In vivo detection, RNA-binding properties and characterization of the RNA-binding domain of the p7 putative movement protein from carnation mottle carmovirus (CarMV). (1/132)

Biochemical and structural characterization studies on the p7 putative movement protein from a Spanish isolate of carnation mottle carmovirus (CarMV) have been conducted. The CarMV p7 gene was fused to a sequence coding for a six-histidine tag and expressed in bacteria, allowing the purification of CarMV p7 and the production of a specific antiserum. This antiserum led to the immunological identification of CarMV p7 in infected leaf tissue from the experimental host Chenopodium quinoa. Putative nucleic acid-binding properties of the CarMV p7 have been explored and demonstrated with both electrophoretic mobility shift and RNA-protein blot in vitro assays using digoxigenin-labeled riboprobes. CarMV p7 did not show preferential binding to any of the different regions of the CarMV genomic RNA tested, suggesting that RNA binding was sequence nonspecific. Quantitative analyses of the data allowed calculation of the apparent dissociation constant of the p7-RNA complex (Kd approximately 0.7 microM) and supported a cooperative type of binding. A small 19-amino-acid synthetic peptide whose sequence corresponds to the putative RNA-binding domain of CarMV p7, at the basic central part of the protein, was synthesized, and it was demonstrated that it binds viral RNA probes. Peptide RNA binding was as stable as p7 binding, although data indicated it was not cooperative, thus suggesting that this cooperative binding requires another motif or motifs within the p7 amino acid sequence. The peptide could be induced to fold into an alpha-helix structure in which amino acids that are conserved among carmovirus p7-like proteins are distributed on one side. This alpha-helix motif could define a new and previously uncharacterized RNA-binding domain for plant virus movement proteins.  (+info)

Mutational analyses of the putative calcium binding site and hinge of the turnip crinkle virus coat protein. (2/132)

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 beta 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.  (+info)

RNA elements required for RNA recombination function as replication enhancers in vitro and in vivo in a plus-strand RNA virus. (3/132)

RNA replication requires cis-acting elements to recruit the viral RNA-dependent RNA polymerase (RdRp) and facilitate de novo initiation of complementary strand synthesis. Hairpins that are hot spots for recombination in the genomic RNA of turnip crinkle virus (TCV) and satellite (sat)-RNA C, a parasitic RNA associated with TCV infections, stimulate RNA synthesis 10-fold from a downstream promoter sequence in an in vitro assay using partially purified TCV RdRp. Artificial hairpins had an inhibitory effect on transcription. RNA accumulation in single cells was enhanced 5- to 10-fold when the natural stem-loop structures were inserted into a poorly accumulating sat-RNA. The effect of the stem-loop structures on RNA replication was additive, with insertion of three stem-loop RNA elements increasing sat-RNA accumulation to the greatest extent (25-fold). These stem-loop structures do not influence the stability of the RNAs in vivo, but may serve to recruit the RdRp to the template.  (+info)

Cap-independent translational enhancement of turnip crinkle virus genomic and subgenomic RNAs. (4/132)

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.  (+info)

Analysis of cis-acting sequences involved in plus-strand synthesis of a turnip crinkle virus-associated satellite RNA identifies a new carmovirus replication element. (5/132)

Satellite RNA C (satC) is a 356-base subviral RNA associated with turnip crinkle virus (TCV). A 3'-proximal element (3'-UCCCAAAGUAU) located 11 bases from the 3' terminus of satC minus strands can function as an independent promoter in an in vitro RNA-dependent RNA polymerase (RdRp) transcription system. Furthermore, in the absence of a 5'-proximal element, the 3'-proximal element is required for complementary strand synthesis in vitro. Site-directed mutagenesis was conducted to investigate the functional significance of this element and the 3' minus-strand terminal sequence "3'-OH-CCCUAU," which contains the minus-strand 3'-end sequence "3'-OH-CC(1-2)(A/U)(A/U)(A/U)" found in all carmovirus RNAs. Single mutations in the 3'-terminal sequence, which we have named the carmovirus consensus sequence (CCS), suppressed satC plus-strand synthesis to undetectable levels in protoplasts while still permitting some minus-strand synthesis. However, single and multiple mutations introduced into the 3'-proximal element had little or no effect on satC accumulation in protoplasts. In vivo genetic selection (SELEX) of the minus-strand 3'-terminal 21 bases revealed that all satC species accumulating in plants contained the 3' CCS. In addition, the 3'-proximal element preferentially contained a sequence similar to the CCS and/or polypurines, suggesting that this element may also contribute to accumulation of satC in vivo.  (+info)

Requirement of a 5'-proximal linear sequence on minus strands for plus-strand synthesis of a satellite RNA associated with turnip crinkle virus. (6/132)

Viral RNA replication begins with specific recognition of cis-acting RNA elements by the viral RNA-dependent RNA polymerase (RdRp) and/or associated host factors. A short RNA element (3'-AACCCCUGGGAGGC) located 41 bases from the 5' end of minus strands of satellite RNA C (satC), a 356-base subviral RNA naturally associated with turnip crinkle virus (TCV), was previously identified as important for plus-strand synthesis using an in vitro RdRp assay (H. Guan, C. Song, A. E. Simon, 1997, RNA 3, 1401-1412). To examine the functional significance of this element in RNA replication, mutations were introduced into the consecutive C residues in the element. A single mutation of the 3'-most C residue resulted in undetectable levels of satC plus strands when transcripts were assayed in protoplasts and suppressed transcription directed by the element in vitro. However, satC minus strands were detectable at 6 h postinoculation (hpi) of protoplasts, accumulating to about 10% of wild-type levels at 24 hpi. This mutation, when in the plus-sense orientation, had little or no effect on minus-strand synthesis from full-length satC plus strands in vitro, suggesting that the 5'-proximal RNA element is required for satC plus-strand synthesis. In addition, in vivo genetic selection revealed a strict requirement for 10 of the 14 nucleotides of the element, indicating that the primary sequence is essential for RNA accumulation.  (+info)

Complete nucleotide sequence and genome organization of hibiscus chlorotic ringspot virus, a new member of the genus Carmovirus: evidence for the presence and expression of two novel open reading frames. (7/132)

The complete nucleotide sequence of hibiscus chlorotic ringspot virus (HCRSV) was determined. The genomic RNA (gRNA) is 3,911 nucleotides long and has the potential to encode seven viral proteins in the order of 28 (p28), 23 (p23), 81 (p81), 8 (p8), 9 (p9), 38 (p38), and 25 (p25) kDa. Excluding two unique open reading frames (ORFs) encoding p23 and p25, the ORFs encode proteins with high amino acid similarity to those of carmoviruses. In addition to gRNA, two 3'-coterminated subgenomic RNA (sgRNA) species were identified. Full-length cDNA clones derived from gRNA and sgRNA were constructed under the control of a T7 promoter. Both capped and uncapped transcripts derived from the full-length genomic cDNA clone were infectious. In vitro translation and mutagenesis assays confirmed that all the predicted ORFs except the ORF encoding p8 are translatable, and the two novel ORFs (those encoding p23 and p25) may be functionally indispensable for the viral infection cycle. Based on virion morphology and genome organization, we propose that HCRSV be classified as a new member of the genus Carmovirus in family Tombusviridae.  (+info)

Members of the Arabidopsis HRT/RPP8 family of resistance genes confer resistance to both viral and oomycete pathogens. (8/132)

Turnip crinkle virus (TCV) inoculation onto TCV-resistant Arabidopsis leads to a hypersensitive response (HR) controlled by the dominant gene HRT. HRT is a member of the class of resistance (R) genes that contain a leucine zipper, a nucleotide binding site, and leucine-rich repeats. The chromosomal position of HRT and its homology to resistance gene RPP8 and two RPP8 homologs indicate that unequal crossing over and gene conversion may have contributed to HRT evolution. RPP8 confers resistance to an oomycete pathogen, Peronospora parasitica. Despite very strong similarities within the HRT/RPP8 family, HRT and RPP8 are specific for the respective pathogens they detect. Hence, the HRT/RPP8 family provides molecular evidence that sequence changes between closely related members of multigene families can generate novel specificities for radically different pathogens. Transgenic plants expressing HRT developed an HR but generally remained susceptible to TCV because of a second gene, RRT, that regulates resistance to TCV. However, several transgenic plants that overexpressed HRT produced micro-HRs or no HR when inoculated with TCV and were resistant to infection. Expression of the TCV coat protein gene in seedlings containing HRT resulted in massive necrosis and death, indicating that the avirulence factor detected by the HRT-encoded protein is the TCV coat protein.  (+info)