A local, interactive network of 3' RNA elements supports translation and replication of Turnip crinkle virus

The majority of the 3' untranslated region (UTR) of Turnip crinkle virus (TCV) was previously identified as forming a highly interactive structure with a ribosome-binding tRNA-shaped structure (TSS) acting as a scaffold and undergoing a widespread conformational shift upon binding to RNA-dependent RNA polymerase (RdRp). Tertiary interactions in the region were explored by identifying two highly detrimental mutations within and adjacent to a hairpin H4 upstream of the TSS that reduce translation in vivo and cause identical structural changes in the loop of the 3' terminal hairpin Pr. Second-site changes that compensate for defects in translation/accumulation and reverse the structural differences in the Pr loop were found in the Pr stem, as well as in a specific stem within the TSS and within the capsid protein (CP) coding region, suggesting that the second-site changes were correcting a conformational defect and not restoring specific base pairing. The RdRp-mediated conformational shift extended upstream through this CP open reading frame (ORF) region after bypassing much of an intervening, largely unstructured region, supporting a connection between 3' elements and coding region elements. These data suggest that the Pr loop, TSS, and H4 are central elements in the regulation of translation and replication in TCV and allow for development of an RNA interactome that maps the higher-order structure of a postulated RNA domain within the 3' region of a plus-strand RNA virus.     

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.     

Structural Plasticity and Rapid Evolution in a Viral RNA Revealed by In Vivo Genetic Selection

Satellite RNAs usually lack substantial homology with their helper viruses. The 356-nucleotide satC of Turnip crinkle virus (TCV) is unusual in that its 3′-half shares high sequence similarity with the TCV 3′ end. Computer modeling, structure probing, and/or compensatory mutagenesis identified four hairpins and three pseudoknots in this TCV region that participate in replication and/or translation. Two hairpins and two pseudoknots have been confirmed as important for satC replication. One portion of the related 3′ end of satC that remains poorly characterized corresponds to juxtaposed TCV hairpins H4a and H4b and pseudoknot ψ₃, which are required for the TCV-specific requirement of translation (V. A. Stupina et al., RNA 14:2379-2393, 2008). Replacement of satC H4a with randomized sequence and scoring for fitness in plants by in vivo genetic selection (SELEX) resulted in winning sequences that contain an H4a-like stem-loop, which can have additional upstream sequence composing a portion of the stem. SELEX of the combined H4a and H4b region in satC generated three distinct groups of winning sequences. One group models into two stem-loops similar to H4a and H4b of TCV. However, the selected sequences in the other two groups model into single hairpins. Evolution of these single-hairpin SELEX winners in plants resulted in satC that can accumulate to wild-type (wt) levels in protoplasts but remain less fit in planta when competed against wt satC. These data indicate that two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts.     

The 3′ end of Turnip crinkle virus contains a highly interactive structure including a translational enhancer that is disrupted by binding to the RNA-dependent RNA polymerase

Precise temporal control is needed for RNA viral genomes to translate sufficient replication-required products before clearing ribosomes and initiating replication. A 3′ translational enhancer in Turnip crinkle virus (TCV) overlaps an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. The higher-order structure in the region was examined through alteration of critical sequences revealing novel interactions between an H-type pseudoknot and upstream residues, and between the TSS and internal and terminal loops of an upstream hairpin. Our results suggest that the TSS forms a stable scaffold that allows for simultaneous interactions with external sequences through base pairings on both sides of its large internal symmetrical loop. Binding of TCV RNA-dependent RNA polymerase (RdRp) to the region potentiates a widespread conformational shift with substantial rearrangement of the TSS region, including the element required for efficient ribosome binding. Degrading the RdRp caused the RNA to resume its original conformation, suggesting that the initial conformation is thermodynamically favored. These results suggest that the 3′ end of TCV folds into a compact, highly interactive structure allowing RdRp access to multiple elements including the 3′ end, which causes structural changes that potentiate the shift between translation and replication.     

Comparison of Turnip Crinkle Virus RNA-Dependent RNA Polymerase Preparations Expressed in Escherichia coli or Derived from Infected Plants

Turnip crinkle virus (TCV) is a small, plus-sense, single-stranded RNA virus of plants. A virus-coded protein, p88, which is required for replication has been expressed and purified from Escherichia coli. In vitro assays revealed that the recombinant p88 has an RNA-dependent RNA polymerase (RdRp) activity and can also bind to RNA. Deletion of the N-terminal region in p88 resulted in a more active RdRp, while further deletions abolished RdRp activity. Comparison of the E. coli-expressed p88, the N-terminal deletion mutant of p88, and a TCV RdRp preparation obtained from infected plants revealed that these preparations show remarkable similarities in RNA template recognition and usage. Both the recombinant and the plant TCV RdRp preparations are capable of de novo initiation on both plus- and minus-strand satC and satD templates, which are small parasitic RNAs associated with TCV infections. In addition, these RdRp preparations can efficiently recognize the related Tomato bushy stunt virus promoter sequences, including the minus- and plus-strand initiation promoters. Heterologous viral and artificial promoters are recognized poorly by the recombinant and the plant TCV RdRps. Further comparison of the single-component recombinant TCV RdRp and the multicomponent plant TCV RdRp will help dissect the functions of various components of the TCV replicase.     

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

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.     

A multifunctional turnip crinkle virus replication enhancer revealed by in vivo functional SELEX

The motif1-hairpin (M1H), located on (-)-strands of Turnip Crinkle Virus (TCV)-associated satellite RNA C (satC), is a replication enhancer and recombination hotspot. Results of in vivo genetic selection (SELEX: systematic evolution of ligands by exponential enrichment), where 28 bases of the M1H were randomized and then subjected to selection in plants, revealed that most winners contained one to three short motifs, many of which in their (-)-sense orientation are found in TCV and satC (-)-strand promoter elements. Ability to replicate in protoplasts correlated with fitness to accumulate in plants with one significant exception. Winner UC, containing only a seven-base replacement sequence, was the second most fit winner, yet replicated no better than a 28-base random replacement sequence. Fitness of satC containing different M1H replacement sequences could be due to enhanced satC replication or enhanced ability to affect TCV movement, since satC interferes with TCV virion accumulation, which is correlated with enhanced movement to younger tissue. Cells inoculated with TCV and UC accumulated fewer virions when compared to other winners that replicated better in protoplasts but were less fit in plants. UC, and other first and second round winners, contained structures that were on average 33% more stable in their (+)-strand orientation, and most formed hairpins with a A-rich sequence at the base. These results suggest that M1H replacement sequences contribute to the fitness of satC by either containing (-)-strand elements that enhance satRNA replication and/or a (+)-strand hairpin flanked with single-stranded sequence that enhances TCV movement.     

In vitro conversion of MVM parvovirus single-stranded DNA to the replicative form by DNA polymerase alpha from Ehrlich ascites tumour cells.

A partially purified preparation of DNA polymerase alpha, obtained from the cytosol of Ehrlich ascites tumour cells, has been found to catalyze the conversion of MVM parvovirus, SS DNA (5 kilobases) to RF in vitro. The reaction initiates at a natural 55 base pair hairpin which exists at the 3' terminus of MVM SS DNA. The SS leads to RF conversion is sensitive to aphidicolin, resistant to ddTTP and is promoted by purine ribonucleoside 5' triphosphates, a phenomenon which could not be explained simply by stabilization effects on the in vitro deoxynucleotide precursor pool. In the absence of rNTPs, nascent complementary strands frequently terminate prematurely at a preferred location, between 1300 and 1700 nucleotides from the initiating 3' hairpin terminus. This in vitro system, involving self-primed parvovirus DNA synthesis, provides a convenient assay for those components of the mammalian replicative DNA polymerase complex which are required for the elongation of nascent DNA chains.     

Magnesium ion-dependent activation of the RecA protein involves the C terminus

Optimal conditions for RecA protein-mediated DNA strand exchange include 6-8 mm Mg(2+) in excess of that required to form complexes with ATP. We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA protein C terminus that activates RecA-mediated DNA strand exchange. In particular, a "closed" (low Mg(2+)) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although single-stranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg(2+) results in an "open" conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure. The removal of 17 amino acid residues at the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg(2+) and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg(2+) levels. Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro. We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations.     

Alternative secondary structures in the 5' exon affect both forward and reverse self-splicing of the Tetrahymena intervening sequence RNA

The natural splice junction of the Tetrahymena large ribosomal RNA is flanked by hairpins that are phylogenetically conserved. The stem immediately preceding the splice junction involves nucleotides that also base pair with the internal guide sequence of the intervening sequence during splicing. Thus, precursors which contain wild-type exons can form two alternative helices. We have constructed a series of RNAs where the stem-loop in the 5' exon is more or less stable than in the wild-type precursor, and tested them in both forward and reverse self-splicing reactions. The presence of a stable hairpin in ligated exon substrates interferes with the ability of the intervening sequence to integrate at the splice junction. Similarly, the presence of the wild-type hairpin in the 5' exon reduces the rate of splicing 20-fold in short precursors. The data are consistent with a competition between unproductive formation of a hairpin in the 5' exon and productive pairing of the 5' exon with the internal guide sequence. The reduction of splicing by a hairpin that is a normal feature of rRNA structure is surprising; we propose that this attenuation is relieved in the natural splicing environment.