Conundrum of the Lack of Defective RNAs (dRNAs) Associated with Tobamovirus Infections: dRNAs That Can Move Are Not Replicated by the Wild-Type Virus; dRNAs That Are Replicated by the Wild-Type Virus Do Not Move

Defective RNAs (dRNAs) are RNA molecules that arise through deletion of internal sequences from the genome of a virus. Since dRNAs generally are incapable of autonomous replication, they depend on trans-acting factors from a helper virus for replication (12, 15, 34, 40). Some dRNAs interfere with replication of the helper virus and thus are referred to as defective interfering (DI) viruses. dRNAs are an intrinsic and common feature among animal RNA viruses (reviewed in references 19, 23, and 27). However, the majority of plant virus genera have not been associated with dRNAs in nature. There is no information concerning the principles of propagation of dRNAs among different viral groups. It has been suggested that certain viral mechanisms that act to suppress the formation and/or accumulation of defective genomes have likely evolved (40). High-multiplicity transfers foster accumulation of dRNAs. Thus, low-multiplicity infections of plant cells resulting from cell-to-cell and long-distance movement of the virus throughout the plants might prevent the survival of dRNAs in populations (3, 40). However, there might also be differences in replication strategies of different dRNAs that affect their ability to compete and survive in populations. Although the Tobamovirus genus is one of the most examined groups of viruses, dRNAs have never been reported in a natural tobamovirus isolate. However, a naturally occurring satellite virus is common in natural populations of tobacco mild green mosaic virus strains in Nicotiana glauca, thus demonstrating that tobamovirus replicases have evolved the ability to support replication of RNA molecules in trans (37). Tobacco mosaic virus (TMV), the type member of the genus, expresses its genetic information from a positive-sense monopartite RNA molecule 6,395 nucleotides (nt) in length (Fig. ​(Fig.1A).1A). The 126-kDa protein is translated from the first open reading frame (ORF) within the genomic RNA, and the 183-kDa protein is expressed via occasional readthrough of an amber stop codon (33). The 126-kDa protein is produced at levels 10 to 20 times higher than those at which the 183-kDa protein is produced. However, mutation of the stop codon to only allow the translation of the 183-kDa protein did not increase accumulation of the 183-kDa protein (18, 21). Both proteins associate at a 1:1 ratio to form a viral replication complex that retains activity in vitro, suggesting that a heterodimer is the active form (39). Nonetheless, the 183-kDa protein alone is sufficient for replication in protoplasts (18, 21). The movement protein (MP) and coat protein (CP) are expressed via individual 3′-coterminal subgenomic RNAs from the two 3′-proximal ORFs. TMV MP and CP are dispensable for replication in protoplasts (18, 21) but are necessary for cell-to-cell and long-distance movement in tobacco (6, 41). FIG. 1 (A) Schematic diagram of the genomic organization of TMV-based helper viruses. Locations of POL domain in TMV and stop codon replacement in 183F are indicated. The movement phenotype of helper virus (right) is indicated as follows: +, wt; (+), ... Tobamovirus-based dRNAs that can be replicated efficiently have been created artificially. However, sequences removed or retained greatly affected their ability to be replicated. Removal of the polymerase (POL) domain from the dRNA genome appears to be a minimum requirement to obtain viable dRNAs from TMV and tomato mosaic virus (20, 25, 26). When sequences in addition to the POL domain were removed, the levels of accumulation of artificial dRNAs varied considerably (20, 26). dRNAs containing the entire 126-kDa protein ORF sequences appeared to require the functional 126-kDa protein in cis for replication (21). Wild-type (wt) TMV and replication-competent TMV mutants can support the artificially constructed dRNAs without notable interference with replication of the helper virus in protoplasts (20). We investigated the performance of two classes of artificially constructed TMV-based dRNAs in planta. dRNAs that accumulated to high levels in protoplasts in combination with wt TMV-like viruses which produce both replicase-associated proteins lacked the ability to move in plants. dRNAs that were able to move in plants were not efficiently replicated by the wt virus. A mutant helper virus deficient in production of the 126-kDa protein replicated and moved the class of dRNAs that encoded a functional 126-kDa protein. Most importantly, the full-length TMV mutant deficient in the 126-kDa protein also generated dRNAs de novo in plants. This is the first report of dRNAs arising de novo within the genus Tobamovirus. These data provide an explanation for the absence of dRNAs associated with tobamovirus infections, namely, that in wild populations dRNAs supported by the helper virus cannot move in plants and that dRNAs that can move are not efficiently replicated by the wt virus.