Biomedical subjects

Jiuchun Zhang

Publications and source records attributed to Jiuchun Zhang.

6 recordsLinked to original sources

PubMed2006

Evolution of virus-derived sequences for high-level replication of a subviral RNA.

Turnip crinkle virus (TCV) and its 356-nt satellite RNA satC share 151 nt of 3'-terminal sequence, which contain 8 positional differences and are predicted to fold into virtually identical structures, including a series of four phylogenetically inferred hairpins. SatC and TCV containing reciprocal exchanges of this region accumulate to only 15% or 1% of wild-type levels, respectively. Step-wise conversion of satC and TCV 3'-terminal sequences into the counterpart's sequence revealed the importance of having the cognate core promoter (Pr), which is composed of a single hairpin that differs in both sequence and stability, and an adjacent short 3'-terminal segment. The negative impact of the more stable TCV Pr on satC could not be attributed to lack of formation of a known tertiary interaction involving the 3'-terminal bases, nor an effect of coat protein, which binds specifically to TCV-like Pr and not the satC Pr. The satC Pr was a substantially better promoter than the TCV Pr when assayed in vitro using purified recombinant TCV RdRp, either in the context of satC or when assayed downstream of non-TCV-related sequence. Poor activity of the TCV Pr in vitro occurred despite solution structure probing indicating that its conformation in the context of satC is similar to the active form of the satC Pr, which is thought to form following a required conformational switch. These results suggest that evolution of satC following its initial formation generated a Pr that can function more efficiently in the absence of additional TCV sequence that may be required for full functionality of the TCV Pr.

Base Sequence↗

PubMed2006

A pseudoknot in a preactive form of a viral RNA is part of a structural switch activating minus-strand synthesis.

RNA can adopt different conformations in response to changes in the metabolic status of cells, which can regulate processes such as transcription, translation, and RNA cleavage. We previously proposed that an RNA conformational switch in an untranslated satellite RNA (satC) of Turnip crinkle virus (TCV) regulates initiation of minus-strand synthesis (G. Zhang, J. Zhang, A. T. George, T. Baumstark, and A. E. Simon, RNA 12:147-162, 2006). This model was based on the lack of phylogenetically inferred hairpins or a known pseudoknot in the "preactive" structure assumed by satC transcripts in vitro. We now provide evidence that a second pseudoknot (Psi(2)), whose disruption reduces satC accumulation in vivo and enhances transcription by the TCV RNA-dependent RNA polymerase in vitro, stabilizes the preactive satC structure. Alteration of either Psi(2) partner caused nearly identical structural changes, including single-stranded-specific cleavages in the pseudoknot sequences and strong cleavages in a distal element previously proposed to mediate the conformational switch. These results indicate that the preactive structure identified in vitro has biological relevance in vivo and support a requirement for this alternative structure and a conformational switch in high-level accumulation of satC in vivo.

Base Sequence↗

PubMed2005

Conformational changes involved in initiation of minus-strand synthesis of a virus-associated RNA.

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.

Base Sequence↗

PubMed2005

Importance of sequence and structural elements within a viral replication repressor.

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.

Base Sequence↗

PubMed2004

Analysis of a viral replication repressor: sequence requirements for a large symmetrical internal loop.

Nearly all members of the Carmovirus genus contain a structurally conserved 3' proximal hairpin (H5) with a large internal symmetrical loop (LSL). H5 has been identified as a repressor of minus-strand synthesis in a satellite RNA (satC), which shares partial sequence similarity with its helper virus Turnip crinkle virus (TCV). Repression was due to sequestration of the 3' end mediated by base pairing between 3' end sequence and the 3' side of the LSL (G. Zhang, J. Zhang and A. E. Simon, J. Virol., in press). Single site mutational analysis and in vivo genetic selection (SELEX) of the 14 base satC H5 LSL indicated specific sequences in the middle and upper regions on both sides of the LSL are necessary for robust satC accumulation in plants and protoplasts. Fitness of wild-type satC and satC LSL mutants to accumulate in plants, however, did not necessarily correlate with the ability of these RNAs to replicate in protoplasts. This suggests that the LSL might be involved in processes in addition to repression of minus-strand synthesis.

3' Untranslated Regions↗

PubMed2004

Repression and derepression of minus-strand synthesis in a plus-strand RNA virus replicon.

Plus-strand viral RNAs contain sequences and structural elements that allow cognate RNA-dependent RNA polymerases (RdRp) to correctly initiate and transcribe asymmetric levels of plus and minus strands during RNA replication. cis-acting sequences involved in minus-strand synthesis, including promoters, enhancers, and, recently, transcriptional repressors (J. Pogany, M. R. Fabian, K. A. White, and P. D. Nagy, EMBO J. 22:5602-5611, 2003), have been identified for many viruses. A second example of a transcriptional repressor has been discovered in satC, a replicon associated with turnip crinkle virus. satC hairpin 5 (H5), located proximal to the core hairpin promoter, contains a large symmetrical internal loop (LSL) with sequence complementary to 3'-terminal bases. Deletion of satC 3'-terminal bases or alteration of the putative interacting bases enhanced transcription in vitro, while compensatory exchanges between the LSL and 3' end restored near-normal transcription. Solution structure analysis indicated that substantial alteration of the satC H5 region occurs when the three 3'-terminal cytidylates are deleted. These results indicate that H5 functions to suppress synthesis of minus strands by sequestering the 3' terminus from the RdRp. Alteration of a second sequence strongly repressed transcription in vitro and accumulation in vivo, suggesting that this sequence may function as a derepressor to free the 3' end from interaction with H5. Hairpins with similar sequence and/or structural features that contain sequence complementary to 3'-terminal bases, as well as sequences that could function as derepressors, are located in similar regions in other carmoviruses, suggesting a general mechanism for controlling minus-strand synthesis in the genus.

Base Sequence↗