Autonomous role of 3'-terminal CCCA in directing transcription of RNAs by Qbeta replicase.

We have studied transcription in vitro by Qbeta replicase to deduce the minimal features needed for efficient end-to-end copying of an RNA template. Our studies have used templates ca. 30 nucleotides long that are expected to be free of secondary structure, permitting unambiguous analysis of the role of template sequence in directing transcription. A 3'-terminal CCCA (3'-CCCA) directs transcriptional initiation to opposite the underlined C; the amount of transcription is comparable between RNAs possessing upstream (CCA)(n) tracts, A-rich sequences, or a highly folded domain and is also comparable in single-round transcription assays to transcription of two amplifiable RNAs. Predominant initiation occurs within the 3'-CCCA initiation box when a wide variety of sequences is present immediately upstream, but CCA or a closely similar sequence in that position results in significant internal initiation. Removal of the 3'-A from the 3'-CCCA results in 5- to 10-fold-lower transcription, emphasizing the importance of the nontemplated addition of 3'-A by Qbeta replicase during termination. In considering whether 3'-CCCA could provide sufficient specificity for viral transcription, and consequently amplification, in vivo, we note that tRNA(His) is the only stable Escherichia coli RNA with 3'-CCCA. In vitro-generated transcripts corresponding to tRNA(His) served as poor templates for Qbeta replicase; this was shown to be due to the inaccessibility of the partially base-paired CCCA. These studies demonstrate that 3'-CCCA plays a major role in the control of transcription by Qbeta replicase and that the abundant RNAs present in the host cell should not be efficient templates.

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.

The valine anticodon and valylatability of Peanut clump virus RNAs are not essential but provide a modest competitive advantage in plants.

The role of valine aminoacylation of the two genomic RNAs of Peanut clump virus (PCV) was studied by comparing the amplification in vivo of RNAs with GAC, GDeltaC, or CCA anticodons in the tRNA-like structure (TLS) present at the 3' end of each viral RNA. The PCV RNA1 TLS of isolate PCV2 possesses a GAC anticodon and is capable of highly efficient valylation, whereas the RNA2 TLS has a GDeltaC anticodon that does not support valylation. The presence in RNA1 of GDeltaC or CCA anticodons that conferred nonvalylatability resulted in about 2- to 4-fold and a 14- to 24-fold reduction, respectively, in RNA accumulations in tobacco BY-2 protoplasts inoculated with the RNA1 variants together with wild-type RNA2(GDeltaC). No differences in RNA levels were observed among protoplasts inoculated with the three variant RNA2s in the presence of wild-type RNA1(GAC). All combinations of valylatable and nonvalylatable RNAs 1 and 2 were similarly infectious in Nicotiana benthamiana plants, and viral RNAs accumulated to similar levels; all input TLS sequences were present unchanged in apical leaves. In direct competition experiments in N. benthamiana plants, however, both RNA1 and RNA2 with GAC valylatable anticodons outcompeted the nonvalylatable variants. We conclude that valylation provides a small but significant replicational advantage to both PCV RNAs. Sequence analysis of the TLS from RNA2 of a second PCV isolate, PO2A, revealed the presence of an intact GAC valine anticodon, suggesting that the differential valylation of the genomic RNAs of isolate PCV2 is not a general characteristic of PCV.

CCA initiation boxes without unique promoter elements support in vitro transcription by three viral RNA-dependent RNA polymerases.

It has previously been observed that the only specific requirement for transcriptional initiation on viral RNA in vitro by the RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus is the CCA at the 3' end of the genome. We now compare the abilities of this RdRp, turnip crinkle virus RdRp, and Qbeta replicase, an enzyme capable of supporting the complete viral replication cycle in vitro, to transcribe RNA templates containing multiple CCA boxes but lacking specific viral sequences. Each enzyme is able to initiate transcription from several CCA boxes within these RNAs, and no special reaction conditions are required for these activities. The transcriptional yields produced from templates comprised of multiple CCA or CCCA repeats relative to templates derived from native viral RNA sequences vary between 2:1 and 0.1:1 for the different RdRps. Control of initiation by such redundant sequences presents a challenge to the specificity of viral transcription and replication. We identify 3'-preferential initiation and sensitivity to structural presentation as two specificity mechanisms that can limit initiation among potential CCA initiation sites. These two specificity mechanisms are used to different degrees by the three RdRps. The finding that three viral RdRps representing two of the three supergroups within the positive-strand RNA viral RdRp phylogeny support substantial transcription in the absence of unique promoters suggests that this phenomenon may be common among positive-strand viruses. A framework is presented arguing that replication of viral RNA in the absence of unique promoter elements is feasible.

Internal and 3' RNA initiation by Qbeta replicase directed by CCA boxes.

RNA initiation by Qbeta replicase directed by the short-sequence CCA at the 3'-end of all RNAs amplified by this enzyme has been studied. Most CCA repeats in an RNA consisting of 12 CCAs serve as independent sites of de novo RNA initiation, with initiation occurring opposite the 3'-C residue of each CCA. Qbeta replicase is thus capable of internal initiation remote from the 3'-end, although predominant initiation occurs close to the 3'-end. The precise 3'-terminal sequence in (CCA)(n)-containing RNAs influences the number and position of active initiation sites near the 3'-terminus. C residues are required at the initiation site, whereas the position of purines (especially A residues) influences the selection of initiation sites. The template activity of (CCA)(n) RNAs is positively correlated with the number of CCA repeats. Three CCA repeats added to the 3'-end of a nontemplate 83-nt RNA are sufficient to activate transcription by Qbeta replicase. These experiments show that CCA boxes can act as strong initiation sites in the absence of specific cis-acting signals derived from Qbeta RNA, although the efficiency of initiation is modulated by surrounding sequence.

Quantitative assessment of EF-1alpha.GTP binding to aminoacyl-tRNAs, aminoacyl-viral RNA, and tRNA shows close correspondence to the RNA binding properties of EF-Tu.

A ribonuclease protection assay was used to determine the equilibrium dissociation constants (Kd) for the binding of various RNAs by wheat germ EF-1alpha.GTP. Aminoacylated fully modified tRNAs and unmodified tRNA transcripts of four specificities (valyl, methionyl, alanyl, and phenylalanyl) from higher plants or Escherichia coli were bound with Kd values between 0.8 and 10 nM. A valylated 3'-fragment of turnip yellow mosaic virus RNA, which has a pseudoknotted amino acid acceptor stem, was bound with affinity similar to that of Val-tRNAVal. Uncharged tRNA and initiator Met-tRNAMet from wheat germ, RNAs that are normally excluded from the ribosomal A site in vivo, bound weakly. The discrimination against wheat germ initiator Met-tRNAMet was almost entirely due to the 2'-phosphoribosyl modification at nucleotide G64, since removal resulted in tight binding by EF-1alpha.GTP. A 44-nucleotide RNA representing a kinked acceptor/T arm obtained by in vitro selection to bacterial EF-Tu formed an Ala-RNA.EF-1alpha.GTP complex with a Kd of 29 nM, indicating that much of the binding affinity for aminoacylated tRNA is derived from interaction with the acceptor/T half of the molecule. The pattern of tRNA interaction observed for EF-1alpha (eEF1A) therefore closely resembles that of bacterial EF-Tu (EF1A).

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.

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.

A turnip yellow mosaic virus RNA-dependent RNA polymerase activity was used to study the template requirements for in vitro minus strand synthesis, which is initiated specifically opposite the 3'-CCA that terminates the 3'-tRNA-like structure. A deletion survey confirmed earlier results suggesting the absence of minus strand promoter elements upstream of the pseudoknotted acceptor stem and 3'-terminus. Reiteration of this 27-nt domain provided two competing initiation sites. By varying the added downstream element, it was shown that the pseudoknotted domain could be functionally replaced by various simple stem/loops, although with some decrease in activity. The addition of varying numbers of consecutive -CCA- triplets to the 3' end of the tRNA-like structure resulted in accurate initiation from each added triplet. A similar spectrum of initiations occurred with an unstructured RNA consisting of 12 consecutive -CCA- triplets and no additional viral sequence. Substitution mutations revealed no influence on minus strand synthesis of the identity of the nucleotide immediately upstream of a -CC- initiation site, but a preference for a purine immediately downstream. The introduction of secondary structure into the linear template showed that the usage of potential -CCR- initiation sites is influenced by nonspecific secondary structure. We conclude that specificity arises from the requirement that a -CCR- sequence be sterically accessible. This mechanism is only applicable to interactions that do not involve RNA unwinding during site selection, but may be used commonly in positive strand RNA virus replication and be applicable to other RNA-protein interactions.

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.

Turnip yellow mosaic virus RNA-dependent RNA polymerase: initiation of minus strand synthesis in vitro.

An RNA-dependent RNA polymerase (RdRp) activity was detergent-solubilized from the chloroplast membranes of Chinese cabbage leaves infected with turnip yellow mosaic virus (TYMV). The template-dependent, micrococcal nuclease-treated activity synthesized full-length minus strands from TYMV RNA and 3'-fragments as short as a 28-nucleotide-long RNA comprising the amino acid acceptor stem of the 3'-tRNA-like structure (TLS). Minus strands were shown to arise by de novo initiation with the insertion of GTP opposite the penultimate (C) residue of the 3'-terminal -CCA. The TYMV RdRp activity was template specific in that poly(A) RNA was not copied, and alfalfa mosaic virus (AIMV) RNA, which does not contain a 3'-TLS, was a very poor template. However, other viral RNAs with a 3'-TLS and in vitro transcripts of tRNAs were copied to varying degrees. Fully modified tRNAs were either inactive or poorly active templates, and AIMV 3'-RNA, even when provided with a 3'-terminal -ACCA, was not copied detectably. A potential role of the acceptor stem pseudoknot as a promoter element was assessed with mutations that drastically altered the structure and sequence of the pseudoknot, revealing only a twofold effect in decreasing template activity. The data show that RNAs with both a tRNA-like conformation and a -CCA 3'-terminus are potential templates for TYMV RdRp and suggest that promoter elements are not limited to the acceptor stem pseudoknot.

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.

Aminoacylation identity switch of turnip yellow mosaic virus RNA from valine to methionine results in an infectious virus.

The turnip yellow mosaic virus genomic RNA terminates at its 3' end in a tRNA-like structure that is capable of specific valylation. By directed mutation, the aminoacylation specificity has been switched from valine to methionine, a novel specificity for viral tRNA-like structures. The switch to methionine specificity, assayed in vitro under physiological buffer conditions with wheat germ methionyl-tRNA synthetase, required mutation of the anticodon loop and the acceptor stem pseudoknot. The resultant methionylatable genomes are infectious and stable in plants, but genomes that lack strong methionine acceptance (as previously shown with regard to valine acceptance) replicate poorly. The results indicate that amplification of turnip yellow mosaic virus RNA requires aminoacylation, but that neither the natural (valine) specificity nor interaction specifically with valyl-tRNA synthetase is crucial.

The turnip yellow mosaic virus tRNA-like structure cannot be replaced by generic tRNA-like elements or by heterologous 3' untranslated regions known to enhance mRNA expression and stability.

The tRNA-like structure (TLS) at the 3' end of the turnip yellow mosaic virus genome was replaced with heterologous tRNA-like elements, and with a poly(A) tail, in order to assess its role. Replacement with the valylatable TLSs from two closely related tymoviruses resulted in infectious viruses. In contrast, no systemic symptoms on plants, and only low viral accumulations in protoplasts, were observed for three chimeric genomes with 3' sequences known to enhance mRNA stability and translatability. One of these chimeras had a poly(A) tail, and the others had the TLS with associated upstream pseudoknot tracts from the 3' ends of brome mosaic and tobacco mosaic viruses. The latter two chimeric RNAs were shown to be appropriately folded by demonstrating their aminoacylation in vitro with tyrosine and histidine, respectively. The results show that enhancement of genome stability or gene expression is not the major role of the turnip yellow mosaic virus TLS. The major role is likely to be replicational, dependent on features present in tymoviral TLSs but not in generic tRNA-like structures.

Identification of the cleavage site recognized by the turnip yellow mosaic virus protease.

The noncapsid protein expressed from ORF-206 of turnip yellow mosaic virus (TYMV) is autocatalytically processed by a papain-like protease, producing N-terminal 150-kDa and C-terminal 70-kDa proteins. By introducing two methionine residues near the N-terminus of the 70-kDa protein, we have obtained N-terminal amino acid sequence of that protein produced from [35S]methionine-labeled in vitro translations. The introduction of methionine residues was demonstrated to not interfere with viral replication or proteolysis, as assayed by inoculating mutant RNA transcripts onto whole plants and protoplasts, as well as by translating the RNAs in a rabbit reticulocyte lysate. This has allowed us to determine that the TYMV protease cleaves between alanine1259 and threonine1260 of the precursor protein p206, yielding proteins of calculated Mr 140,618 and 66,037, which will be referred to henceforth as p141 and p66, respectively. The sequence context around the cleavage site is LNGA/TP.

Coding density of the turnip yellow mosaic virus genome: roles of the overlapping coat protein and p206-readthrough coding regions.

More than one-third of the turnip yellow mosaic virus (TYMV) genome simultaneously encodes two ORFs. We have investigated the functions of the overlapping coat protein ORF and readthrough domain of ORF-206 in the 3' region of the genome. TYMC-206 RNA, in which a second stop codon has been positioned to prevent ORF-206 readthrough, induced infections in protoplasts and plants that were indistinguishable from wild type. ORF-206 readthrough is thus nonessential. Nevertheless, TYMV-221 RNA, in which the ORF-206 stop codon was replaced with a tyrosine codon to force readthrough, was infectious to protoplasts, suggesting that a role for ORF-206 readthrough under certain conditions is possible. TYMV RNA variants that produce truncated or no coat protein were used to show that the coat protein is dispensable for local movement but necessary for systemic spread of virus in plants. Studies in protoplasts showed that (-) RNA levels are normal in the absence of coat protein, but (+) strand levels are decreased about 10-fold relative to wild-type infections. A mutant with a short C-terminal coat protein extension that formed virions less stable than normal demonstrated the protective role of capsids toward genomic RNA. The evolutionary implications of the dense information content of the TYMV genome are discussed.

Cloning and sequencing of the major rainbow trout constitutive cytochrome P450 (CYP2K1): identification of a new cytochrome P450 gene subfamily and its expression in mature rainbow trout liver and trunk kidney.

We have cloned and sequenced a full-length cDNA encoding the major constitutive rainbow trout liver cytochrome P450 that we had earlier designated as cytochrome P450 LMC2 (Arch. Biochem. Biophys. 268, 227, 1989). The 1859-bp cDNA was isolated from a male rainbow trout liver cDNA library and contained an open reading frame encoding for a protein of 504 amino acids and having a calculated molecular weight of 56,795. From nucleotide and amino acid sequence comparisons, the trout P450 LMC2 has been assigned to a new cytochrome P450 gene subfamily designated P4502K1 or CYP2K1. Marked differences in the sex- and tissue-specific expression of this gene were found at both the transcriptional and translational level in sexually mature rainbow trout liver and trunk kidney. Transcriptional expression investigated by Northern analysis of total RNA using a 440-bp 3'-terminal cDNA probe (2K1,7c) showed sexual and organ differences. Mature male trunk kidney expressed 2K1,7c-hybridizable mRNA to a substantially greater extent than did female trunk kidney, with multiple mRNA bands appearing in approximately the 2.8- and 1.9-kb region. While the livers of some males displayed separate 2.8-kb hybridizable bands, such bands were generally undetectable in female liver samples. By contrast, 1.9-kb mRNA bands were found at generally similar concentrations in the livers of both sexes. Taking into consideration the individual biological variabilities, it was apparent that mature male trout expressed the 2.8-kb mRNA much more strongly in trunk kidney than in liver. Translational expression, analyzed by Western blotting of microsomes separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with rabbit polyclonal antibody and two different monoclonal antibodies prepared against P450 LMC2, demonstrated corresponding sex- and organ-related differences in P450 protein expression. Southern analysis of sexually mature male trout genomic DNA suggested that CYP2K1 is not a single copy gene, thus providing additional evidence for the complexity of the CYP2K1 system in rainbow trout.

Preferential replication of defective turnip yellow mosaic virus RNAs that express the 150-kDa protein in cis.

The turnip yellow mosaic virus genome encodes two proteins (the 150-kDa and 70-kDa proteins) that are proteolytically released from a single precursor and which are essential for RNA replication. Genomes with mutations in either of these coding regions were defective for independent replication in turnip protoplasts. The replication in trans of genomes with mutations in each region was studied by coinoculation with either a helper genome that carries a deletion in the coat protein gene, or with a second defective RNA that carries a mutation in the region encoding the other essential protein. Inefficient trans-replication of the defective RNAs was observed in most cases. In contrast, a defective RNA with a large deletion in the 70-kDa protein coding region could be replicated efficiently in trans, demonstrating that the cis-preference of replication can be overcome in some cases. Defective RNAs encoding wild type 150-kDa protein and defective 70-kDa protein were more efficiently replicated in trans than those encoding defective 150-kDa protein and wild type 70-kDa protein. The results suggest a model in which the 150-kDa and 70-kDa proteins form a relatively stable complex in cis on the viral RNA template.

Identification of the essential cysteine and histidine residues of the turnip yellow mosaic virus protease.

The nonstructural protein expressed from ORF-206 of turnip yellow mosaic virus is proteolytically processed to produce N-terminal 150-kDa and C-terminal 70-kDa proteins. Through the use of linker insertion and deletion analyses coupled to in vitro translation, we have delimited the protease domain to residues 731-885 encoded by the 150-kDa coding region of ORF-206. The effects of substitutions of conserved residues within this region were studied in two assays: a direct assay for proteolysis occurring during in vitro translation and an assay for viral replication in turnip protoplasts. Replication was shown to be dependent on proteolytic maturation by the failure of a mutant with an inactive protease cleavage/recognition site to amplify in protoplasts. Substitutions at Cys783 and His869 were the only ones that resulted in undetectable signals in both assays, indicating that these are probable active sites residues, most likely of a papain-like protease. Domains putatively encoding similar proteases were detected in the genomes of other tymoviruses and viruses related to tymoviruses. The putative active site cysteine residues of these domains are followed by an aliphatic amino acid, whereas an aromatic amino acid at this position is typical of cellular and previously characterized viral papain-like proteases.

Cis-preferential replication of the turnip yellow mosaic virus RNA genome.

The largest open reading frame of the turnip yellow mosaic virus RNA genome encodes a 206-kDa protein that is cleaved to yield N-terminal 150-kDa (p150) and C-terminal 70-kDa (p70) proteins. Using a genomic cDNA clone capable of generating infectious transcripts in vitro, we have introduced substitution, frameshift, and in-frame deletion mutations into the regions encoding both proteins. None of the mutant RNAs was able to replicate independently in turnip protoplasts, indicating that p150 and p70 are both essential. The replication in protoplasts of most of these defective RNAs was poorly supported in trans by a coinoculated helper genome with a deletion in the coat protein gene; replication could also be supported in trans by certain defective RNAs, but this complementation was likewise inefficient in most cases. The replication in trans was more efficient for defective RNAs encoding wild-type p150 and defective p70 than for those encoding defective p150 and wild-type p70. One defective RNA with a large deletion in the p70 coding region was able to replicate efficiently, both when inoculated with the helper genome and when inoculated with a second complementing defective RNA that supplied a wild-type p70. Thus, the cis preference of replication can be overcome in some cases. A model in which p150 and p70 form a complex with the 3' end of the RNA is proposed to explain the cis-preferential replication of turnip yellow mosaic virus RNA.

Increased viral yield and symptom severity result from a single amino acid substitution in the turnip yellow mosaic virus movement protein.

Turnip yellow mosaic virus is a positive-strand RNA virus that produces light green or yellow-green mosaic symptoms in Chinese cabbage plants. We have characterized a strain that produces nearly uniform yellow-green chlorosis in systemically infected Chinese cabbage leaves. The increased symptom severity is due to the single nucleotide substitution U1888-->C, which results in a tyrosine to histidine substitution in the movement protein encoded by ORF-69. Coding by the overlapping ORF-206 is not affected. The mutation results in fourfold higher accumulations of viral products in systemically infected Chinese cabbage leaves but does not affect viral replication in isolated protoplasts. These results suggest that the increased viral yield and symptom severity result from improved viral spread in the host plant. These effects were specific to Chinese cabbage, since neither viral yield nor symptoms in turnips were affected by the U1888-->C mutation.