein-free RNA. If rp-complex is used in reassembly, the same subunits remain bound to RNA on subsequent dissociation; if free RNA is used, rp-complex is regenerated. In both cases, the assembly is selective for viral RNA in competition experiments with heterologous RNA. Electron microscopy shows that assembly proceeds by continuous growth of a shell from an initiating structure, rather than by formation of distinct intermediates. We suggest that rp-complex is the initiating structure, suggest a model based on the organization of the TCV particle, and propose a mechanism for TCV assembly.     

Conjoined Use of EM and NMR in RNA Structure Refinement

More than 40% of the RNA structures have been determined using nuclear magnetic resonance (NMR) technique. NMR mainly provides local structural information of protons and works most effectively on relatively small biomacromolecules. Hence structural characterization of large RNAs can be difficult for NMR alone. Electron microscopy (EM) provides global shape information of macromolecules at nanometer resolution, which should be complementary to NMR for RNA structure determination. Here we developed a new energy term in Xplor-NIH against the density map obtained by EM. We conjointly used NMR and map restraints for the structure refinement of three RNA systems — U2/U6 small-nuclear RNA, genome-packing motif (Ψ^CD)₂ from Moloney murine leukemia virus, and ribosome-binding element from turnip crinkle virus. In all three systems, we showed that the incorporation of a map restraint, either experimental or generated from known PDB structure, greatly improves structural precision and accuracy. Importantly, our method does not rely on an initial model assembled from RNA duplexes, and allows full torsional freedom for each nucleotide in the torsion angle simulated annealing refinement. As increasing number of macromolecules can be characterized by both NMR and EM, the marriage between the two techniques would enable better characterization of RNA three-dimensional structures.     

R protein activation: another player revealed

Resistance proteins play an integral role in plant innate immunity by perceiving pathogens and triggering defense responses. In this issue of Cell Host & Microbe, Kang et al. uncover CRT1, an ATPase essential for resistance to turnip crinkle virus in Arabidopsis mediated by the Resistance (R) protein HRT. CRT1 interacts with an array of R proteins in vivo, suggesting that it plays a role in R protein activation.     

Structure of turnip crinkle virus. III. Identification of a unique coat protein dimer

The minor structural protein (p80), found in about one copy per virion in turnip crinkle virus (TCV), is shown by amino acid analysis and peptide mapping to be a covalent dimer of the major coat protein (p40). The covalent linkage occurs near the N termini of the crosslinked chains. These data suggest that TGV and related viruses contain 178 copies of p40 (89 non-covalent dimers) and one copy of p80 (covalent dimer of two additional p40 chains). The presence of p80 in the salt-stable RNA-protein complex formed when TCV dissociates, as described in an accompanying paper, indicates that the covalent modification affects binding to RNA. We suggest that p80 might be the final dimer to be incorporated into the shell and that it might also be the site for initiation of uncoating.     

Structure and assembly of turnip crinkle virus. VI. Identification of coat protein binding sites on the RNA

Structural studies of turnip crinkle virus have been extended to include the identification of high-affinity coat protein binding sites on the RNA genome. Virus was dissociated at elevated pH and ionic strength, and a ribonucleoprotein complex (rp-complex) was isolated by chromatography on Sephacryl S-200. Genomic RNA fragments in the rp-complex, resistant to RNase A and RNase T1 digestion and associated with tightly bound coat protein subunits, were isolated using coat-protein-specific antibodies. The identity of the protected fragments was determined by direct RNA sequencing. These approaches allowed us to study the specific RNA-protein interactions in the rp-complex obtained from dissociated virus particles. The location of one protected fragment downstream from the amber terminator codon in the first and largest of the three viral open reading frames suggests that the coat protein may play a role in the regulation of the expression of the polymerase gene. We have also identified an additional cluster of T1-protected fragments in the region of the coat protein gene that may represent further high-affinity sites involved in assembly recognition.     

Effects of turnip crinkle virus infection on the structure and function of mitochondria and expression of stress proteins in turnips

We have investigated the effect of turnip crinkle virus (TCV) infection on mitochondrial structure and function in turnips ( Brassica rapa cultivar Just Right ). TCV infection resulted in plants with small, mottled leaves with severely crinkled edges, and in a 46% reduction in storage root mass. TCV infection resulted in specific vesicularization of mitochondrial outer membranes where TCV replication is thought to occur, with no apparent affect on other cellular membrane systems. Immunoblot analysis of mitochondrial proteins from storage roots indicated that the TCV p28 protein, which is essential for viral replication, was associated with mitochondria and that mitochondrial heat shock protein 70 and cpn60 levels increased upon TCV infection. Isolation of mitochondrial outer membranes further showed TCV p28 protein enrichment in the outer membrane as compared with total mitochondrial proteins or total cellular proteins. Analysis of mitochondrial electron transport chain activities indicated that TCV infection resulted in a 54% decrease in exogenous NADH-dependent oxygen uptake and a 8% decrease in succinate-dependent oxygen uptake. Together these results indicate that TCV infection induces a stress response in mitochondria and a reduction in the ability of mitochondria to supply adenosine 5'-triphosphate to the cell.