The infectivities of turnip yellow mosaic virus genomes with altered tRNA mimicry are not dependent on compensating mutations in the viral replication protein.

Five highly infectious turnip yellow mosaic virus (TYMV) genomes with sequence changes in their 3'-terminal regions that result in altered aminoacylation and eEF1A binding have been studied. These genomes were derived from cloned parental RNAs of low infectivity by sequential passaging in plants. Three of these genomes that are incapable of aminoacylation have been reported previously (J. B. Goodwin, J. M. Skuzeski, and T. W. Dreher, Virology 230:113-124, 1997). We now demonstrate by subcloning the 3' untranslated regions into wild-type TYMV RNA that the high infectivities and replication rates of these genomes compared to their progenitors are mostly due to a small number of mutations acquired in the 3' tRNA-like structure during passaging. Mutations in other parts of the genome, including the replication protein coding region, are not required for high infectivity but probably do play a role in optimizing viral amplification and spread in plants. Two other TYMV RNA variants of suboptimal infectivities, one that accepts methionine instead of the usual valine and one that interacts less tightly with eEF1A, were sequentially passaged to produce highly infectious genomes. The improved infectivities of these RNAs were not associated with increased replication in protoplasts, and no mutations were acquired in their 3' tRNA-like structures. Complete sequencing of one genome identified two mutations that result in amino acid changes in the movement protein gene, suggesting that improved infectivity may be a function of improved viral dissemination in plants. Our results show that the wild-type TYMV replication proteins are able to amplify genomes with 3' termini of variable sequence and tRNA mimicry. These and previous results have led to a model in which the binding of eEF1A to the 3' end to antagonize minus-strand initiation is a major role of the tRNA-like structure.

Transfer RNA mimicry among tymoviral genomic RNAs ranges from highly efficient to vestigial.

Three tRNA-associated properties of a representative set of tymoviral RNAs have been quantitatively assessed using higher plant (wheat germ) proteins: aminoacylation, EF-1alpha*GTP binding, and 3'-adenylation of 3'-CC forms of the RNAs by CTP, ATP:tRNA nucleotidyltransferase. The RNAs fall into three classes differing in the extent of tRNA mimicry. Turnip yellow mosaic (TYMV) and kennedya yellow mosaic virus RNAs had activities in all three properties similar to those of a higher plant tRNAValtranscript, and thus are remarkable tRNA mimics. Although the isolated approximately 83 nt long tRNA-like structures showed high activity in these assays, in the case of TYMV, the 6318 nt long TYMV RNA was an even better substrate for valylation. Eggplant mosaic virus RNA, which has a differently constructed acceptor stem pseudoknot, differed from the above tymoviral RNAs in binding more weakly to EF-1alpha*GTP. Erysimum latent virus RNA, which lacks an identifiable anticodon domain, could not be valylated and had very low 3'-adenylation activity. The range of tRNA mimicry within the tymovirus genus thus ranges from extremely highly developed to minimal. The implications on the role of the tRNA mimicry in viral biology are discussed.

Transfer RNA mimicry in a new group of positive-strand RNA plant viruses, the furoviruses: differential aminoacylation between the RNA components of one genome.

Recent sequencing of the genomes of several furoviruses--fungus-transmitted rod-shaped positive-strand plant viruses--has suggested the presence of tRNA-like structures (TLSs) at the 3' ends of the genomic RNAs. We show here that the genomic RNAs of soil-borne wheat mosaic virus (SBWMV), beet soil-borne virus (BSBV), potato mop-top virus (PMTV), peanut clump virus (PCV), and Indian peanut clump virus (IPCV) all possess functional TLSs that are capable of high-efficiency valylation. While the SBWMV, BSBV, and PMTV TLSs are similar to those found in tymoviruses, the PCV and IPCV TLSs harbor an insertion of about 40 nucleotides between the two halves of the TLS. The valylated SBWMV and BSBV RNAs formed tight complexes with wheat germ EF-1 alpha.GTP (Kd = 2 to 11 nM), whereas valylated PMTV, PCV, and IPCV RNAs bound EF-1 alpha.GTP weakly (Kd > or = 50 nM). The TLS of PCV RNA2 differs from PCV RNA1 in lacking the major valine identity nucleotide in the anticodon and consequently is capable of only very inefficient valylation. This is the first case of differential aminoacylation between the RNA components of one genome.

Characterization of chimeric turnip yellow mosaic virus genomes that are infectious in the absence of aminoacylation.

Previous experiments have characterized the chimeric genome TYMC-TMVPSK, in which the 3'-tRNA-like structure of turnip yellow mosaic virus (TYMV) was replaced by 3' sequences from tobacco mosaic virus. This genome accumulated in turnip protoplasts to a level about 3% of wild type, but was not infectious on plants. In the present study, TYMV sequences introduced into the anticodon loop and amino acid acceptor arm of the 3' region of this chimera led to three- to fourfold increases in viral accumulation. Two such modified chimeric genomes gave rise to stable infections in plants. After further passaging in plants and the accumulation of minor sequence changes in the 3' terminal region, the resultant viruses, TYMC-XX and TYMC-YY, were highly infectious. Viral accumulations in protoplasts were about 40% of wild type on the basis of coat protein levels, and virion yields in plants were about 0.1 mg/g leaf. Extensive assays failed to detect aminoacylation of these genomic RNAs in vitro, but they were active substrates for wheat germ. CCA nucleotidyltransferase. In separate experiments, the 3'-tRNA-like structure of TYMV RNA was replaced by the 3' terminal 96 nucleotides from erysimum latent tymovirus RNA, resulting in a genome that was infectious to plants (isolate TYMC-H). This chimeric virus produced similar symptoms and virion yield in plants as TYMC-XX and -YY, although accumulations of coat protein in protoplasts were 13% of wild type. The viral RNA was a poor substrate for CCA nucleotidyltransferase and could not be aminoacylated. TYMC-XX, -YY, and -H are the first TYMV replicons known to amplify efficiently and infect plants in the absence of aminoacylation. Their viability suggests that other properties can compensate for the absence of aminoacylation.