Riboswitching on RNA virus replication

Abstract

Positive-strand RNA viruses direct different virus-specific processes during their infection of host cells. Fundamental events such as viral RNA genome replication are controlled by viral regulatory RNA elements (REs). Here, we have investigated the possibility of specifically modulating the action of a viral RE using RNA aptamer technology. Through rational design, a tombusvirus RE, which has the structure of a perfect RNA stem loop in the plus-strand RNA genome, was replaced with a theophylline-binding RNA aptamer sequence, an imperfect stem loop. The aptamer-RE hybrid was designed so that, upon binding theophylline, it would become more stable and structurally mimic the functional RE (i.e., represent a ligand-inducible RE riboswitch). Initial experiments were conducted with a small noncoding virus genome-derived RNA replicon, and the results showed that replication was inducible, up to Ϸ10-fold, in a theophylline-specific and dose-dependent manner. A similar level of theophylline-dependent induction was also observed when a full-length viral genome containing an RE riboswitch was tested. Analysis of this engineered viral genome revealed that this RE, located in the 5 untranslated region, specifically mediates efficient accumulation of plus-strands of the virus genome. Therefore, in addition to allowing for modulation of virus reproduction, the RE riboswitch system also provided insight into RE function. The ability to chemically induce a viral process via modulation of virus genome structure could be useful for basic and applied aspects of research.aptamer ͉ gene regulation ͉ riboswitch ͉ RNA structure ͉ tombusvirus R NA aptamers are RNA molecules that are capable of specifically binding to cognate ligands. Small moleculebinding RNA aptamers were first identified through artificial selection by using systematic evolution of ligands by exponential enrichment (i.e., SELEX) (1). Surprisingly, some of these RNA aptamers showed favorable binding affinities and high levels of specificity (1). These observations suggested that natural RNA aptamers might exist and function in nature. Indeed, naturally occurring RNA aptamers, present as components of riboswitches, were subsequently identified in bacteria and shown to be involved in regulating translation or transcription of cellular mRNA (2). These riboswitches contain two mutually exclusive structures, one functional and the other nonfunctional, and ligand binding to the aptamer domain within the riboswitch acts to stabilize one of the two structures (2). In this way, the presence or absence of the ligand dictates which RNA structure dominates and, thus, the activity of the functional RNA module.Engineered RNA-based regulation of gene expression in living cells is now a rapidly expanding area of biotechnology. RNA aptamers that bind to small-molecule ligands (e.g., theophylline) have been instrumental in the development of novel systems that allow for ligand-dependent regulation of mRNA translational activity (3-10), ribozyme activity (10, 11), or other cellular proc...

A Hepatitis C Virus cis -Acting Replication Element Forms a Long-Range RNA-RNA Interaction with Upstream RNA Sequences in NS5B

“…At least on the input genome, before a pool of progeny genomes have been generated, these are mutually exclusive processes. In certain examples, additional functions ascribed to the RNA genome include subversion of the innate immune response, temporal and spatial control of the replication process, and encapsidation (12,28,35,37). Functional specificity is provided by the evolutionary conservation of binding determinants, often in a structural context.…”

Higher-Order RNA Structural Requirements and Small-Molecule Induction of Tombusvirus Subgenomic mRNA Transcription

“…In a previous study, we showed that the activity of a viral SL structure, termed SL5 (representing an essential viral RNA replication element) could be modulated exogenously by replacing it with a small-molecule-binding RNA aptamer (17). This was accomplished by replacing SL5 (a perfect RNA hairpin), located in the 5Ј untranslated region of the TBSV genome, with a modified theophylline-binding aptamer, which is an imperfect RNA hairpin containing internal loops.…”

Rational design of artificial riboswitches based on ligand-dependent modulation of internal ribosome entry in wheat germ extract and their applications as label-free biosensors

“…If the PSIV IRES does not function well in a certain eukaryote, other types of IRES whose activity is high in that organism and whose secondary structures are known (such as loops and/or pseudoknots required for efficient translation) could be used as the target of an aIRES to construct similar IRES-based riboswitches with the same procedure as for the PSIV IRES-based riboswitches. These riboswitches using the IRES originally present in various viruses are also useful for research regarding such viruses (Wang and White 2007). In vivo applications of the IRESbased riboswitches are now in progress.…”