The RNA replication enhancer element of tombusviruses contains two interchangeable hairpins that are functional during plus-strand synthesis

Abstract

Replication of the RNA genomes of tombusviruses, which are small plus-sense RNA viruses of plants, may be regulated by cis-acting elements, including promoters and replication enhancers that are present in the RNA templates. Using a partially purified RNA-dependent RNA polymerase (RdRp) preparation (P. D. Nagy and J. Pogany, Virology 276:279-288, 2000), we demonstrate that the minus-strand templates of tombusviruses contain a replication enhancer, which can upregulate RNA synthesis initiating from the minimal plus-strand initiation promoter by 10- to 20-fold in an in vitro assay. Dissection of the sequence of the replication enhancer element revealed that the two stem-loop structures present within the approximately 80-nucleotide-long enhancer region have interchangeable roles in upregulating RNA synthesis. The single-stranded sequence located between the two stem-loops also plays an important role in stimulation of RNA synthesis. We also demonstrate that one of the two hairpins, both of which are similar to the hairpin of the minus-strand initiation promoter, can function as a promoter in vitro in the presence of short cytidylate-containing initiation sites. Overall, the in vitro data presented are consistent with previous in vivo results (D. Ray and K. A. White, Virology 256:162-171, 1999) and they firmly establish the presence of a replication enhancer on the minus-stranded RNA of tombusviruses.

FIG. 1.

Enhancement of template activity of tombusvirus RdRp by conserved regions of a TBSV-associated DI RNA. (A) Schematic representation of the RNA genome of a prototypical DI RNA (DI-72) and the RNA constructs tested. Region I (169 nucleotides) is derived from the 5′ nontranslated region of the genomic TBSV RNA. Region II is 239 nucleotides and originated from the coding region (the p92 ORF), while region III (82 nucleotides) represents the end of the p22 ORF plus part of the 3′ noncoding region. Region IV is 131 nucleotides long and is derived from the very 3′-terminal noncoding region of the TBSV RNA. The previously characterized 11-nucleotide-long cPR11 promoter (represented by a triangle) was fused to the 3′ end of each segment, both plus and minus stranded, to generate seven constructs. The triangles point leftward in the plus-stranded constructs and rightward in the minus-stranded constructs. Note that region I(−) originally contained cPR11 at its 3′ end, and therefore no additional cPR11 was fused to that construct [R1(−)/cPR11]. Also, region IV(+) was not tested because that segment already contains a promoter sequence (termed gPR). (B) Relative template activities of the above RNA constructs in an in vitro tombusvirus RdRp assay. The radiolabeled RdRp products, synthesized by in vitro transcription with CNV RdRp, were analyzed on denaturing gels, quantified with a phosphoimager and normalized to the number of templated urilydates ([³²P]UTP was used for labeling in the RdRp reaction). RNA templates were used in equal molar amounts. Half of the RdRp products were treated with single-strand-specific RNase to confirm the double-stranded nature of the RdRp products (not shown) (16). The template activity of the construct MDV(−)/cPR11 (Fig. 2A), which contained a 221-nucleotide-long minus-stranded satellite RNA sequence derived from the unrelated bacteriophage Qβ (1) fused to cPR11 at the 3′ end, was set at 100% in these studies. Each experiment was repeated two or three times.

FIG. 2.

Region III(−) enhances RNA synthesis by the CNV RdRp when present on a heterologous template. (A) Schematic representation of the constructs tested in in vitro CNV RdRp assays. cPR11 is shown with a triangle, while the minus-stranded MDV RNA is represented with a black box. The 82-nucleotide-long region III(−) sequence is shown with a gray box. The sequences are shown in the 3′ to 5′ orientation because they represent minus-stranded sequences. (B) Representative denaturing gel analyses of radiolabeled RNA products synthesized by in vitro transcription with CNV RdRp. Arrows point to the RdRp products generated by de novo initiation from the 3′ terminus, while asterisks depict products that were generated by self-priming from the 3′ end. Note that the de novo products are RNase insensitive (R, RNase-treated; —, untreated samples), while the self-primed products are partially RNase sensitive. The self-primed products move faster in the untreated samples due to their highly stable secondary structure (hairpin structures [16]). The positions of the molecular size markers are shown on the right (in nucleotides). The relative efficiency of template activities (only the de novo products that are pointed at by the arrows were measured) is shown at the bottom. Each experiment was repeated three times.

FIG. 3.

Replacing region III(−) sequences with artificial sequences reduces template activities. (A) The actual sequences of the constructs tested are shown in the 3′ to 5′ orientation. The predicted secondary structures, based on the M-fold program (12), are shown: the two stem-loop structures, SL1-III(−) and SL2-III(−), and the cPR11 promoter are boxed. A 6-nucleotide-long region, termed the bridge, within the single-stranded portion of region III(−) that may interact with the cPR11 promoter is shown in a black box. (B) Representative denaturing gel analyses of radiolabeled RNA products synthesized by in vitro transcription with CNV RdRp. Arrows point to the RdRp products generated by initiation from the 3′ terminus. Note that the RdRp products obtained with the GC-rich templates migrate slightly aberrantly under the condition used, possibly due to their unusual structure. (C) Relative template activities of the above RNA constructs in an in vitro tombusvirus RdRp assay. The results were normalized as described in the legend to Fig. 1.

FIG. 4.

Region III replication enhancer can enhance RNA synthesis from the minus-strand initiation promoter in vitro. (A) Schematic representation of the constructs tested in the in vitro CNV RdRpassays. The 19-nucleotide-long core minus-strand initiation promoter, gPR, is shown with a black triangle, while the minus-stranded MDV sequence (see Fig. 2) is indicated with a black box. Sequences representing region III(+) and region III(−) are shown with light and dark gray boxes, respectively. The constructs are drawn in the 3′ to 5′ orientation. (B) Representative denaturing gel analyses of radiolabeled RNA products synthesized by in vitro transcription with CNV RdRp. Arrows point to the RdRp products generated by de novo initiation from the 3′ terminus, while asterisks depict products that were generated by self-priming from the 3′ end. See the legend to Fig. 2 for details. (C) Relative template activities of the constructs shown in panel A (only the de novo products that were pointed at by the arrows were measured). See the legend to Fig. 1 for further details.

FIG. 5.

SL2-III(−) hairpin functions as a replication enhancer in the in vitro CNV RdRp assay. (A) Schematic representation of the series of deletion constructs generated from R3(−)/cPR11 (Fig. 1). The actual sequence and predicted secondary structure of R3(−)/cPR11 are shown on the top (see Fig. 3 for detailed description of the individual sequence elements) in the 3′ to 5′ orientation. Sequences present in the constructs are indicated with gray bars. The gray triangle represents the cPR11 promoter sequence. The names of the constructs indicate the lengths of deletions. (B) Representative denaturing gel analyses of radiolabeled RNA products synthesized by in vitro transcription with CNV RdRp. Arrows point to the RdRp products generated by initiation from the 3′ terminus. (C) Relative template activities of the above RNA constructs in the in vitro tombusvirus RdRp assay. The results were normalized as described in the legend to Fig. 1.

FIG. 6.

SL1-III(−) hairpin functions as a replication enhancer in the in vitro CNV RdRp assay. (A) Schematic representation of the series of deletion constructs generated from construct R3(−)Δ10/cPR, which was derived from construct Δ10 (Fig. 5A) by a 2-nucleotide deletion in the cPR11 promoter sequence (termed cPR11Δ2; boxed). Note that cPR11Δ2 is as active as cPR11 in the presence of SL1-III(−) (see Fig. 8). The actual sequence and predicted secondary structure of construct R3(−)Δ10/cPR are shown on the top (see Fig. 3 for detailed description of the individual sequence elements) in the 3′ to 5′ orientation. Sequences present in the constructs are indicated with gray bars. The names of the constructs indicate the lengths of deletions. (B) Representative denaturing gel analyses of radiolabeled RNA products synthesized by in vitro transcription with CNV RdRp. Arrows point to the RdRp products generated by initiation from the 3′ terminus. (C) Relative template activities of the above RNA constructs in the in vitro tombusvirus RdRp assay. The results are normalized as described in the legend to Fig. 1.

FIG. 7.

Secondary structure of the SL1-III(−) hairpin plays a role in stimulation of RNA synthesis in the in vitro CNV RdRp assay. (A) The actual sequence and predicted secondary structure of construct Δ10/Δ22 (see Fig. 6) are shown on the top in the 3′ to 5′ orientation. Note that letters in a black box represents a 2-nucleotide deletion version of cPR11 (see Fig. 8), and the SL1-III(−) region is boxed with a dotted line. The 11 constructs are identical to Δ10/Δ22 except for the sequences shown, which replaced SL1-III(−) in each construct. Gray letters indicate mutated nucleotides, while black letters represent nucleotides present in SL1-III(−). (B) Representative denaturing gel analyses of radiolabeled RNA products synthesized by in vitro transcription with CNV RdRp. Arrows point to the RdRp products generated by initiation from the 3′ terminus. Note that a few RNAs move slightly aberrantly in the gels under the conditions used due to their unusual sequence or structure (for example, S-GC in lane 10). (C) Relative template activities of the above RNA constructs in the in vitro tombusvirus RdRp assay. The results were normalized as described in the legend to Fig. 1.

FIG. 8.

Region III(−) replication enhancer facilitates RNA synthesis in the presence of an initiation sequence. (A) The actual sequence and predicted secondary structure of construct Δ10 [see Fig. 5A; indicated here as R3(−)Δ10] is shown in the 3′ to 5′ orientation. The individual sequence elements are shown as in Fig. 3. The five constructs are identical to construct R3(−)Δ10 except for the deletions within the cPR11 region (the deleted sequences are indicated by dashes). (B) Representative denaturing gel analyses of radiolabeled RNA products synthesized by in vitro transcription with CNV RdRp. Arrows point to the RdRp products generated by initiation from the 3′ terminus. (C) Relative template activities of the above RNA constructs in the in vitro tombusvirus RdRp assay. The results were normalized as described in the legend to Fig. 1.