Short internal sequences involved in replication and virion accumulation in a subviral RNA of turnip crinkle virus.
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cis-acting sequences and structural elements in untranslated regions of viral genomes allow viral RNA-dependent RNA polymerases to correctly initiate and transcribe asymmetric levels of plus and minus strands during replication of plus-sense RNA viruses. Such elements include promoters, enhancers, and transcriptional repressors that may require interactions with distal RNA sequences for function. We previously determined that a non-sequence-specific hairpin (M1H) in the interior of a subviral RNA (satC) associated with Turnip crinkle virus is required for fitness and that its function might be to bridge flanking sequences (X. Sun and A. E. Simon, J. Virol. 77:7880-7889, 2003). To establish the importance of the flanking sequences in replication and satC-specific virion repression, segments on both sides of M1H were randomized and subjected to in vivo functional selection (in vivo SELEX). Analyses of winning (functional) sequences revealed three different conserved elements within the segments that could be specifically assigned roles in replication, virion repression, or both. One of these elements was also implicated in the molecular switch that releases the 3' end from its interaction with the repressor hairpin H5, which is possibly involved in controlling the level of minus-strand synthesis. (+info)
The nuclear localization of the Arabidopsis transcription factor TIP is blocked by its interaction with the coat protein of Turnip crinkle virus.
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We have previously reported that TIP, an Arabidopsis protein, interacts with the coat protein (CP) of Turnip crinkle virus (TCV) in yeast cells and that this interaction correlated with the resistance response in the TCV-resistant Arabidopsis ecotype Dijon-17. TIP was also able to activate transcription of reporter genes in yeast cells, suggesting that it is likely a transcription factor. We have now verified the physical interaction between TIP and TCV CP in vitro and showed that CP mutants unable to interact with TIP in yeast cells bind TIP with much lower affinity in vitro. Secondly, we have performed gel shift experiments demonstrating that TIP does not bind to DNA in a sequence-specific manner. The subcellular localization of TIP was also investigated by transiently expressing green fluorescence protein (GFP)-tagged TIP in Nicotiana benthamiana plant cells, which showed that GFP-tagged TIP localizes primarily to nuclei. Significantly, co-expression of TCVCP and GFP-TIP prevented the nuclear localization of TIP. Together, these results suggest that TIP might be a transcription factor involved in regulating the defense response of Arabidopsis to TCV and that its normal role is compromised by interaction with the invading viral CP. (+info)
Importance of sequence and structural elements within a viral replication repressor.
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Efficient replication of plus-strand RNA viruses requires a 3' proximal core promoter and an increasingly diverse inventory of supporting elements such as enhancers, repressors, and 5' terminal sequences. While core promoters have been well characterized, much less is known about structure-functional relationships of these supporting elements. Members of the genus Carmovirus family Tombusviridae contain a hairpin (H5) proximal to the core promoter that functions as a repressor of minus-strand synthesis in vitro through an interaction between its large symmetrical internal loop (LSL) and 3' terminal bases. Turnip crinkle virus satellite RNA satC with the H5 of carmovirus Japanese iris necrosis virus or Cardamine chlorotic fleck virus (CCFV) did not accumulate to detectable levels even though 3' end base-pairing would be maintained. Replacement of portions of the satC H5 with analogous portions from CCFV revealed that the cognate LSL and lower stem were of greater importance for satC accumulation than the upper stem. In vivo selex of the H5 upper stem and terminal GNRA tetraloop revealed considerable plasticity in the upper stem, including the presence of three- to six-base terminal loops, allowed for H5 function. In vivo selex of the lower stem revealed that both a stable stem and specific base pairs contributed to satC fitness. Surprisingly, mutations in H5 had a disproportionate effect on plus-strand accumulation that was unrelated to the stability of the mutant plus-strands. In addition, fitness to accumulate in plants did not always correlate with enhanced ability to accumulate in protoplasts, suggesting that H5 may be multifunctional. (+info)
Double-spanning plant viral movement protein integration into the endoplasmic reticulum membrane is signal recognition particle-dependent, translocon-mediated, and concerted.
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The current model for cell-to-cell movement of plant viruses holds that transport requires virus-encoded movement proteins that intimately associate with endoplasmic reticulum membranes. We have examined the early stages of the integration into endoplasmic reticulum membranes of a double-spanning viral movement protein using photocross-linking. We have discovered that this process is cotranslational and proceeds in a signal recognition particle-dependent manner. In addition, nascent chain photocross-linking to Sec61alpha and translocating chain-associated membrane protein reveal that viral membrane protein insertion takes place via the translocon, as with most eukaryotic membrane proteins, but that the two transmembrane segments of the viral protein leave the translocon and enter the lipid bilayer together. (+info)
Heterologous RNA replication enhancer stimulates in vitro RNA synthesis and template-switching by the carmovirus, but not by the tombusvirus, RNA-dependent RNA polymerase: implication for modular evolution of RNA viruses.
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The viral RNA plays multiple roles during replication of RNA viruses, serving as a template for complementary RNA synthesis and facilitating the assembly of the viral replicase complex. These roles are coordinated by cis-acting regulatory elements, such as promoters and replication enhancers (REN). To test if these RNA elements can be used by related viral RNA-dependent RNA polymerases (RdRp), we compared the potential stimulatory effects of homologous and heterologous REN elements on complementary RNA synthesis and template-switching by the tombus- (Cucumber necrosis virus, CNV), carmovirus (Turnip crinkle virus, TCV) and hepatitis C virus (HCV) RdRps in vitro. The CNV RdRp selectively utilized its cognate REN, while discriminating against the heterologous TCV REN. On the contrary, RNA synthesis by the TCV RdRp was stimulated by the TCV REN and the heterologous tombusvirus REN with comparable efficiency. The heterologous REN elements also promoted in vitro template-switching by the TCV and HCV RdRps. Based on these observations, we propose that REN elements could facilitate intervirus recombination and post-recombinational amplification of new recombinant viruses. (+info)
Conformational changes involved in initiation of minus-strand synthesis of a virus-associated RNA.
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Synthesis of wild-type levels of turnip crinkle virus (TCV)-associated satC complementary strands by purified, recombinant TCV RNA-dependent RNA polymerase (RdRp) in vitro was previously determined to require 3' end pairing to the large symmetrical internal loop of a phylogenetically conserved hairpin (H5) located upstream from the hairpin core promoter. However, wild-type satC transcripts, which fold into a single detectable conformation in vitro as determined by temperature-gradient gel electrophoresis, do not contain either the phylogenetically inferred H5 structure or the 3' end/H5 interaction. This implies that conformational changes are required to produce the phylogenetically inferred H5 structure for its pairing with the 3' end, which takes place subsequent to the initial conformation assumed by the RNA and prior to transcription initiation. The DR region, located 140 nucleotides upstream from the 3' end and previously determined to be important for transcription in vitro and replication in vivo, is proposed to have a role in the conformational switch, since stabilizing the phylogenetically inferred H5 structure decreases the negative effects of a DR mutation in vivo. In addition, high levels of aberrant transcription correlate with a specific conformational change in the Pr while maintaining the same conformation of the 3' terminus. These results suggest that a series of events that promote conformational changes is needed to expose the 3' terminus to the RdRp for accurate synthesis of wild-type levels of complementary strands in vitro. (+info)
Host-induced avirulence of hibiscus chlorotic ringspot virus mutants correlates with reduced gene-silencing suppression activity.
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Post-transcriptional gene silencing (PTGS) and virus-encoded gene-silencing suppressors are defence and counterdefence strategies developed by host and pathogens during evolution. Using a green fluorescence protein-based transient suppression system, the coat protein (CP) of Hibiscus chlorotic ringspot virus (HCRSV) was identified as a strong gene-silencing suppressor. CP suppressed sense RNA-induced but not dsRNA-induced local and systemic PTGS. This is different from another virus in the genus Carmovirus, Turnip crinkle virus (TCV), the CP of which strongly suppresses dsRNA-induced PTGS. HCRSV CP domain deletion mutants lost their suppression function, indicating that the complete CP is essential for suppression of PTGS. When CP was expressed from a Potato virus X (PVX) vector, it was able to enhance the symptom severity and to increase the accumulation of PVX RNA. Here, it is proposed that HCRSV CP suppresses PTGS at the initiation step, which is different from TCV CP. In addition, a previous study demonstrated that CP mutants resulting from serial passage of HCRSV in its local lesion host also showed a significantly reduced suppression function, indicating that host-induced mutations that lead to avirulence of HCRSV in kenaf correlate with its reduced ability to suppress PTGS. (+info)
Analyses of subgenomic promoters of Hibiscus chlorotic ringspot virus and demonstration of 5' untranslated region and 3'-terminal sequences functioning as subgenomic promoters.
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Hibiscus chlorotic ringspot virus (HCRSV), which belongs to the genus Carmovirus, generates two 3'-coterminal subgenomic RNAs (sgRNAs) of 1.4 kb and 1.7 kb. Transcription start sites of the two sgRNAs were identified at nucleotides (nt) 2178 and 2438, respectively. The full promoter of sgRNA1, a 118-base sequence, is localized between positions +6 and -112 relative to its transcription start site (+1). Similarly, a 132-base sequence, from +6 to -126, defines the sgRNA2 promoter. Computer analysis revealed that both sgRNA promoters share a similar two-stem-loop (SL1 + SL2) structure, immediately upstream of the transcription start site. Mutational analysis of the primary sequence and secondary structures showed further similarities between the two subgenomic promoters. The basal portion of SL2, encompassing the transcription start site, was essential for transcription activity in each promoter, while SL1 and the upper portion of SL2 played a role in transcription enhancement. Both the 5' untranslated region (UTR) and the last 87 nt at the 3' UTR of HCRSV genomic RNA are likely to be the putative genomic plus-strand and minus-strand promoters, respectively. They function well as individual sgRNA promoters to produce ectopic subgenomic RNAs in vivo but not to the same levels of the actual sgRNA promoters. This suggests that HCRSV sgRNA promoters share common features with the promoters for genomic plus-strand and minus-strand RNA synthesis. To our knowledge, this is the first demonstration that both the 5' UTR and part of the 3' UTR can be duplicated and function as sgRNA promoters within a single viral genome. (+info)