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 approximately 10-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.

Fig. 1.

Schematic structures of the TBSV genome and a viral replicon. (A) Linear representation of the TBSV RNA genome. Encoded viral proteins are depicted as boxes with their molecular masses (in thousands) prefixed by “p.” The 5′ UTR is delineated. Initiation sites for sg mRNA transcription are labeled sg1 and sg2, and corresponding structures of the two sg mRNAs are represented by arrows above the genome. (B) The TBSV-derived replicon shown is composed of four noncontiguous regions (I–IV) that correspond to different segments of the viral genome (delineated by vertical dotted lines). Note that region I in the replicon is the 5′ UTR from the viral genome. The horizontal line joining the four regions represents genomic segments that are not present in the replicon.

Fig. 2.

Designing and testing a ligand-inducible viral replicon. (A) RNA secondary structure of the 5′ UTR of the TBSV RNA genome (which also corresponds to region I of the replicon). The 5′-proximal T-shaped domain (TSD) and 3′-proximal downstream domain (DSD) are indicated. These domains are separated by SL5, which is outlined by shading. A tertiary interaction, PK-TD1, is indicted, and substitutions in Rep-TD1c are shown in the box. (B) Structure of theophylline (*, N7). (C) A theophylline-binding RNA aptamer (1). (D) The structures of putative RE riboswitches that were used to replace SL5 in Rep-TD1c are shown at the top. Sequence differences in the closing stems are boxed. Replicons were cotransfected with helper TBSV genome sg1T100 into plant-cell protoplasts, incubated in the absence (−) or presence (+) of 0.3 mM theophylline, and their accumulation levels quantified by Northern blot analysis at 22 h after cotransfection. The values at the bottom represent means that were normalized to that for uninduced Rep-TD1c (set at 100%) with standard deviations from three separate experiments.

Fig. 3.

Analysis of an RE riboswitch in a viral replicon. (A) The effect of increasing concentrations of ligand (theophylline or caffeine) on the relative levels of Rep-2A or Rep-2Am accumulation in plant-cell protoplasts at 22 h after cotransfection. (B) Modified RE riboswitch in Rep-2Am (the substitution is circled). (C) Induction of Rep-2A accumulation at various times after cotransfection. For both A and C, the graphed values represent means that were normalized to that for uninduced Rep-TD1c control (set at 100%) with standard deviations from three separate experiments. cont., control.

Fig. 4.

Screening and identification of an RE riboswitch for induction of TBSV genome (Gen) accumulation. (A) Structures of putative RE riboswitches that were introduced in place of SL5 in the TBSV genome PK-TD1cT. Sequence differences in the closing stems are boxed. RE-riboswitch-containing viral genomes were transfected into plant-cell protoplasts, incubated in the absence or presence of theophylline (0.5 mM), and accumulation levels were monitored 22 h after transfection by Northern blotting. Gen-65A (shaded) was identified as the most responsive to theophylline. (B) Dose-dependent theophylline induction of Gen-65A. Protoplasts were transfected with Gen-65A and incubated in the presence of different concentrations of theophylline. Gen-65A accumulation levels were quantified by Northern blot analysis at 22 h after transfection. The graphed values represent means that were normalized to that for the uninduced PK-TD1cT genome control (set at 100%) with standard deviations from three separate experiments. cont., control.

Fig. 5.

Analysis of viral RNA accumulation in Gen-65A infections. (A) A representative Northern blot showing accumulation of plus strands for TBSV viral RNAs. The concentration of theophylline that transfected plant-cell protoplasts were incubated in posttransfection is indicated at the top, and the positions of the genome (g) and sg mRNAs (sg1 and sg2) are indicated to the left. (B) A representative Northern blot showing accumulation of minus strands for TBSV viral RNAs, as described in A. (C) Schematic representation of TBSV genome replication and sg-mRNA transcription and the effect of disrupting SL5 RE activity. The TBSV genome is shown in the middle. Synthesis of progeny genomes, i.e., replication, proceeds via the synthesis of a full-length complementary minus-strand RNA of the genome that then serves as the template for progeny genome production (as shown in the upper half). In contrast, transcription of sg mRNAs involves premature termination of minus-strand synthesis followed by use of the 3′-truncated minus strand generated as a template for sg-mRNA transcription (as shown in the lower half). A defective SL5 RE (depicted by “X”) specifically inhibits the accumulation of progeny genomes, suggesting a defect in plus-strand genome synthesis.