Artificial Defective Interfering RNAs Derived from Brome Mosaic Virus

Abstract

Naturally occurring defective interfering RNAs (DIRNAs) greatly reduce the accumulation of their helper virus in vivo, but are rarely associated with plant positive-strand RNA viruses. Deletion mutants pRNA-2 M/S and pRNA-2 E/S, derived from brome mosaic virus (BMV) genomic RNA-2, replicated in a manner dependent on BMV RNA-1 and -2, and effectively interfered with their accumulation in barley protoplasts. Based on their mode of replication, these mutant RNAs have been termed parasitic RNAs (pRNAs). When present with RNA-1 and -2 at low inoculum amounts, pRNA-2 M/S and pRNA-2 E/S reduced the level of replication of RNA-2, the parental RNA, by 37 % and 64 %, respectively. Greater amounts of pRNA in the inoculum completely eliminated the replication of both RNA-I and -2. Mutations that prevented translation of truncated proteins from the pRNAs did not affect interference, but those that reduced pRNA replication decreased their ability to interfere with genomic RNA replication. At a molar pRNA: genomic RNA inoculum ratio of 1.5: 1, pRNA-2 E/S reduced the accumulation of all helper virus RNAs by > 60%. This occurred in the presence of wild-type RNA-3 or ASGP RNA-3, a deletion mutant of RNA-3 that lacks the subgenomic promoter necessary for coat protein expression, demonstrating that the interference mediated by the pRNAs was not effected by encapsidation. These data indicate that the expression of pRNAs that function as artificial DIRNAs in transgenic plants may be an approach for inducing resistance to virus infection which is applicable to a wide range of plant viruses.

Non-replicating deletion mutants of brome mosaic virus RNA-2 interfere with viral replication

“…5d) showing minus-strand progeny. Under these conditions, RNAs-3 and -4 constitute 80 ~ of the total plus-strand progeny (Marsh et al, 1991a). In the inoculations containing NRI RNA-2 Nc/S, both the preferential debilitation of minus-strand synthesis and the characteristic reduction in accumulation of the parental genomic RNA-2 are evident in the presence of wt RNA-3 or ASGP RNA-3 (Fig.…”

“…Although RNA-3 is not required for replication of RNAs-1 and -2 in protoplasts, its presence markedly affects the ratio (symmetry) of plus :minus strand R N A accumulation (Marsh et al, 1991a). Whereas progeny plus:minus ratios are close to unity in the absence of RNA-3 or in the presence of a mutant (ASGP RNA-3) lacking the subgenomic promoter core, they are approximately 100:1 in the presence of wt RNA-3.…”

Rna-Rna Recombination and Evolution in Virus-Infected Plants

“…Based on the sequence of DI RNAs associated with animal viruses, their generation can be explained by errors made during RNA replication (45). DI RNAs are ubiquitous among animal RNA viruses and have been found in several plant viruses including bromoviruses (75,98), carmoviruses (69,104), furoviruses (14), hordeiviruses (49), potexviruses (113,114), rhabdo viruses (48), tombusviruses (21,44,51,61), and tomato spotted wilt virus, a bunyavirus (94). Essentially, these events can be defined as recombination events that occur either intrarno-lecularly (detaching-reinitiation) or intermolecularly (copying back, looping out).…”

Resistance to plant viruses: Obtaining genes by non-conventional approaches

“…Although not yet demonstrated in transgenic plants for RNA viruses, in vitro studies with turnip yellow mosaic virus (Morch et al ., 1987) and BMV (Marsh et al ., 1991) show promise for this approach .…”