Covariation in the capsid protein of hibiscus chlorotic ringspot virus induced by serial passaging in a host that restricts movement leads to avirulence in its systemic host.
(25/132)Hibiscus chlorotic ringspot virus (HCRSV) from naturally infected Hibiscus rosa-sinensis L. loses virulence in its experimental systemic host Hibiscus cannabinus L. (kenaf) after serial passages in a local lesion host Chenopodium quinoa. Here we report the genetic changes responsible for the loss of virulence at the molecular level. A remarkable covariation of eight site-specific amino acids was found in the HCRSV capsid protein (CP) after serial passages in C. quinoa: Val(49)-->Ile, Ile(95)-->Val, Lys(270)-->Arg, Gly(272)-->Asp, Tyr(274)-->His, Ala(311)-->Asp, Asp(334)-->Ala, and Ala(335)-->Thr. Covariation of at least three of the eight amino acids, Val(49), Ile(95), and Lys(270), caused the virus to become avirulent in kenaf. Interestingly, the nature of the covariation was consistent and reproducible at each serial passage. These data indicate that the nonsynonymous substitutions of amino acids in the HCRSV CP after serial passages in C. quinoa are not likely to be random events but may be due to host-associated positive selection or accelerated genetic drift. The observed interdependence among the three amino acids leading to avirulence in kenaf may have implications for structural or functional relationships in this virus-host interaction. (+info)
The coat protein of turnip crinkle virus suppresses posttranscriptional gene silencing at an early initiation step.
(26/132)Posttranscriptional gene silencing (PTGS), or RNA silencing, is a sequence-specific RNA degradation process that targets foreign RNA, including viral and transposon RNA for destruction. Several RNA plant viruses have been shown to encode suppressors of PTGS in order to survive this host defense. We report here that the coat protein (CP) of Turnip crinkle virus (TCV) strongly suppresses PTGS. The Agrobacterium infiltration system was used to demonstrate that TCV CP suppressed the local PTGS as strongly as several previously reported virus-coded suppressors and that the action of TCV CP eliminated the small interfering RNAs associated with PTGS. We have also shown that the TCV CP must be present at the time of silencing initiation to be an effective suppressor. TCV CP was able to suppress PTGS induced by sense, antisense, and double-stranded RNAs, and it prevented systemic silencing. These data suggest that TCV CP functions to suppress RNA silencing at an early initiation step, likely by interfering the function of the Dicer-like RNase in plants. (+info)
Spatio-temporal analysis of the RNAs, coat and movement (p7) proteins of Carnation mottle virus in Chenopodium quinoa plants.
(27/132)Time-course and in situ hybridization analyses were used to study the spatio-temporal distribution of Carnation mottle virus (CarMV) in Chenopodium quinoa plants. Genomic and subgenomic RNAs of plus polarity accumulated linearly with time, whereas the corresponding minus strands reached a peak during infection in inoculated leaves. Analyses of serial tissue sections showed that plus polarity strands were localized throughout the infection area, whereas minus strands were localized at the borders of the chlorotic lesions. The accumulation kinetics of the coat protein (CP) and the p7 movement protein (MP) as well as their subcellular localization were also studied. Unlike most MPs, CarMV p7 showed a non-transient expression and a mainly cytosolic location. However, as infection progressed the presence of p7 in the cell wall fraction increased significantly. These results are discussed on the basis of a recent model proposed for the mechanism of cell-to-cell movement operating in the genus Carmovirus. (+info)
Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana.
(28/132)All of the protein products of Turnip crinkle virus (TCV; Tombusviridae, Carmovirus) were tested for their ability to suppress RNA silencing of a reporter gene after transient expression in Agrobacterium-infiltrated Nicotiana benthamiana leaves. Only the capsid protein, P38, showed suppression activity, although this was not obvious when P38 was expressed as part of a TCV infection of the same tissues. When P38 was expressed from a PVX vector, symptoms with enhanced severity that correlated with increased PVX RNA accumulation were observed. This contradiction between ectopic expression of P38 and TCV infection could be accounted for if the active determinant of suppressor activity within P38 was sequestered within the capsid protein structure. The N-terminal 25 amino acids were shown to be important for this activity. This region forms part of the unexposed R-domain that interacts with the RNA within the virus particle. This observation throws light on some of the complex biology exhibited by TCV. (+info)
Synergism of the 3'-untranslated region and an internal ribosome entry site differentially enhances the translation of a plant virus coat protein.
(29/132)The use of internal ribosome entry sites (IRESs) is one of the unorthodox mechanisms exploited by viruses to initiate the translation of internal genes. Herein, we report a plant virus exploiting an IRES and its 3'-untranslated region (UTR) to express its internal genes, notably the 3'-proximal viral coat protein gene. Hibiscus chlorotic ringspot virus (HCRSV), a positive-strand non-polyadenylated RNA virus, was demonstrated to harbor a unique 100-nucleotide (nt) IRES, located 124 nt upstream of the coat protein gene, that could function in wheat germ extract, rabbit reticulocyte lysate, and mammalian cells. In comparison with other known IRESs of picornaviruses and eukaryotic mRNAs, this 100-nt IRES is distinctively short and simple. The IRES activity was tested in homologous and heterologous bicistronic constructs, and the expression of the 3'-proximal gene was enhanced when the 3'-UTR was present. When the IRES element was bisected, each half still possessed IRES activity and could initiate internal translation on its own. Site-directed mutagenesis and deletion analyses revealed that the primary sequence within the 5' half was crucial for IRES activity, whereas the primary sequence of the second half and a GNRA motif were non-essential. To our knowledge, this is the first report describing a mechanism whereby an IRES, located in the 3' portion of the virus genome, co-operates with the 3'-UTR to enhance gene expression differentially. (+info)
Fitness of a turnip crinkle virus satellite RNA correlates with a sequence-nonspecific hairpin and flanking sequences that enhance replication and repress the accumulation of virions.
(30/132)satC, a satellite RNA associated with Turnip crinkle virus (TCV), enhances the ability of the virus to colonize plants by interfering with stable virion accumulation (F. Zhang and A. E. Simon, unpublished data). Previous results suggested that the motif1-hairpin (M1H), a replication enhancer on minus strands, forms a plus-strand hairpin flanked by CA-rich sequence that may be involved in enhancing systemic infection (G. Zhang and A. E. Simon, J. Mol. Biol. 326:35-48, 2003). In this study, sequence and structural requirements of the M1H were further assayed by replacing the 28-base M1H with 10 random bases and then subjecting the pool of satellite RNA to functional selection in plants. Unlike previous results with 28-base replacement sequences (G. Zhang and A. E. Simon, J. Mol. Biol. 326:35-48, 2003), only a few of the 10-base SELEX (systematic evolution of ligands by exponential enrichment) assay winners contained short motifs in their minus-sense orientation that were similar to TCV replication elements. However, all second- and third-round winning replacement sequences folded into hairpins flanked by CA-rich sequence predicted to be more stable on plus strands than minus strands. Plus strands of several of the most fit satellite RNAs contained insertions of CA-rich sequence at the base of their hairpins whose presence correlated with enhanced replication and reduced detection of virions. Deletion of the M1H resulted in no detectable virions despite very low satellite accumulation. These results support the hypothesis that a sequence-nonspecific plus-strand hairpin brings together flanking CA-rich sequences in the M1H region that confers fitness to satC by reducing the accumulation of stable virions. (+info)
Mechanism of RNA recombination in carmo- and tombusviruses: evidence for template switching by the RNA-dependent RNA polymerase in vitro.
(31/132)RNA recombination occurs frequently during replication of tombusviruses and carmoviruses, which are related small plus-sense RNA viruses of plants. The most common recombinants generated by these viruses are either defective interfering (DI) RNAs or chimeric satellite RNAs, which are thought to be generated by template switching of the viral RNA-dependent RNA polymerase (RdRp) during the viral replication process. To test if RNA recombination is mediated by the viral RdRp, we used either a purified recombinant RdRp of Turnip crinkle carmovirus or a partially purified RdRp preparation of Cucumber necrosis tombusvirus. We demonstrated that these RdRp preparations generated RNA recombinants in vitro. The RdRp-driven template switching events occurred between either identical templates or two different RNA templates. The template containing a replication enhancer recombined more efficiently than templates containing artificial sequences. We also observed that AU-rich sequences promote recombination more efficiently than GC-rich sequences. Cloning and sequencing of the generated recombinants revealed that the junction sites were located frequently at the ends of the templates (end-to-end template switching). We also found several recombinants that were generated by template switching involving internal positions in the RNA templates. In contrast, RNA ligation-based RNA recombination was not detected in vitro. Demonstration of the ability of carmo- and tombusvirus RdRps to switch RNA templates in vitro supports the copy-choice models of RNA recombination and DI RNA formation for these viruses. (+info)
Cell-to-Cell, but not long-distance, spread of RNA silencing that is induced in individual epidermal cells.
(32/132)A Turnip crinkle virus (TCV)-based system was devised to discriminate cell-to-cell and systemic long-distance spread of RNA silencing in plants. Modified TCV-GFPDeltaCP, constructed by replacing the coat protein (CP) gene with the green fluorescent protein (GFP) gene, replicated in single epidermal cells but failed to move from cell to cell in Nicotiana benthamiana. Mechanical inoculation of TCV-GFPDeltaCP induced effective RNA silencing in single epidermal cells which spread from cell to cell to form silenced foci on inoculated leaves, but no long-distance systemic spread of RNA silencing occurred. Agroinfiltration of TCV-GFPDeltaCP was, however, able to induce both local and systemic RNA silencing. TCV coinfection arrested TCV-GFPDeltaCP-mediated local induction of RNA silencing. Possible mechanisms involved in cell-to-cell and long-distance spread of RNA silencing are discussed. (+info)