Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (−)-sense genomic RNA

Abstract

The genomes of many (+)-stranded RNA viruses, including plant viruses and alphaviruses, consist of polycistronic RNAs whose internal genes are expressed via subgenomic messenger RNAs. The mechanism(s) by which these subgenomic mRNAs arise are poorly understood. Based on indirect evidence, three models have been proposed: (1) internal initiation by the replicase on the (-)-strand of genomic RNA, (2) premature termination during (-)-strand synthesis, followed by independent replication of the subgenomic RNA and (3) processing by nuclease cleavage of genome-length RNA. Using an RNA-dependent RNA polymerase (replicase) preparation from barley leaves infected with brome mosaic virus (BMV) to synthesize the viral subgenomic RNA in vitro, we now provide evidence that subgenomic RNA arises by internal initiation on the (-)-strand of genomic RNA. We believe that this also represents the first in vitro demonstration of a replicase from a eukaryotic (+)-stranded RNA virus capable of initiating synthesis of (+)-sense RNA.

Sequence-specific recognition of a subgenomic RNA promoter by a viral RNA polymerase

“…1B, lanes [1][2][3][4][5][6][7][8][9] and are the same size (13 and 14 nt) as T7 size markers. However, no product was synthesized from the ϩ1 C͞G proscript (Fig.…”

“…The BMV genome is comprised of three RNAs designated 1, 2, and 3, and a subgenomic RNA4 which is initiated from (Ϫ)-strand RNA3. Enriched BMV RdRp preparations from infected barley can, in a highly specific manner, synthesize (Ϫ)-strand RNA from (ϩ)-strand templates and subgenomic (ϩ)-strand products from (Ϫ)-strand templates (4)(5)(6).…”

Specific site selection in RNA resulting from a combination of nonspecific secondary structure and -CCR- boxes: Initiation of minus strand synthesis by turnip yellow mosaic virus RNA-dependent RNA polymerase

“…Specific RNA recognition by proteins underlies many biological processes that demand high-fidelity performance+ In the best-described examples, highly specific outcomes are the result of the recognition of RNA elements that comprise short sequence-specific features placed in a defined structural framework+ Thus, specific aminoacylation of transfer RNAs by the cognate aminoacyl-tRNA synthetase requires the accurate placement of a few identity nucleotides within the generic tRNA structure that is approximately 76 nt long (Pallanck et al+, 1995)+ Shorter elements (ca+ 20-30 nt long) recognized by combined structural and sequence information are the helix/loop combinations bound by the HIV Tat protein (Puglisi et al+, 1992), phage R17/MS2 coat protein (Valegård et al+, 1994), and U1A spliceosomal protein (Oubridge et al+, 1994)+ This is not the only way in which proteins recognize specific sites on RNA, however+ For instance, evidence exists that the phage T4 translational repressor, regA, recognizes a consensus sequence of some 12-15 linear nucleotides in unstructured RNA (Szewczak et al+, 1991;Brown et al+, 1997)+ Here, as a result of studying the replication of a positive strand RNA virus, we report a further strategy for specific RNA recognition that requires a combination of a very short specific-sequence and adjacent non-or low-specificity secondary structure+ Positive strand RNA viruses replicate via two sequential transcriptional steps that synthesize full-length minus and plus sense genomes; additionally, in some viruses, internal initiation on the minus strand (Miller et al+, 1985) results in the synthesis of subgenomic RNAs that are collinear with the 39-region of the genomic RNA and that serve as mRNAs expressing downstream cistrons (Buck, 1996)+ Specific initiation sites are used for each of these transcriptional events, and a breakdown of the fidelity of initiation site selection would lead to truncated genomes and viral proteins+ The cisrequired promoter elements controlling these specific strand initiations have been studied in a number of viruses by in vivo approaches using deleted genomes and by in vitro approaches using viral RNA-dependent RNA polymerase (RdRp) preparations (Buck, 1996)+ These studies have generally supported the view that accurate transcription is controlled by the detection of specific features of either sequence or a combination of sequence and structure (e+g+, Miller et al+, 1986;Dreher & Hall, 1988;Levis et al+, 1990;Cui & Porter, 1995;Song & Simon, 1995;Miranda et al+, 1997;Siegel et al+, 1997)+ Against this backdrop of apparently specific recognition of defined, discrete promoter elements, we have studied the features directing minus strand synthesis by the turnip yellow mosaic virus (TYMV) RdRp+ Minus strand synthesis initiates specifically opposite the penultimate resi...…”

Replication of tobacco mosaic virus RNA

“…There is no evidence that subgenomic RNAs are self-replicating. TMV subgenomic RNAs are likely to be synthesized by internal initiation on genome-length negative-strand RNA templates, which requires subgenomic promoters containing sequences not found in the subgenomic RNA itself, as shown for brome mosaic virus (Miller et al 1985;Marsh et al 1988;Siegel et al 1997;Adkins et al 1998). Subgenomic double-stranded RNAs are therefore probably dead-end products formed as a result of synthesis of a negative strand on a subgenomic positive-strand RNA template.…”